Compositions and methods for detecting head and neck cancer - Patents.com

Abstract

To provide a composition and method for detecting head and neck cancer.SOLUTION: The present invention provides a method of detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual and includes a step of obtaining a sample from the individual, and a step of measuring the amount of expression product from a gene including at least one gene from among those in table 4 and / or table 6. In one embodiment, the gene is at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, and HSD17B6.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 608,296, filed December 20, 2017. The disclosure of U.S. Provisional Patent Application No. 62 / 608,296 is incorporated herein by reference in its entirety. [Background technology]

[0002] background Head and neck cancer is a common disease. The majority of head and neck cancers are histologically of squamous cell type and are therefore classified as head and neck squamous cell carcinoma (HNSCC). HNSCC is the sixth most common cancer in the world and the third most common in developing countries.

[0003] The biological mechanisms behind HNSCC are unclear, and few, if any, biomarkers provide reliable indications of this condition. Nevertheless, lifestyle adjustments to avoid triggering the onset of symptoms and / or promoting further disease progression would be helpful for individuals susceptible to HNSCC. Thus, there is a need to develop and evaluate biomarkers for HNSCC. Summary of the Invention [Means for solving the problem]

[0004] Abstract The present disclosure can be embodied in a variety of ways.

[0005] In one embodiment, a method is disclosed for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one gene in Table 4 and / or Table 6 in the sample. In one embodiment, a method is disclosed for detecting biomarkers associated with HNSCC in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one of the following genes in the sample: CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, and HSD17B6. In another embodiment, a method is disclosed for detecting biomarkers associated with HNSCC in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one of at least one of the human papillomavirus (HPV) E6 or E7 genes. Additionally and / or alternatively, the method may comprise measuring at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In one embodiment, the normalization gene may be RPL30 or another normalization gene. Alternatively, measurements of the expression of various combinations of these genes may be performed.

[0006] In one embodiment, a panel of the disclosed biomarkers is used. In one embodiment, the present disclosure includes a composition for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising a reagent for quantifying the expression level of at least one gene in Table 4 and / or Table 6, and / or at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6, and / or at least one of HPV E6 and E7 genes. Additionally and / or alternatively, the composition may include at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In one embodiment, the normalization gene may be RPL30 or another normalization gene. The composition may include, in certain embodiments, a primer and / or a probe of any one of these genes, and the primer and / or probe is labeled with a detectable moiety as described herein.

[0007] Other embodiments include systems for performing the methods and / or using the compositions disclosed herein.

[0008] Other features, objects, and advantages of the disclosure herein will be apparent in the detailed description, drawings, and claims that follow. It should be understood, however, that the detailed description, drawings, and claims, while indicating embodiments of the disclosed methods, compositions, and systems, are provided by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: obtaining a sample from the individual; measuring the amount of an expression product from a gene comprising at least one gene in Table 4 and / or Table 6; A method comprising: (Item 2) 2. The method of claim 1, wherein the genes include at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6. (Item 3) 2. The method of claim 1, further comprising measuring the amount of an expression product from at least one of the HPV E6 and / or HPV E7 genes. (Item 4) 2. The method of claim 1, further comprising measuring the amount of a normalization gene. (Item 5) 2. The method of claim 1, wherein the measuring step comprises measuring mRNA. (Item 6) 2. The method of claim 1, wherein the measuring step comprises an immunoassay. (Item 7) 2. The method of claim 1, comprising measuring the expression of at least four of the genes. (Item 8) 2. The method of claim 1, wherein the sample comprises serum, tissue, FFPE, saliva or plasma. (Item 9) 1. A method for detecting susceptibility to head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: obtaining a sample from said individual; measuring the amount of at least one expression product from at least one gene in Table 4 and / or Table 6; comparing the expression of at least one gene in Table 4 and / or Table 6 in the sample with a control value of the expression product of the gene, wherein a difference between gene expression in the individual and the control value indicates that the individual is likely to have HNSCC or is predisposed to developing HNSCC (i.e., is at high risk of HNSCC). (Item 10) 10. The method of claim 9, wherein the genes include at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6. (Item 11) 10. The method of claim 9, further comprising: measuring the amount of at least one expression product of at least one HPV E6 and / or E7 gene; and comparing the expression of the at least one HPV E6 and / or E7 gene in the sample to a control value of expression. (Item 12) 10. The method of claim 9, further comprising measuring the amount of a normalization gene. (Item 13) 10. The method of claim 9, wherein said measuring comprises measuring mRNA. (Item 14) 10. The method of claim 9, wherein the measuring comprises measuring a protein. (Item 15) 16. The method of claim 9, comprising measuring the expression of at least four of the genes. 10. The method of claim 9, wherein the sample comprises serum tissue, FFPE, saliva or plasma. (Item 17) A composition for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual comprising a reagent for quantifying said expression level of at least one gene in Table 4 and / or Table 6. (Item 18) 18. The composition of claim 17, wherein the at least one gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6. (Item 19) 18. The composition of claim 17, further comprising at least one reagent for quantifying the expression level of at least one of HPV E6 and / or E7. (Item 20) 18. The composition of item 17, further comprising at least one normalization gene. (Item 21) 18. The composition of claim 17, wherein the reagent detects mRNA. (Item 22) 18. The composition of claim 17, wherein the reagent comprises at least one primer and / or probe for any one of these genes, and the at least one primer and / or probe is labeled with a detectable moiety. (Item 23) 18. The composition of claim 17, wherein the reagent detects a protein. (Item 24) A kit comprising reagents for quantifying the expression level of at least one gene in Table 4 and / or Table 6. [Brief description of the drawings]

[0009] The present invention may be better understood with reference to the following non-limiting drawings.

[0010] [Figure 1] FIG. 1 shows the results of an experiment using a TCGA dataset and random forest analysis to identify differentially expressed genes in HNSCC according to an embodiment of the present disclosure.

[0011] [Diagram 2] FIG. 2 illustrates that evaluation of a gene panel containing multiple markers can improve assay performance according to various embodiments of the present disclosure.

[0012] [Diagram 3] Figure 3 shows the regional gene expression of four markers of the present disclosure (SH3BGRL2, CAB39L, NRG2 and ADAM12) in both normal and HNSCC tissues. In Figure 3, for each plot, the three datasets at the left end of the x-axis (larynx, oral cavity and oropharynx) are from normal tissues, and the four datasets at the right end of the x-axis (hypopharynx, larynx, oral cavity and oropharynx) are from HNSCC tissues.

[0013] [Figure 4] Figure 4 shows regional gene expression of four additional markers of the present disclosure (LOXL2, COL13A1, HSD17B6 and GRIN2D) in both normal and HNSCC tissues according to various embodiments of the present disclosure. In Figure 4, for each plot, the three datasets at the left end of the x-axis (Larynx, Oral Cavity and Oropharynx) are from normal tissues, and the three datasets at the right end of the x-axis (Hypopharynx, Larynx, Oral Cavity and Oropharynx) are from HNSCC tissues.

[0014] [Diagram 5] FIG. 5 shows a comparison of all HNSCC markers between HNSCC samples and normals for all data, and between HNSCC in the oral cavity (OC) or oropharynx (OP) and normals, according to an embodiment of the present disclosure.

[0015] [Figure 6A] Figure 6, panel A (i.e., Figure 6A) shows differential expression of the TCGA gene set according to embodiments of the present disclosure. Panel B (i.e., Figure 6B) shows the median rank of 36 genes (darker symbols in the figure) from a random forest analysis using various embodiments of the present disclosure. For increased gene expression in HNSCC, the cutoff is the 5th percentile of HNSCC and the 95th percentile of normal (e.g., Figure 6B, inset for GRIN2D). [Figure 6B] Figure 6, panel A (i.e., Figure 6A) shows differential expression of the TCGA gene set according to embodiments of the present disclosure. Panel B (i.e., Figure 6B) shows the median rank of 36 genes (darker symbols in the figure) from a random forest analysis using various embodiments of the present disclosure. For increased gene expression in HNSCC, the cutoff is the 5th percentile of HNSCC and the 95th percentile of normal (e.g., Figure 6B, inset for GRIN2D).

[0016] [Figure 7]FIG. 7 shows differentially expressed markers (darker symbols in the figure) identified from a literature search, according to various embodiments of the present disclosure.

[0017] [Figure 8] FIG. 8 is a diagram illustrating normalized markers from a literature search, according to various embodiments of the present disclosure.

[0018] [Figure 9A] FIG. 9 Panel A (i.e., FIG. 9A) shows the identification of normalized genes using the TCGA dataset. The left panel shows the entire dataset. The middle panel shows genes with a median fold change in gene expression between normal and cancer <2 [positive or negative] with an interquartile range (IQR) <2 (IQR=75th percentile expression / 25th percentile expression). The right panel shows the expression levels in normal and HNSCC of genes in the TCGA database to identify genes with similar median expression levels to the panel of interest according to various embodiments of the present disclosure. Panel B (i.e., FIG. 9B) shows a differential plot of KHDRBS1 according to various embodiments of the present disclosure. [Figure 9B] FIG. 9 Panel A (i.e., FIG. 9A) shows the identification of normalized genes using the TCGA dataset. The left panel shows the entire dataset. The middle panel shows genes with a median fold change in gene expression between normal and cancer <2 [positive or negative] with an interquartile range (IQR) <2 (IQR=75th percentile expression / 25th percentile expression). The right panel shows the expression levels in normal and HNSCC of genes in the TCGA database to identify genes with similar median expression levels to the panel of interest according to various embodiments of the present disclosure. Panel B (i.e., FIG. 9B) shows a differential plot of KHDRBS1 according to various embodiments of the present disclosure.

[0019] [Figure 10]FIG. 10 shows a comparison of droplet digital PCR (ddPCR) data and TCGA RNASeq data for the levels of gene expression of potential marker genes, ADAM12 and SH3BGRL2, in cancer tissue (i.e., tongue squamous cell carcinoma) compared to normal tissue (i.e., oral mucosa) according to various embodiments of the present disclosure.

[0020] [Figure 11] Figure 11 shows additional ddPCR data for three formalin-fixed paraffin-embedded patient samples (DA1081983; DR1041686; DA0063595) and one URNA control sample (derived from cell cultured cancer tissue) using ddPCR. Either duplicate or triplicate sampling was performed according to various embodiments of the present disclosure.

[0021] [Figure 12] FIG. 12 shows the concentration dependence of expression of SH3BGRL2 (a potential cancer marker) (·) compared to expression of KHDRBS1 (×) (a potential normalizer gene) in three different patient samples (RNA1, 3, and 5) and a URNA control, showing a relatively constant ratio up to the assay limit of 1 copy / μL (dotted line), in accordance with various embodiments of the present disclosure.

[0022] [Figure 13] 13 shows the expression of potential cancer marker SH3BGRL2 in formalin-fixed paraffin-embedded (FFPE) samples compared to URNA controls (left panel); the ratio of SH3BGRL2 / KHDRBS1 ddPCR products in cancer and normal tissues (middle panel); and the distribution of reported gene expression for these two markers in the TCGA database (right panel) according to various embodiments of the present disclosure. In this figure, x's are samples from cancer patients and circles (white or black) are normal tissue samples.

[0023] [Figure 14]FIG. 14 shows the analysis of various patient samples for SH3BGRL2 using either a singleplex assay format (single) (i.e., containing only SH3BGRL2 primers) or a duplex assay format (duplex) (containing SH3BGRL2 and KHDRBS1 primers) according to various embodiments of the present disclosure.

[0024] [Figure 15] FIG. 15 shows expression of five biomarkers (SH3BGRL2, KRT4, EMP1, LOXL2 and ADAM12) and housekeeping gene KHDRBS1 by duplex ddPCR from 22 benign (circles) and 8 carcinoma (x) FFPE samples according to various embodiments of the present disclosure.

[0025] [Figure 16] Figure 16 shows the expression of five biomarkers by ddPCR (left panel) after normalization to the housekeeping gene KHDRBS1 from 22 benign and 8 carcinoma FFPE samples compared to RNASeq data from HNSCC TCGA (right panel) for the same biomarkers and housekeeping genes, with the median fold change in expression for each biomarker summarized (table) according to various embodiments of the present disclosure. In this figure, N=normal tissue, C=cancer tissue.

[0026] [Figure 17] FIG. 17 shows ddPCR Score algorithm results (left panel) and receiver operating characteristic (ROC) analysis (right panel) of normalized ddPCR expression used to distinguish cancer from normal FFPE samples according to various embodiments of the present disclosure.

[0027] [Figure 18]FIG. 18 shows correlation of E6 and E7 HPV16 expression by ddPCR in p16 positive FFPE HNSCC samples (top panel) and p16 negative FFPE HNSCC samples (bottom panel); and normalized ddPCR expression levels of E6 and E7 from p16 positive samples (right plot), according to various embodiments of the present disclosure.

[0028] [Figure 19] FIG. 19 shows RNA yield (μg RNA / 2 mL saliva) and A260 / A280 ratios from 15 saliva samples in tabular form (left table) and box plots (right plots) according to various embodiments of the present disclosure.

[0029] [Figure 20] FIG. 20 shows duplex ddPCR expression of five biomarkers (LOXL2, SH3BGRL2, CRISP3, EMP1, and KRT4) and housekeeping gene RPL30 from 15 saliva samples (left plot) and separately from five duplex ddPCR reactions (right plot), according to various embodiments of the present disclosure.

[0030] [Figure 21] FIG. 21 shows expression by ddPCR of five biomarkers after normalization to housekeeping gene RPL30 from 15 saliva samples (left panel) compared to RNASeq data from HNSCC TCGA for the same biomarkers (right panel), along with the median fold increase in expression of each biomarker relative to LOXL2 summarized (table), according to various embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description Terms and Definitions In order that this disclosure may be more readily understood, certain terms are first defined. Further definitions of the following terms, as well as other terms, are set forth throughout the specification.

[0032] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including both maximum and minimum values); that is, all subranges begin with a minimum value of 1 or more (e.g., 1 to 6.1) and end with a maximum value of 10 or less (e.g., 5.5 to 10). Moreover, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0033] It is further noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "and / or" is typically used to refer to at least one or the other. In some instances, the term "and / or" is used synonymously with the term "or." The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to." The term "such as" is used herein to mean, and is used synonymously with, the phrase "such as but not limited to."

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Experts are directed to Current Protocols in Molecular Biology (Ausubel), in particular, for definitions and terms in the art.

[0035] Antibody: As used herein, the term "antibody" refers to a polypeptide consisting of one or more polypeptides substantially encoding immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as various immunoglobulin variable region genes. Light chains are generally classified as either kappa or lambda. Heavy chains are generally classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "light chain variable chain" (VL) and "heavy chain variable chain" (VH) refer to these light and heavy chains, respectively. An antibody can be specific for a particular antigen. The antibody or its antigen can be either an analyte or a binding partner. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody under the disulfide bond of the hinge region to produce F(ab)'2, which is a dimer of Fab, itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond of the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially an Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). While various antibody fragments have been defined in terms of the digestion of an intact antibody, one of skill in the art will appreciate that such Fab' fragments can be synthesized de novo either chemically or by using recombinant DNA methodology.Thus, the term "antibody" as used herein also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. In some embodiments, the antibody is a single chain antibody, e.g., a single chain Fv (scFv) antibody in which the heavy and light variable chains are linked together (directly or via a peptide linker) to form a contiguous polypeptide. Single chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers and can be expressed from nucleic acids that contain VH- and VL-encoding sequences that are directly linked or linked by a peptide-encoding linker. (See, e.g., Huston et al., (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883, the entire contents of which are incorporated herein by reference). Several structures exist for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778.

[0036] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies and chimeric antibodies, such as those generated by combinatorial mutagenesis and phage display. The term "antibody" also includes antibody mimetics or peptidomimetics. Peptidomimetics are compounds based on or derived from peptides and proteins. The peptidomimetics of the present disclosure can be obtained by structural modification of known peptide sequences, generally using non-natural amino acids, conformational constraints, isosteric substitutions, etc.

[0037] Allele: As used herein, the term "allele" refers to different versions of the nucleotide sequence at the same genetic locus (eg, gene).

[0038] Allele-specific primer extension (ASPE): As used herein, the term "allele-specific primer extension (ASPE)" refers to a mutation detection method using a primer that hybridizes to a corresponding DNA sequence and is extended upon successful hybridization of the 3'-terminal nucleotide of such primer. In general, an extension primer having a 3'-terminal nucleotide that forms a perfect match with the target sequence is extended to form an extension product. Modified nucleotides can be incorporated into the extension product, and such nucleotides can effectively label the extension product for detection purposes. Alternatively, the extension primer may instead contain a 3'-terminal nucleotide that forms a mismatch with the target sequence. In this case, primer extension will not occur unless the polymerase used for extension inadvertently has exonuclease activity.

[0039] Amplification: As used herein, the term "amplification" refers to methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. The target nucleic acid may be either DNA or RNA. Generally, the sequence amplified in this manner comprises an "amplicon." Amplification may be accomplished in a variety of ways, including, but not limited to, polymerase chain reaction ("PCR"), transcription-based amplification, isothermal amplification, rolling circle amplification, and the like. Amplification may be performed with relatively similar amounts of each primer in a primer pair to generate a double-stranded amplicon. However, asymmetric PCR may be used to amplify primarily or exclusively single-stranded products as is well known in the art (e.g., Poddar, Molec.And Cell.Probes 14:25-32 (2000)). This may be achieved by using each pair of primers to greatly reduce the concentration of one primer in the pair compared to the other primer (e.g., 100-fold difference). Amplification by asymmetric PCR is usually linear. Those skilled in the art will appreciate that different amplification methods may be used together.

[0040] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0041] Approximately: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or smaller) of the stated reference value, unless otherwise stated or otherwise clear from the context (unless such number exceeds 100% of possible values). The term "about" is used to indicate that the value includes the inherent variation of error of the device, the method used to determine the value, or the variation that exists between samples.

[0042] Associated with a condition or disease of interest: As used herein, "associated with a condition or disease of interest" means that the variant is found more frequently in patients with the condition or disease of interest than in non-symptomatic or non-disease controls. Generally, the statistical significance of such association can be determined by assaying multiple patients.

[0043] Biological sample: As used herein, the term "biological sample" or "sample" encompasses any sample obtained from a biological source. Biological samples may include, by way of non-limiting example, blood, amniotic fluid, serum, plasma, liquid or tissue biopsy, urine, feces, epithelial samples, skin samples, buccal swabs, sperm, amniotic fluid, cultured cells, bone marrow samples, and / or chorionic villi. Convenient biological samples may be obtained, for example, by scraping cells from the surfaces of the buccal cavity. The term biological sample encompasses samples that have been treated to release or make available nucleic acids or proteins for detection as described herein. The term biological sample also includes cell-free nucleic acids that may be present in the sample (e.g., plasma or amniotic fluid). For example, biological samples may include cDNA obtained by reverse transcription of RNA from cells in the biological sample. Biological samples may be obtained from stages of life, such as fetuses, young adults, adults, etc. Fixed or frozen tissues may also be used.

[0044] Biomarker: As used herein, the term "biomarker" or "marker" refers to one or more nucleic acids, polypeptides, and / or other biomolecules (e.g., cholesterol, lipids) that can be used alone or in combination with other biomarkers to diagnose or aid in the diagnosis or prognosis of a disease or condition of interest; monitor the progression of a disease or condition of interest; and / or monitor the effectiveness of a treatment for a condition or disease of interest.

[0045] Binder: As used herein, the term "binder" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction may be non-covalent, e.g., as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it may be covalent. The term "soluble binder" refers to a binder that is not bound (i.e., covalently or non-covalently bound) to a solid support.

[0046] Carrier: The term "carrier" refers to a person who has no symptoms but carries a mutation that can be passed on to their children. Generally, for autosomal recessive genetic disorders, a carrier has one allele that contains the disease causing mutation and a second allele that is normal or not associated with the disease.

[0047] Coding and non-coding sequences: As used herein, the term "coding sequence" refers to a sequence of a nucleic acid or its complement, or a portion thereof, that can be transcribed and / or translated to produce an mRNA of a polypeptide or a fragment thereof and / or a polypeptide or a fragment thereof. A coding sequence comprises exons in genomic DNA or in the premature primary RNA transcript, which are joined together by the biochemical machinery of the cell to produce a mature mRNA. The antisense strand is the complement of such a nucleic acid, from which the encoded sequence can be deduced. As used herein, the term "non-coding sequence" refers to a sequence or a portion thereof of a nucleic acid or its complement that is not transcribed in vivo into amino acids, or that does not interact or attempt to position an amino acid with a tRNA. Non-coding sequences include intron sequences, both in genomic DNA or in the premature primary RNA transcript, and gene associated sequences, such as promoters, enhancers, silencers, etc.

[0048] Complement: As used herein, the terms "complement," "complementary," and "complementarity" refer to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, the sequence 5'-GCGGTCCCA-3' has a complementary sequence of 5'-TGGGACCGC-3'. A complementary sequence can also be a sequence of RNA that is complementary to a DNA sequence. Certain bases not commonly found in natural nucleic acids may be included in complementary nucleic acids, including, but not limited to, inosine, 7-deazaguanine, locked nucleic acids (LNA) and peptide nucleic acids (PNA). Complementarity need not be perfect; a stable duplex may contain mismatched base pairs, degenerate or unmatched bases. Those skilled in the art of nucleic acid technology can empirically determine duplex stability, taking into account numerous variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0049] Conserved: As used herein, the term "conserved residue" refers to amino acids that are the same among multiple proteins with the same structure and / or function. Regions of conserved residues may be important to the structure or function of a protein. Thus, adjacent conserved residues identified in a three-dimensional protein may be important to the structure or function of the protein. To find conserved residues, or conserved regions of 3D structure, comparisons of sequences of the same or similar proteins from different species or from individuals of the same species can be made.

[0050] Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. In general, controls are used to increase the consistency of an experiment by isolating a variable in order to draw conclusions about such variable. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to obtain a basis for comparison. In one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control" (i.e., the variable being tested) is not applied. In some embodiments, a control is a historical control (i.e., a test or assay previously performed, or a control of a previously known amount or outcome). In some embodiments, a control is or includes a stored record, printed or otherwise. A control may be a positive or negative control.

[0051] A "control" or "predetermined standard" of a biomarker refers to the level of expression of the biomarker in a healthy subject, or the expression level of said biomarker in non-diseased or non-symptomatic tissue from the same subject. A control or predetermined standard expression level or protein amount of a given biomarker can be established by prospective and / or retrospective statistical studies using only routine experimentation. Such predetermined standard expression level and / or protein level (amount) can be determined by those skilled in the art using well-known methods. A positive control is a sample (or reagent) that provides a predetermined amount of measured signal.

[0052] Crude: As used herein, the term "crude" when used in reference to a biological sample refers to a sample that is in a substantially unpurified state. For example, a crude sample can be a cell lysate or a biopsy tissue sample. A crude sample can be present in a dissolved state or as a dry preparation.

[0053] Deletion: As used herein, the term "deletion" includes a mutation that removes one or more nucleotides from a naturally occurring nucleic acid.

[0054] Disease or condition of interest: As used herein, the disease or condition of interest is head and neck cancer, and in some embodiments, more specifically HNSCC.

[0055] Detection: As used herein, the terms "detection," "detected," or "detecting" include "measurement," "measured," or "measuring," and vice versa.

[0056] Detectable moiety: As used herein, the term "detectable moiety" or "detectable biomolecule" or "reporter" refers to a molecule that can be measured in a quantitative assay. For example, a detectable moiety may include an enzyme that can be used to convert a substrate into a product that can be measured (e.g., a visible product). Alternatively, the detectable moiety may be a radioisotope that can be quantified. Alternatively, the detectable moiety may be a fluorophore. Alternatively, the detectable moiety may be a luminescent molecule. Alternatively, other detectable molecules may be used.

[0057] Epigenetic: As used herein, epigenetic elements can change gene expression by mechanisms other than changes in the basic DNA sequence. Such elements can include paramutation, imprinting, gene silencing, X-chromosome inactivation, position effect, reprogramming, transvection, maternal effect, histone modification, and elements that regulate heterochromatin.

[0058] Epitope: As used herein, the term "epitope" refers to a fragment or portion of a molecule or molecular compound (eg, a polypeptide or protein complex) that contacts a particular antibody or antibody-like protein.

[0059] Exon: As used herein, an exon is a nucleic acid sequence found in mature or processed RNA after other parts of the RNA (e.g., intervening regions known as introns) have been removed by RNA splicing. Thus, an exon sequence usually codes for a protein or a portion of a protein. An intron is a portion of an RNA that is removed from the surrounding exon sequence by RNA splicing.

[0060] Expression and expressed RNA: As used herein, expressed RNA is RNA that codes for a protein or polypeptide ("coding RNA"), or any other RNA that is transcribed but not translated ("non-coding RNA"). The term "expression" is used herein to mean the process by which a polypeptide is produced from DNA. This process involves the transcription of a gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which it is used, "expression" can refer to the production of RNA, protein, or both.

[0061] Measurement of protein quantity and / or expression of the biomarkers of the present disclosure may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or its corresponding protein. Non-limiting examples of such methods include immunological methods for detection of secreted proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In certain embodiments, expression of a marker gene is assessed using an antibody (e.g., a radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody) that specifically binds to a protein corresponding to the marker gene, such as a protein encoded by an open reading frame corresponding to the marker gene or a protein that has undergone all or some of its normal post-translational modifications, an antibody derivative (e.g., an antibody conjugate bound to a substrate or to a protein or ligand of a protein-ligand pair {e.g., biotin-streptavidin}), or an antibody fragment (e.g., a single chain antibody, an isolated antibody hypervariable domain, etc.). In certain embodiments, the reagent may be directly or indirectly labeled with a detectable substance. The detectable substance may, for example, be selected from the group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Methods of labeling antibodies are well known in the art.

[0062] In another embodiment, the expression of the marker gene is evaluated by preparing mRNA / cDNA (i.e., transcribed polynucleotide) from cells in the sample and hybridizing the mRNA / cDNA with a reference polynucleotide that is the complement of the polynucleotide containing the marker gene and fragments thereof. The cDNA can be amplified, if desired, using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide; preferably, it is not amplified.

[0063] Family history: As used herein, the term "family history" generally refers to the occurrence of events (e.g., disease-related disorders or mutation carriers) related to an individual's immediate family, including parents and siblings. Family history may also include grandparents and other relatives.

[0064] Flanking: As used herein, the term "flanking" means that the primer hybridizes to the target nucleic acid adjacent to the region of interest to be amplified on the target. Those skilled in the art will understand that the preferred primers are a pair of primers, one on each strand of the target double-stranded DNA molecule, that hybridize 5' (upstream) from the region of interest, so that nucleotides are added to the 3' end of the primer by a suitable DNA polymerase. For example, a primer flanking a mutant sequence does not actually anneal to the mutant sequence, but rather to a sequence adjacent to the mutant sequence. In some cases, primers flanking an exon are usually designed to anneal to a sequence adjacent to the exon (e.g., an intron sequence) rather than to the exon sequence. However, in some cases, the amplification primers may be designed to anneal to an exon sequence.

[0065] Gene: As used herein, a gene is a unit of heredity. Generally, a gene is a portion of DNA that codes for a protein or functional RNA. A gene is a locatable region of a genomic sequence that corresponds to a unit of heredity. A gene may be associated with regulatory regions, transcriptional regions, and other functional sequence regions.

[0066] Genotype: As used herein, the term "genotype" refers to the genetic makeup of an organism. More specifically, the term refers to the identity of the alleles present in an individual. "Genotyping" an individual or DNA sample refers to determining the nature, in terms of nucleotide bases, of the two alleles that an individual possesses at known polymorphic sites.

[0067] Genetic regulatory element: As used herein, a genetic regulatory element or regulatory sequence is a segment of DNA to which regulatory proteins, such as transcription factors, bind to regulate gene expression. Such regulatory regions are often located upstream of the gene that they regulate.

[0068] Healthy individual: As used herein, the term "healthy individual" or "control" refers to a subject who has not been diagnosed with the condition and / or disease of interest.

[0069] Heterozygous: As used herein, the term "heterozygous" or "HET" refers to an individual who carries two different alleles of the same gene. As used herein, the term "heterozygous" encompasses "compound heterozygous" or "compound heterozygous mutant". As used herein, the term "compound heterozygous" refers to an individual who carries two different alleles. As used herein, the term "compound heterozygous mutant" refers to an individual who carries two different copies of an allele, and such alleles are characterized as mutant forms of the gene.

[0070] Homozygous: As used herein, the term "homozygous" refers to an individual who carries two copies of the same allele. As used herein, the term "homozygous mutant" refers to an individual who carries two copies of the same allele, such alleles being characterized as mutant forms of the gene.

[0071] Housekeeping or normalization gene: As used herein, a "housekeeping gene" is a gene that is generally constitutively expressed in all cells because it provides basic functions necessary for the maintenance of all cell types. A housekeeping or normalization gene is measured simultaneously with a gene of interest to account for variation due to sample-to-sample variation. Such sample-to-sample variation may reflect variations in experimental variables such as, but not limited to, RNA isolation, reverse transcription, PCR efficiency, etc. Normalization includes reporting the ratio of the gene of interest to the housekeeping gene. See, for example, Bustin, SA et al., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, Apr; 55(4): 611-622.

[0072] Hybridize: As used herein, the term "hybridize" or "hybridization" refers to the process in which two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Oligonucleotides or probes suitable for hybridization generally contain 10-100 nucleotides in length (e.g., 18-50, 12-70, 10-30, 10-24, 18-36 nucleotides in length). Nucleic acid hybridization techniques are well known in the art. Those skilled in the art know how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired level of complementarity will stably hybridize, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular See Biology. John Wiley & Sons, Secaucus, NJ.

[0073] Identity or percent identity: As used herein, the term "identity" or "percent identity" refers to the sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning the two sequences and refers to the number of identical residues (i.e., amino acids or nucleotides) at positions shared by the compared sequences. Sequence alignment and comparison can be performed using standard algorithms in the art (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482-489; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444-2448), or computerized versions of these algorithms published as BLAST and FASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI). ENTREZ, available through the National Institutes of Health, Bethesda, MD, can also be used for sequence comparison. In other examples, percent identity can be determined using commercially available software, such as GenomeQuest. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the internet site of the National Center for Biotechnology Information) can be used. In one embodiment, the percent identity of two sequences can be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, Calif.) sequence alignment software package, can be used.

[0074] As used herein, the term at least 90% identical thereto includes sequences that are in the range of 90-100% identity with the indicated sequence, including all ranges therebetween. Thus, the term at least 90% identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence. Similarly, the term "at least 70% identical" includes sequences that are in the range of 70-100% identity, including all ranges therebetween. The determination of percent identity is determined using the algorithm described herein.

[0075] Insertion or Addition: As used herein, the term "insertion" or "addition" refers to a change in an amino acid or nucleotide sequence that results in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.

[0076] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism, but in an artificial environment, such as a test tube or reaction vessel, cell culture, and the like.

[0077] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal.

[0078] Isolated: As used herein, the term "isolated" refers to (1) a substance and / or entity that is separated from at least some of the elements (whether in nature and / or in an experimental setting) with which it is associated when originally created, and / or (2) a substance and / or entity that is generated, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of other elements with which it is originally associated. In some embodiments, an isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other elements. As used herein, the term "isolated cell" refers to a cell that is not contained in a multicellular organism.

[0079] Labeled: The terms "labeled" and "labeled with a detectable agent or moiety" are used interchangeably herein to specify that an entity (e.g., a nucleic acid probe, an antibody, etc.) can be measured, for example, by detection of the label (e.g., visual detection, such as radioactivity) after binding to another entity (e.g., a nucleic acid, a polypeptide, etc.). The detectable agent or moiety may be selected to generate a signal that can be measured and whose intensity is related (e.g., proportional) to the amount of bound entity. A wide variety of systems for labeling and / or detecting proteins and peptides are known in the art. Labeled proteins and peptides can be prepared by incorporation or conjugation of a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. A label or labeling moiety may be directly detectable (i.e., it does not require further reaction or manipulation to become detectable, e.g., a fluorophore is directly detectable) or indirectly detectable (i.e., it becomes detectable by reaction or binding with another entity that is detectable, e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody that contains a reporter such as a fluorophore). Suitable detectable agents include, but are not limited to, radionucleotides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, aptamer beacons, and the like.

[0080] MicroRNA: As used herein, microRNA (miRNA) is a short (20-24 nucleotide) non-coding RNA involved in the post-transcriptional regulation of gene expression. MicroRNAs can affect both mRNA stability and translation. For example, microRNAs can bind to complementary sequences in the 3'UTR of target mRNAs, causing gene silencing. miRNAs are transcribed by RNA polymerase II as part of a capped and polyadenylated primary transcript (pri-miRNA), which may or may not be protein-coding. The primary transcript can be cleaved by the Drosha ribonuclease III enzyme to generate an approximately 70 nucleotide stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate mature miRNAs and antisense miRNA star (miRNA). * Mature miRNAs can be incorporated into an RNA-induced silencing complex (RISC), which can recognize target mRNAs through imperfect base pairing with the miRNA, most commonly resulting in translational inhibition or destabilization of the target mRNA.

[0081] Multiplex PCR: As used herein, the term "multiplex PCR" refers to the simultaneous amplification of two or more regions, each primed with a separate primer pair.

[0082] Multiplex ASPE: As used herein, the term "multiplex ASPE" refers to an assay that combines multiplex PCR with allele-specific primer extension (ASPE) to detect genetic polymorphisms. In general, multiplex PCR is used to first amplify regions of DNA that serve as target sequences for ASPE primers. See the definition of allele-specific primer extension.

[0083] Mutation and / or variant: As used herein, the terms mutation and variant are used interchangeably to describe changes in a nucleic acid or protein sequence. The term "mutant" as used herein refers to a mutated or potentially non-functional form of a gene.

[0084] Nucleic acid: As used herein, a "nucleic acid" is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogs.

[0085] Obtain or acquire: As used herein, the term "obtain" or "obtain" includes obtaining directly or receiving indirectly (i.e., from a third party).

[0086] Polypeptide or protein: As used herein, the terms "polypeptide" and / or "protein" refer to a polymer of amino acids, not a specific length. Thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide and / or protein. "Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may include partial or full-length proteins. The term "peptide" is used to indicate less than full-length proteins or very short proteins, unless the context indicates otherwise.

[0087] As known in the art, "proteins," "peptides," "polypeptides," and "oligopeptides" are chains of amino acids (generally L-amino acids) whose alpha carbons are linked by peptide bonds formed by a condensation reaction between the carboxyl group on the alpha carbon of one amino acid and the amino group on the alpha carbon of another amino acid. Generally, the amino acids constituting a protein are numbered sequentially, starting with the amino terminal residue and increasing toward the carboxy terminal residue of the protein. Amino acid residue abbreviations are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0088] As used herein, a "domain" of a polypeptide or protein includes a region along a polypeptide or protein that comprises an independent unit. A domain may be defined in terms of structure, sequence and / or biological activity. In one embodiment, a polypeptide domain may include a region of a protein that folds in a manner that is substantially independent from the rest of the protein. Domains may be identified using domain databases such as, but not limited to, PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

[0089] Primer: As used herein, the term "primer" refers to a short, single-stranded oligonucleotide capable of hybridizing to a complementary sequence in a nucleic acid sample. Generally, a primer serves as the initiation point for template-dependent DNA synthesis. Deoxyribonucleotides can be added to a primer by DNA polymerase. In some embodiments, such addition of deoxyribonucleotides to a primer is also known as primer extension. As used herein, the term primer includes all forms of primers that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like. A "primer pair" or "primer set" for a PCR reaction generally refers to a set of primers that includes a "forward primer" and a "reverse primer." As used herein, a "forward primer" refers to a primer that anneals to the antisense strand of dsDNA. A "reverse primer" anneals to the sense strand of dsDNA.

[0090] Genetic polymorphism: As used herein, the term "genetic polymorphism" refers to the coexistence of two or more forms of a gene or portions thereof.

[0091] Portion and Fragment: As used herein, the terms "portion" and "fragment" are used interchangeably to refer to a polypeptide, nucleic acid, or other molecular construct.

[0092] Sample: As used herein, the term "sample" refers to material obtained from an individual or subject or patient. Samples can be from any biological source, including any bodily fluid (e.g., whole blood, plasma, serum, saliva, ocular lens fluid, sweat, urine, milk, etc.), tissue or extract, cell, cell-free nucleic acid, formalin-fixed paraffin-embedded (FFPE) tissue, etc.

[0093] Sense and antisense strands: As used herein, the term "sense strand" refers to the strand of double-stranded DNA (dsDNA) that contains at least a portion of the coding sequence of a functional protein. As used herein, the term "antisense strand" refers to the strand of dsDNA that is the reverse complement of the sense strand.

[0094] Significant difference: As used herein, the term "significant difference" is well within the knowledge of a person skilled in the art and is empirically determined with reference to each particular biomarker. For example, a significant difference in the expression of a biomarker in a subject with a disease or condition group of interest compared to a healthy subject is a statistically significant difference in protein abundance.

[0095] Analogous or Homologous: As used herein, the term "analogous" or "homologous" when referring to an amino acid or nucleotide sequence refers to a polypeptide that has a degree of homology or identity with a wild-type amino acid sequence. Homology comparisons can be performed by eye, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology between two or more sequences (e.g., Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA, 80:726-730). For example, homologous sequences can be construed to include amino acid sequences that are at least 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 97% identical, or 98% identical to each other in alternative embodiments.

[0096] Specific: As used herein, the term "specific" refers to an oligonucleotide or primer that is capable of hybridizing to a target of interest under suitable hybridization or washing conditions and does not substantially hybridize to nucleic acids that are not of interest. Higher levels of sequence identity are preferred, including at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity. In some embodiments, a specific oligonucleotide or primer comprises at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more bases of sequence identity with the portion of the nucleic acid that it hybridizes or amplifies when the oligonucleotide and the nucleic acid are aligned.

[0097] As known in the art, conditions for hybridizing nucleic acid sequences to each other can be described as ranging from low stringency to high stringency. Generally, highly stringent hybridization conditions refer to washing the hybrids with a low salt buffer at high temperature. Hybridization can be performed using standard hybridization solutions in the art, such as 0.5M NaHPO4, 7% sodium dodecyl sulfate (SDS), filtering the bound DNA at 65°C, washing with 0.25M NaHPO4, 3.5% SDS, followed by washing with 0.1×SSC / 0.1% SDS at temperatures ranging from room temperature to 68°C, depending on the length of the probe (see, for example, Ausubel, FM et al., Short Protocols in Molecular Biology, 4). th(See, Ed., Chapter 2, John Wiley & Sons, NY). For example, high stringency washes include washing in 6×SSC / 0.05% sodium pyrophosphate at 37° C. for 14-base oligonucleotide probes, or 48° C. for 17-base oligonucleotide probes, or 55° C. for 20-base oligonucleotide probes, or 60° C. for 25-base oligonucleotide probes, or 65° C. for nucleotide probes of about 250 nucleotides in length. The nucleic acid probe may be, for example, [γ- 32 with radioactive nucleotides by end-labeling with [P]ATP or by random primer labeling with [α- 32 Alternatively, the probe may be labeled by incorporation of a biotinylated or fluorescein-labeled nucleotide and the probe may be detected using streptavidin or an anti-fluorescein antibody.

[0098] siRNA: As used herein, siRNA (small inhibitory RNA) is an essentially double-stranded RNA molecule consisting of about 20 complementary nucleotides. siRNA is generated by degradation of a larger double-stranded (ds) RNA molecule. siRNA can suppress gene expression by essentially splitting its corresponding mRNA into two by interaction of siRNA with mRNA, causing degradation of the mRNA. siRNA can also interact with DNA to promote chromatin silencing and heterochromatin expansion.

[0099] Subject: As used herein, the term "subject" refers to a human or any non-human animal. A subject may be a patient, which refers to a human who visits a health care provider for diagnosis or treatment of a disease. Humans include prenatal and postnatal forms. Also, as used herein, the terms "individual," "subject," or "patient" include all warm-blooded animals. In one embodiment, the subject is a human. In one embodiment, the individual is a subject at increased risk of developing HNSCC.

[0100] Substantially: As used herein, the term "substantially" refers to a qualitative condition that indicates the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed perfectly or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.

[0101] Substantially complementary: As used herein, the term "substantially complementary" refers to two sequences that can hybridize under stringent hybridization conditions. One of skill in the art will understand that substantially complementary sequences need not hybridize over their entire length. In some embodiments, "stringent hybridization conditions" refers to hybridization conditions that are at least as stringent as the following: overnight hybridization in 50% formamide, 5X SSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5X Denhart's solution at 42°C; washing with 2X SSC, 0.1% SDS at 45°C; and washing with 0.2X SSC, 0.1% SDS at 45°C. In some embodiments, stringent hybridization conditions should not allow hybridization of two nucleic acids that differ by more than 2 bases within a stretch of 20 contiguous nucleotides.

[0102] Substitution: As used herein, the term "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively, as compared to a naturally occurring molecule.

[0103] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or is exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0104] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be exhibiting symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0105] Solid support: The term "solid support" or "support" refers to a structure that provides a substrate onto which a biomolecule can be bound. For example, a solid support can be a well of an assay (i.e., a microtiter plate, etc.), or a solid support can be a location on an array, or a mobile support such as a bead.

[0106] Upstream and downstream: As used herein, the term "upstream" refers to the residue that is N-terminal to the second residue if the molecule is a protein, or 5' to the second residue if the molecule is a nucleic acid. Also, as used herein, the term "downstream" refers to the residue that is C-terminal to the second residue if the molecule is a protein, or 3' to the second residue if the molecule is a nucleic acid. All protein, polypeptide and peptide sequences disclosed herein are written from the N-terminal amino acid to the C-terminal acid, and all nucleic acid sequences disclosed herein are written from the 5' end of the molecule to the 3' end of the molecule.

[0107] overview The disclosure herein provides novel mutations identified in specific genes that are associated with diseases and / or condition clusters of interest and can be used to more accurately diagnose disorders associated with the genes and / or condition clusters of interest.

[0108] In some embodiments, the sample contains nucleic acid. In some embodiments, the testing step includes sequencing. In some embodiments, the testing step includes hybridization. In some embodiments, the hybridization is performed using one or more oligonucleotide probes specific to the region of the biomarker of interest. In some embodiments, for detection of mutations, the hybridization is performed under sufficiently stringent conditions to not allow a single nucleotide mismatch. In some embodiments, the hybridization is performed using a microarray. In some embodiments, the testing step includes restriction enzyme digestion. In some embodiments, the testing step includes PCR amplification. In some embodiments, the testing step includes reverse transcriptase PCR (rtPCR). In some embodiments, the PCR amplification is digital PCR amplification. In some embodiments, the testing step includes primer extension. In some embodiments, the primer extension is single-base primer extension. In some embodiments, the testing step includes performing multiplex allele-specific primer extension (ASPE).

[0109] In some embodiments, the sample contains proteins. In some embodiments, the testing step includes amino acid sequencing. In some embodiments, the testing step includes performing an immunoassay using one or more antibodies that specifically recognize the biomarker of interest. In some embodiments, the testing step includes protease digestion (e.g., trypsin digestion). In some embodiments, the testing step further includes performing 2D gel electrophoresis.

[0110] In some embodiments, the testing step includes determining the presence of one or more biomarkers using mass spectrometry. In some embodiments, the mass spectrometry format is selected from among matrix-assisted laser desorption / ionization, time-of-flight (MALDI-TOF), electrospray (ES), IR-MALDI, ion cyclotron resonance (ICR), Fourier transform, and combinations thereof.

[0111] In some embodiments, the sample is obtained from cells, tissues (e.g., FFPE tissues), whole blood, mouthwash, plasma, serum, urine, stool, saliva, umbilical cord blood, chorionic villus sample, chorionic villus sample culture, amniotic fluid, amniotic fluid culture, transcervical lavage, or combinations thereof. In certain embodiments, the sample can be either a liquid or tissue biopsy. In some embodiments, the sample comprises cell-free nucleic acid (e.g., DNA) that can be present in biological samples such as blood, plasma, serum, or amniotic fluid.

[0112] In some embodiments, the testing step comprises determining the identity of the nucleotide and / or amino acid at a predetermined position of the biomarker, hi some embodiments, the presence of a mutation is determined by comparing the identity of the nucleotide and / or amino acid at the predetermined position with a control.

[0113] In embodiments, the method may include performing an assay (eg, nucleic acid sequencing) on ​​multiple individuals to determine the statistical significance of the association.

[0114] In another aspect, the disclosure provides reagents for detecting a biomarker of interest, such as, but not limited to, a nucleic acid probe that specifically binds to the biomarker (e.g., a mutation in a DNA sequence, mRNA, protein), or an array containing one or more probes that specifically bind to the biomarker. In some embodiments, the disclosure provides an antibody that specifically binds to the biomarker. In some embodiments, the disclosure provides a kit comprising one or more such reagents. In some embodiments, the one or more reagents are provided in the form of a microarray. In some embodiments, the kit further comprises a reagent for primer extension. In some embodiments, the kit further comprises a control representative of a healthy individual. In some embodiments, the kit further comprises instructions on how to determine whether an individual has a symptom or disease of interest based on the biomarker of interest.

[0115] In some examples, the amount of one or more biomarkers may be detected in certain embodiments by: (a) detecting the amount of a polypeptide or protein that is regulated by said one or more biomarkers; (b) detecting the amount of a polypeptide or protein that regulates said biomarkers; or (c) detecting the amount of a metabolite of the biomarker.

[0116] In yet another aspect, the disclosure herein provides a computer readable medium encoding information corresponding to detection of a biomarker.

[0117] Methods and compositions for diagnosing HNSCC The embodiments of the present disclosure include methods and compositions for diagnosing the presence of HNSCC or the increased risk of developing it.The methods and compositions of the present disclosure can be used to obtain or provide genetic information from a subject, and objectively diagnose the presence of HNSCC or the increased risk of developing it for that subject or other subjects.The methods and compositions can be embodied in a variety of ways.

[0118] In one embodiment, a method is disclosed for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one gene in Table 4 and / or Table 6 in the sample. In one embodiment, a method is disclosed for detecting biomarkers associated with HNSCC in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one of the following genes in the sample: CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6. In one embodiment, a method is disclosed for detecting biomarkers associated with HNSCC in an individual, comprising obtaining a sample from the individual; and measuring the expression level of at least one human papillomavirus (HPV) E6 or E7 gene. Alternatively, various combinations of genes may be evaluated. The measured expression of any of these genes may be compared to a control value in certain embodiments. In various embodiments, the difference between the expression of the gene in an individual and the control value indicates that the individual may have (i.e., is diagnostic of the presence of) or is prone to develop (i.e., is at high risk of) HNSCC. The control value may be derived from one or more samples of normal (non-cancerous tissue) or may be derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include the measurement of at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used.

[0119] Additionally and / or alternatively, a method for detecting susceptibility to head and neck squamous cell carcinoma (HNSCC) in an individual is disclosed, comprising obtaining a sample from the individual; measuring the amount of expression products from genes comprising at least one gene in Table 4 and / or Table 6 in the sample; and comparing the expression of at least one gene in Table 4 and / or Table 6 in the sample to a control value of expression. In various embodiments, a difference between the expression of the genes in the individual and the control value indicates that the individual may have (i.e., is diagnostic of the presence of) or is prone to (i.e., at high risk of) developing HNSCC. The control value may be derived from one or more samples of normal (non-cancerous tissue) or may be derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include measuring at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used.

[0120] Additionally and / or alternatively, disclosed are methods of detecting susceptibility to HNSCC in an individual, comprising obtaining a sample from the individual; measuring in the sample an amount of at least one expression product from at least one gene including at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6; and comparing expression of at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6 in the sample to a control value for expression of each of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6. Additionally and / or alternatively, a method for detecting susceptibility to HNSCC in an individual is disclosed, comprising obtaining a sample from the individual; measuring the amount of at least one expression product from at least one of the genes, including HPV E6 and / or HPV E7, in the sample; and comparing the expression of at least one of the HPV E6 and / or HPV E7 expression products in the sample with a control value of the expression of each of HPV E6 and / or HPV E7. In various embodiments, a difference between the expression of the gene in the individual and the control value indicates that the individual may have (i.e., is diagnostic of the presence of) or is prone to (i.e., at high risk of) developing HNSCC. The control value may be derived from one or more samples of normal (non-cancerous tissue) or may be derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include the measurement of at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used.

[0121] In certain embodiments, the measuring comprises measuring RNA (e.g., mRNA). Alternatively, the measuring can comprise an immunoassay.

[0122] In some cases, increasing the number of biomarkers improves the statistical power of this method.For example, in certain embodiments, this method can include measuring the expression of at least 2, 3, 4, 5 or more biomarkers.In some cases, at least 4 biomarkers are measured.

[0123] A variety of samples may be assayed. In certain embodiments, the sample includes serum, plasma, saliva, or tissue (e.g., FFPE tissue).

[0124] The disclosed method also includes a method of identifying markers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising identifying at least one marker that is increased or decreased in expression in HNSCC but not expressed in HNSCC disease compared to normal controls.As disclosed herein, such a method may include statistical evaluation of markers that show differential expression in HNSCC compared to normal tissue.Or, the method may include biomarkers that discriminate HNSCC compared to normal based on other biological criteria (e.g., mutated genes, copy number differences and translocations, DNA methylation, and / or microRNA).Or, other biological aspects of the biomarker may be evaluated.

[0125] As disclosed herein, various methods can be used to measure the biomarker of interest. In one embodiment, measuring comprises measuring mRNA. In one embodiment, measuring comprises measuring peptide or polypeptide biomarker. For example, in one embodiment, measuring comprises immunoassay. Or, measuring can comprise flow cytometry. Or, as discussed in detail herein, nucleic acid methods can be used.

[0126] In yet another embodiment, a method for treating HNSCC is disclosed. The method for treating may include: obtaining a sample from an individual; measuring the amount of expression products from genes including at least one gene in Table 4 and / or Table 6 in the sample; comparing the expression of at least one gene in Table 4 and / or Table 6 in the sample with a control value of expression; and treating the individual for HNSCC if the difference between the gene expression in the individual and the control value indicates that the individual may have HNSCC (i.e., is diagnostic of the presence) or is prone to develop HNSCC (i.e., is at high risk). The control value may be derived from one or more samples of normal (non-cancerous tissue) or may be derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include measuring at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used.

[0127] For example, in certain embodiments, a method of treatment includes obtaining a sample from an individual; measuring in the sample the amount of at least one expression product from at least one gene including at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6; and determining in the sample the amount of at least one expression product from at least one gene including at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6. or HSD17B6 to a control value of expression of each of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6; and treating the individual for HNSCC if a difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., is diagnostic of the presence of) or is predisposed to developing (i.e., is at high risk of) HNSCC.

[0128] The method of treatment may further include measuring the amount of at least one expression product from a gene including at least one of HPV E6 and / or HPV E7 in the sample; comparing the expression of at least one of the HPV E6 and / or HPV E7 expression products in the sample to a control value for the expression of each of HPV E6 and / or HPV E7; and treating the individual for HNSCC if a difference between the gene expression in the individual and the control value indicates that the individual may have HNSCC (i.e., is diagnostic of the presence) or is susceptible to developing HNSCC (i.e., is at high risk).

[0129] In various embodiments of the method of treatment, the reference value may be derived from one or more samples of normal (non-cancerous tissue) or may be derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include the measurement of at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used.

[0130] As described above, yet another embodiment includes a composition for detecting a biomarker associated with HNSCC in an individual. In certain embodiments, the composition includes a reagent for quantifying the level of at least one disclosed biomarker in a biological sample. For example, as described in detail herein, the composition may include a reagent for measuring mRNA. Or, the composition may include a reagent for measuring a peptide or polypeptide biomarker. In one embodiment, the composition includes a reagent for performing an immunoassay. Or, the composition may include a reagent for performing flow cytometry. Or, as described in detail herein, the composition may include a reagent for determining the presence of a specific sequence and / or expression level of a nucleic acid. As described in detail herein, the reagent may be labeled with a detectable moiety.

[0131] Accordingly, other embodiments of the present disclosure include compositions for detecting biomarkers associated with HNSCC in an individual comprising a reagent for quantifying the expression level of at least one gene in Table 4 and / or Table 6. Additionally and / or alternatively, other embodiments of the present disclosure include compositions for detecting biomarkers associated with HNSCC in an individual comprising a reagent for quantifying the expression level of at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6. Additionally and / or alternatively, embodiments of the present disclosure include compositions for detecting biomarkers associated with HNSCC in an individual comprising a reagent for quantifying the expression level of at least one of HPV The present invention includes a composition for detecting biomarkers associated with HNSCC in an individual, comprising a reagent for quantifying the expression level of at least one of E6 and / or HPV E7. Additionally and / or alternatively, the composition may include a reagent for detecting at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1. In other embodiments, RPL30 or other normalization genes may be used. For example, in some embodiments, the reagent detects mRNA. Alternatively, the reagent may detect protein.

[0132] Thus, the composition may, in certain embodiments, comprise a primer (e.g., a primer pair) and / or a probe for any one of these genes, the primer and / or the probe being labeled with a detectable moiety as described herein. Additionally and / or alternatively, the primer and / or the probe may comprise an array in which the primer and / or the probe are immobilized on a surface. In other embodiments, the reagent may comprise a reagent for measuring the peptides and / or proteins expressed from the disclosed genes. For example, the composition may comprise a reagent for performing an immunoassay. In some embodiments, these reagents may comprise an array as described in detail herein. The reagent may be labeled with a detectable moiety as described in detail herein.

[0133] Other embodiments include systems for carrying out the methods disclosed herein and / or using the compositions disclosed herein. For example, other aspects of the present disclosure include kits that include the compositions disclosed herein or kits for carrying out the methods disclosed herein. For example, other embodiments include systems such as kits that contain at least some of the compositions disclosed herein and / or reagents for carrying out the methods disclosed herein. Such systems or kits may include computer-readable media that include instructions and / or other information for carrying out the methods and / or using the compositions disclosed herein.

[0134] Various types of samples can be used in any of the methods, compositions or systems disclosed herein.In certain embodiments, the sample comprises serum, plasma, saliva or tissue (e.g., FFPE tissue).Or other sample types can be used.

[0135] Peptide, Polypeptide and Protein Assays In certain embodiments, the biomarker of interest is detected at the protein level (or peptide or polypeptide level), i.e., the gene product is analyzed. For example, the protein or fragments thereof can be analyzed by amino acid sequencing or immunoassay using one or more antibodies that specifically recognize one or more epitopes present on the biomarker of interest or, in some cases, specific for the mutation of interest. The protein can be analyzed by protease digestion (e.g., trypsin digestion), and in some embodiments, the digested protein product can be further analyzed by 2D gel electrophoresis.

[0136] Antibody-Based Detection Methods Specific antibodies that recognize the biomarker of interest can be used in any of a variety of ways known in the art. Antibodies against a particular epitope, polypeptide, and / or protein can be generated using any of a variety of ways known in the art. For example, the epitope, polypeptide, or protein against which an antibody is desired can be produced and injected into an animal, typically a mammal (e.g., donkey, mouse, rabbit, horse, chicken, etc.), and the antibodies produced by the animal can be collected from the animal. Monoclonal antibodies can also be generated by generating hybridomas that express the antibody of interest in an immortalized cell line.

[0137] In some embodiments, the antibody is labeled with a detectable moiety described herein.

[0138] Antibody detection methods are well known in the art and include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and Western blot. Some such methods are suitable for implementation in an array format.

[0139] For example, in some embodiments, the biomarker of interest is detected using a first antibody (or antibody fragment) that specifically recognizes the biomarker. The antibody may be labeled with a detectable moiety (e.g., a chemiluminescent molecule), an enzyme, or a second binding agent (e.g., streptavidin). Alternatively, the first antibody may be detected using a second antibody as known in the art.

[0140] In certain embodiments, the method may further include adding a capture support, which includes at least one capture support binder that recognizes and binds the biomarker so as to immobilize the biomarker on the capture support. In certain embodiments, the method may further include adding a second binder that can specifically recognize and bind at least a portion of the plurality of binder molecules and / or biomarkers on the capture support. In one embodiment, the binder that can specifically recognize and bind at least a portion of the plurality of binder molecules and / or biomarkers on the capture support is a soluble binder (e.g., a second antibody). The second binder may be labeled (e.g., with an enzyme) such that binding of the biomarker of interest is measured by adding a substrate for the enzyme and quantifying the amount of product formed.

[0141] In one embodiment, the capture solid support can be an assay well (i.e., a microtiter plate, etc.). Alternatively, the capture solid support can be a location on an array, or a mobile support, such as a bead, or the capture support can be a filter.

[0142] In some examples, the biomarker can be complexed with a first binding agent (e.g., a primary antibody specific for the biomarker and labeled with a detectable moiety) and a second binding agent (e.g., a secondary antibody or a second primary antibody that recognizes the primary antibody), which forms a complex with a third binding agent (e.g., biotin), which can then interact with a capture support (e.g., magnetic beads) that has a reagent (e.g., streptavidin) that recognizes the third binding agent linked to the capture support. The complex (labeled primary antibody:biomarker:second primary antibody-biotin:streptavidin-beads) is then captured using a magnet (e.g., a magnetic probe) to measure the amount of the complex.

[0143] A variety of binding agents may be used in the disclosed methods. For example, the binding agent attached to the capture support or the second antibody may be either an antibody or an antibody fragment that recognizes the biomarker. Alternatively, the binding agent may include a protein that binds to a non-protein target (i.e., a protein that specifically binds to a small molecule biomarker, or a receptor that binds to a protein, etc.).

[0144] In certain embodiments, the solid support may be treated with a passivation agent. For example, in certain embodiments, the biomarker of interest may be captured on a passivated surface (i.e., a surface that has been treated to reduce non-specific binding). One such passivation agent is BSA. Additionally and / or alternatively, if the binding agent used is an antibody, the solid support may be coated with Protein A, Protein G, Protein A / G, Protein L, or another agent that binds with high affinity to the binding agent (e.g., an antibody). These proteins bind to the Fc domain of the antibody and thus can direct the binding of the antibody that recognizes one or more proteins of interest.

[0145] Nucleic Acid Assays In certain embodiments, the biomarkers disclosed herein are detected at the nucleic acid level. In one embodiment, the disclosure includes a method for diagnosing the presence of, or an increased risk of developing, a condition or disease of interest (e.g., HNSCC) in a subject.

[0146] The method may include obtaining nucleic acid from a tissue or body fluid sample from a subject, and performing an assay to identify whether there is overexpression of a gene of interest. For example, the overexpression of a particular gene product may be quantified using reverse transcriptase PCR (RT-PCR). Alternatively, droplet digital PCR (ddPCR) may be used.

[0147] Alternatively, the method may include obtaining nucleic acid from a tissue or fluid sample from the subject, and performing an assay to identify whether the subject's nucleic acid has a variant sequence (i.e., a mutation). In certain embodiments, the method may include comparing the variant to known variants associated with the condition or disease of interest, and determining whether the variant is a variant previously identified as associated with the condition or disease of interest. Alternatively, the method may include identifying the variant as a new variant that has not been previously characterized. If the variant is a new variant, the method may further include performing an analysis to determine whether the mutation is expected to be deleterious to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using the variant profile (i.e., a compilation of the mutations identified in the subject) to diagnose the presence of the syndrome or disease of interest, or an increased risk of developing the syndrome or disease of interest.

[0148] Nucleic acid analysis can be performed on genomic DNA, messenger RNA, and / or cDNA. In various embodiments, the nucleic acid includes genes, RNA, exons, introns, gene regulatory elements, expressed RNA, siRNA, or epigenetic elements. Regulatory elements including splice sites, transcription factor binding, AI editing sites, microRNA binding sites, and functional RNA structure sites can also be evaluated for mutations (i.e., variants). Thus, for each of the disclosed methods and compositions, the variants can include nucleic acid sequences that encompass at least one of the following: (1) A-to-I editing sites; (2) splice sites; (3) conserved functional RNA structures; (4) effective transcription factor binding sites (TFBS); (5) microRNA (miRNA) binding sites; (6) polyadenylation sites; (7) known regulatory elements; (8) miRNA genes; (9) ROI-encoded small nucleolar RNA genes; and / or (10) ultraconserved elements across placental mammals.

[0149] In many embodiments, the nucleic acid is extracted from the biological sample. In some embodiments, the nucleic acid is analyzed without being amplified. In some embodiments, the nucleic acid is amplified using techniques known in the art (e.g., generation of cDNA amplified using polymerase chain reaction (PCR)), and the amplified nucleic acid is used for subsequent analysis. Multiplex PCR may be used, in which multiple sets of primer pairs are used to amplify several amplicons (e.g., from different genomic regions) at once. For example, the nucleic acid may be analyzed by sequencing, hybridization, PCR amplification, restriction enzyme digestion, primer extension, such as single-base primer extension or multiplex allele-specific primer extension (ASPE), or DNA sequencing. In some embodiments, the nucleic acid is amplified such that the size of the amplification product of the wild-type allele is different from the size of the mutant allele. Thus, the presence or absence of a particular mutant allele can be determined by detecting the difference in size of the amplification product, for example on an electrophoretic gel. For example, deletions or insertions of gene regions may be particularly suitable for use with size-based approaches.

[0150] Certain exemplary nucleic acid analysis methods are described in detail below.

[0151] Analysis of mRNA In certain embodiments, mRNA is analyzed using real-time and / or reverse transcriptase PCR, using methods known in the art and / or commercially available reagents and / or kits. "Real-time PCR" or rPCR is a method for detecting and measuring the product generated during each cycle of PCR, which is proportional to the amount of template nucleic acid before the start of PCR. The information obtained, such as an amplification curve, can be used to determine the presence of target nucleic acid and / or to quantify the initial amount of target nucleic acid sequence. The term "real-time PCR" is used to describe a subset of PCR technology that allows detection of PCR products throughout the entire PCR reaction or in real time.

[0152] In some examples, rPCR is real-time reverse transcriptase (RT) PCR (rRT-PCR). Alternatively, droplet digital PCR may be used. Reverse transcriptase PCR is used when the starting material is RNA and / or mRNA. RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase. In rRT-PCR, the cDNA is then used as a template for the qPCR reaction. rRT-PCR can be performed in a one-step method that combines reverse transcription and PCR in a single tube and buffer, using reverse transcriptase together with DNA polymerase. In one-step rRT-PCR, both RNA and DNA targets are amplified using sequence-specific targets. The term "quantitative PCR" encompasses all PCR-based techniques that allow for quantitative or semi-quantitative determination of the target nucleic acid sequence initially present.

[0153] The principle of real-time PCR (rPCR) is generally described, for example, in Held et al., “Real Generally, rPCR measures a signal at each amplification cycle. Some rPCR techniques rely on fluorophores that emit a signal upon the completion of every amplification cycle. An example of such a fluorophore is a fluorescent dye, such as SYBR Green, that fluoresces at a given wavelength when bound to double-stranded DNA. Thus, an increase in double-stranded DNA during each amplification cycle leads to an increase in fluorescence intensity due to the accumulation of PCR products. Another example of a fluorophore used for detection in rPCR is a sequence-specific fluorescent reporter probe, described elsewhere in this document. Such a probe is An example of a probe is the TAQMAN® probe. The use of sequence-specific reporter probes provides highly specific detection of target sequences and allows quantification even in the presence of non-specific DNA amplification. Fluorescent probes can also be used in multiplex assays to detect several genes in the same reaction, based on specific probes labeled with different colors. For example, multiplex assays can use several sequence-specific probes labeled with various fluorophores, including but not limited to FAM, JA270, CY5.5, and HEX, in the same PCR reaction mixture.

[0154] rPCR relies on the detection of a measurable parameter, such as fluorescence, during the course of the PCR reaction. The amount of the measurable parameter is proportional to the amount of PCR product, thereby allowing the increase in PCR product to be observed in "real time". Some rPCR methods allow for quantification of the input DNA template based on the observable progress of the PCR reaction. Data analysis and processing are discussed below. A "growth curve" or "amplification curve" in the context of a nucleic acid amplification assay is a graph of a function where the independent variable is the number of amplification cycles and the dependent variable is an amplification-dependent measurable parameter, such as the fluorescence emitted by a fluorophore, that is measured at each cycle of amplification. As discussed above, the amount of amplified target nucleic acid can be detected using a fluorophore-labeled probe. Generally, the amplification-dependent measurable parameter is the amount of fluorescence emitted by the probe upon hybridization or upon hydrolysis of the probe by the nuclease activity of a nucleic acid polymerase. The increase in fluorescence emission is measured in real time and is directly related to the increase in target nucleic acid amplification. In some instances, the change in fluorescence (dR n ) is expressed by the formula dR n =R n+ -R n- Calculated using R n+ is the fluorescence emission of the product at each time point, and R n- is the baseline fluorescence emission. dR n The values ​​are plotted against cycle number to produce an amplification plot. In a typical polymerase chain reaction, the growth curve contains a segment of exponential growth followed by a plateau, which results in an S-shaped amplification plot when a linear scale is used. Growth curves are characterized by the "crossing point" value or "C p This is characterized by a "threshold" or "cycle threshold" (C t ), is the cycle number at which a measurable parameter of a given magnitude is achieved. For example, when using fluorophore-labeled probes, the threshold (C t ) is the fluorescence emission (dR nC is the PCR cycle number at which the C exceeds a selected threshold, which is typically 10 times the standard deviation of the baseline (although this threshold level can be modified as desired). t The lower the value, the faster the amplification is completed and the C t The higher the value, the slower the amplification will be completed. If the amplification efficiency is similar, C t Lower values ​​reflect higher starting amounts of target nucleic acid, C t Higher values ​​reflect lower starting amounts of target nucleic acid. A control nucleic acid of known concentration is used to generate a "standard curve" or "control" C values ​​at various known concentrations of the control nucleic acid. t To generate a set of values, the C t By comparing the values ​​it is possible to determine the absolute amount of the target nucleic acid in the sample.

[0155] Allele-specific amplification In some embodiments, for example when the biomarker for a disease and / or condition group of interest is a mutation, the biomarker is detected using an allele-specific amplification assay. This approach includes PCR amplification of specific alleles (PASA) (Sarkar et al., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama et al., 1989 J. Lab. Clin. Med. 114:105-113), allele-specific PCR (ASPCR) (Wu et al., 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractory mutation system (ARMS) (Newton et al., 1989 Nucleic Acids Res. 17:2503-2516). The entire contents of each of these references are incorporated herein. This method is applicable to single base substitutions as well as microdeletions / insertions.

[0156] For example, for PCR-based amplification methods, amplification primers can be designed to be able to distinguish between different alleles (e.g., between wild-type and mutant alleles). Thus, the presence or absence of an amplification product can be used to determine whether a genetic mutation is present in a given nucleic acid sample. In some embodiments, allele-specific primers can be designed such that the presence of an amplification product indicates a genetic mutation. In some embodiments, allele-specific primers can be designed such that the absence of an amplification product indicates a genetic mutation.

[0157] In some embodiments, two complementary reactions are used. One reaction uses a primer specific for the wild-type allele (the "wild-type specific reaction") and the other reaction uses a primer for the mutant allele (the "mutation specific reaction"). The two reactions may use a common second primer. PCR primers specific for a particular allele (e.g., the wild-type allele or the mutant allele) usually perfectly match one allelic variant of the target but not the other allelic variant (e.g., the mutant allele or the wild-type allele). The mismatch may be located at / near the 3' end of the primer, leading to preferential amplification of the perfectly matched allele. Whether an amplification product can be detected from one or both reactions indicates the presence or absence of the mutant allele. Detection of an amplification product only from the wild-type specific reaction indicates the presence of only the wild-type allele (e.g., homozygosity for the wild-type allele). Detection of an amplification product only from the mutation specific reaction indicates the presence of only the mutant allele (e.g., homozygosity for the mutant allele). Detection of amplification products from both reactions indicates (e.g., a heterozygote). Herein, this approach is also referred to as "allele-specific amplification (ASA)."

[0158] Allele-specific amplification can also be used to detect duplications, insertions, or rearrangements by using primers that partially hybridize across the junction. The extent of junction overlap can be varied to allow specific amplification.

[0159] The amplification products can be examined by methods known in the art, including visualizing (e.g., with one or more dyes) bands of nucleic acid that have migrated (e.g., by electrophoresis) through a gel to separate the nucleic acids by size.

[0160] Allele-specific primer extension In some embodiments, an allele-specific primer extension (ASPE) approach is used to detect genetic mutations. ASPE uses allele-specific primers in the extension reaction that can distinguish between alleles (e.g., mutant and wild-type alleles), so that extension products are obtained only in the presence of a particular allele (e.g., mutant or wild-type alleles). The extension products can be detectable or can be made detectable, for example, by using labeled deoxyribonucleotides in the extension reaction. Any of a variety of labels are suitable for use in these methods, including, but not limited to, radioactive labels, fluorescent labels, chemiluminescent labels, enzyme labels, and the like. The nucleotides are labeled with an entity that can then be bound (directly or indirectly) by a biotin molecule that can be bound to a detectable label, for example, a streptavidin-linked fluorescent dye. In some embodiments, the reaction is performed multiplexed, for example, using many allele-specific primers in the same extension reaction.

[0161] In some embodiments, the extension products are hybridized to a solid or semi-solid support, such as a bead, matrix, gel, etc., among others. For example, the extension products may be tagged with a specific nucleic acid sequence (e.g., included as part of an allele-specific primer), and an "anti-tag" (e.g., a nucleic acid sequence complementary to the tag in the extension product) may be attached to the solid support. The extension products may be captured and detected on the solid support. For example, the beads may be sorted and detected.

[0162] Single nucleotide primer extension In some embodiments, single nucleotide primer extension (SNuPE) assays are used, in which the primer is designed to extend only one nucleotide. In such methods, the identity of the nucleotide immediately downstream of the 3' end of the primer is known, and the mutant allele is different compared to the wild-type allele. SNuPE can be performed using an extension reaction in which only a specific type of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP, labeled dGTP, or labeled dTTP). Thus, the presence of a detectable extension product can be used as an indication of the identity of the nucleotide at the position of interest (e.g., the position immediately downstream of the 3' end of the primer), and therefore, can be used as an indication of the presence or absence of a mutation at that position. SNuPE is disclosed in U.S. Patent No. 5,888,819; U.S. Patent No. 5,846,710; U.S. Patent No. 6,280,947; U.S. Patent No. 6,482,595; U.S. Patent No. 6,503,718; U.S. Patent No. 6,919,174; Piggee, C. et al., Journal of Chromatography A 781 (1997), p. 367-375 ("Capillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detection"); Hoogendoorn, B. et al., Human Genetics (1999) 104:89-93, ("Genotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid Chromatography" (2004), the contents of each of which are incorporated herein by reference in their entireties.

[0163] In some embodiments, primer extension can be combined with mass spectrometry to accurately and quickly detect the presence or absence of mutation.See U.S. Patent No. 5,885,775 to Haff et al. (analysis of single nucleotide polymorphism analysis by mass spectrometry); U.S. Patent No. 7,501,251 to Koster (DNA diagnosis based on mass spectrometry); the teachings of both are incorporated herein by reference.Suitable mass spectrometry formats include, but are not limited to, matrix-assisted laser desorption / ionization, time-of-flight (MALDI-TOF), electrospray (ES), IR-MALDI, ion cyclotron resonance (ICR), Fourier transform, and combinations thereof.

[0164] Oligonucleotide ligation assay In some embodiments, oligonucleotide ligation assay ("OLA" or "OL") is used. OLA uses two oligonucleotides designed to hybridize to adjacent sequences of a single strand of a target molecule. Typically, one oligonucleotide is biotinylated and the other is detectably labeled, for example, with a streptavidin-linked fluorescent moiety. When the exact complementary sequence is found in the target molecule, the oligonucleotides hybridize with their ends adjacent, creating a ligation substrate that can be captured and detected. See, for example, Nickerson et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927, Landegren, U. et al. (1988) Science 241:1077-1080 and U.S. Patent No. 4,998,617, the entire contents of which are incorporated herein by reference in their entirety.

[0165] Hybridization approach In some embodiments, nucleic acid is analyzed by hybridization using one or more oligonucleotide probes specific to the biomarker of interest, under sufficiently stringent conditions that do not allow single nucleotide mismatch.In certain embodiments, suitable nucleic acid probes can distinguish between normal and mutant genes.Therefore, for example, those skilled in the art can use the probe of the present invention to determine whether an individual is homozygous or heterozygous for a particular allele.

[0166] Nucleic acid hybridization techniques are well known in the art. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least desired levels of complementarity will stably hybridize, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ.

[0167] In some embodiments, probe molecules that hybridize to mutant or wild-type sequences can be used to detect such sequences in the amplification products by liquid phase or, more preferably, solid phase hybridization, which can be accomplished, for example, by attaching the probe to a microchip.

[0168] Nucleic acid probes may include ribonucleic acid and / or deoxyribonucleic acid. In some embodiments, the nucleic acid probes provided are oligonucleotides (i.e., "oligonucleotide probes"). Generally, oligonucleotide probes are long enough to specifically bind to homologous regions of a gene of interest, but short enough that a one nucleotide difference between the probe and the nucleic acid sample being tested disrupts hybridization. Generally, the size of an oligonucleotide probe ranges from approximately 10 to 100 nucleotides. In some embodiments, the oligonucleotide probes are 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-35, 15-30, 18-30, or 18-26 nucleotides in length. As will be appreciated by those of skill in the art, the optimal length of an oligonucleotide probe may depend on the particular method and / or conditions in which the oligonucleotide probe may be used.

[0169] In some embodiments, the nucleic acid probe is useful as a primer, for example, for nucleic acid amplification and / or extension reaction. For example, in certain embodiments, the gene sequence evaluated for variants includes exon sequences. In certain embodiments, exon sequences and additional flanking sequences (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more nucleotides of UTR and / or intron sequences) are analyzed in the assay. Alternatively, intron sequences or other non-coding regions can be evaluated for potentially harmful mutations. Alternatively, parts of these sequences can be used. Such variant gene sequences can include sequences that have at least one of the mutations described herein.

[0170] Other embodiments of the present disclosure provide isolated gene sequences that contain mutations associated with symptoms and / or diseases of interest. Such gene sequences can be used to objectively diagnose the presence of HNSCC or increased risk of developing it for a subject. In certain embodiments, the isolated nucleic acid can contain non-variant sequences or any one or combination of variant sequences. For example, in certain embodiments, the gene sequence includes exon sequences. In certain embodiments, exon sequences and additional flanking sequences (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more nucleotides of UTR and / or intron sequences) are analyzed in the assay. Alternatively, intron sequences or other non-coding regions may be used. Or, portions of these sequences may be used. In certain embodiments, the gene sequence includes exon sequences from at least one of the biomarker genes disclosed herein.

[0171] In some embodiments, the nucleic acid probe is labeled with a detectable moiety as described herein.

[0172] array Various methods mentioned herein can be adapted for use as arrays, which allows a set of biomarkers to be analyzed and / or detected in a single experiment.For example, multiple mutations involving biomarkers can be analyzed simultaneously.In particular, methods involving the use of nucleic acid reagents (e.g., probes, primers, oligonucleotides, etc.) are particularly suitable for adaptation to array-based platforms (e.g., microarrays).In some embodiments, the array contains one or more probes that are specific for detecting mutations in biomarkers of interest.

[0173] In one embodiment, a panel of the disclosed biomarkers is used. In one embodiment, the present disclosure includes a composition for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising a reagent for quantifying the expression level of at least one gene in Table 4 and / or Table 6, and / or at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6, and / or at least one of HPV E6 and E7 genes. Additionally and / or alternatively, the composition may include at least one normalization (e.g., housekeeping) gene. In one embodiment, the normalization gene may be KHDRBS1 and / or RPL30 or other normalization gene. The composition may include, in certain embodiments, a primer and / or a probe of any one of these genes, and the primer and / or probe is labeled with a detectable moiety as described herein.

[0174] DNA sequencing In certain embodiments, diagnosis of a biomarker of interest is performed by detecting variations in the sequence, genomic location or configuration, and / or genomic copy number of a nucleic acid or panel of nucleic acids by sequencing.

[0175] In some embodiments, the method may include obtaining nucleic acid from a tissue or body fluid sample from a subject and sequencing at least a portion of the nucleic acid to obtain a sample nucleic acid sequence of at least one gene. In certain embodiments, the method may include comparing the variant with known variants associated with HNSCC to determine whether the variant is a variant previously identified as associated with HNSCC. Alternatively, the method may include identifying the variant as a new variant that has not been previously characterized. If the variant is a new variant, or in some cases a previously characterized (i.e., identified) variant, the method may further include performing an analysis to determine whether the mutation is predicted to be deleterious to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using the variant profile (i.e., a compilation of variants identified in the subject) to diagnose the presence of HNSCC or an increased risk of developing HNSCC.

[0176] For example, in certain embodiments, next-generation (massively parallel sequencing) may be used. Alternatively, Sanger sequencing may be used. Alternatively, a combination of next-generation (massively parallel sequencing) and Sanger sequencing may be used. Additionally and / or alternatively, sequencing includes at least one single molecule sequencing-by-synthesis method. Thus, in certain embodiments, multiple DNA samples are analyzed in pools to identify samples that exhibit variation. Additionally and / or alternatively, in certain embodiments, multiple DNA samples are analyzed in pools to identify individual samples that exhibit the same variation in at least two pools.

[0177] One traditional method of performing sequencing is by chain termination and gel separation, as described in Sanger et al., 1977, Proc Natl Acad Sci USA, 74:5463-67. Another traditional sequencing method involves chemical degradation of nucleic acid fragments. See Maxam et al., 1977, Proc. Natl. Acad. Sci., 74:560-564. Methods based on sequencing by hybridization have also been developed. See, e.g., Harris et al., U.S. Patent Publication No. 20090156412. Each of these references is incorporated herein by reference in its entirety.

[0178] In other embodiments, nucleic acid sequencing is achieved by massively parallel sequencing (also known as "next-generation sequencing") of single molecule or a group of nearly identical molecules derived from a single molecule by amplification by methods such as PCR. Massively parallel sequencing is described, for example, in Lapidus et al., U.S. Patent No. 7,169,560; Quake et al., U.S. Patent No. 6,818,395; Harris, U.S. Patent No. 7,282,337; and Braslavsky et al., PNAS (USA), 100:3960-3964 (2003), the contents of each of which are incorporated herein by reference.

[0179] In next generation sequencing, PCR or whole genome amplification can be performed on the nucleic acid to obtain sufficient amount of nucleic acid for analysis.In some forms of next generation sequencing, amplification is not required, since this method can evaluate DNA sequence from unamplified DNA.Once determined, the sequence and / or genomic arrangement and / or genomic copy number of the nucleic acid from the test sample is compared with a standard reference obtained from one or more individuals who were not known to be affected by HNSCC at the time the sample was collected.Any difference between the sequence and / or genomic arrangement and / or genomic arrangement and / or copy number of the nucleic acid from the test sample and the standard reference is considered as a variant.

[0180] In the next generation (massively parallel sequencing), all regions of interest are sequenced together and the origin of each read sequence is determined by comparison (alignment) with a reference sequence. Regions of interest can be enriched together in one reaction or enriched separately and then combined before sequencing. In a particular embodiment, and as described in more detail in the examples herein, DNA sequences derived from coding exons of genes included in the assay are enriched by bulk hybridization of randomly fragmented genomic DNA to specific RNA probes. The same adapter sequence binds to the ends of all fragments, allowing enrichment of fragments captured in all hybridizations by PCR with one primer pair in one reaction. Regions that were not captured very efficiently by hybridization are amplified by PCR with specific primers. In addition, PCR with specific primers can be used to amplify exons where similar sequences ("pseudo-exons") are present elsewhere in the genome.

[0181] In certain embodiments where massively parallel sequencing is used, PCR products are ligated to form long stretches of DNA that are sheared (e.g., by acoustic energy) into short fragments. This step ensures that the ends of the fragments are distributed throughout the region of interest. A stretch of dA nucleotides is then added to the 3' end of each fragment, which allows the fragments to be bound to a flat surface coated with oligo(dT) primers ("flow cell"). Each fragment can then be sequenced by extending the oligo(dT) primers with fluorescently labeled nucleotides. During each sequencing cycle, only one type of nucleotide (A, G, T, or C) is added, and only one nucleotide is incorporated with a chain-terminating nucleotide. For example, during the first sequencing cycle, fluorescently labeled dCTP can be added. This nucleotide is incorporated only into the growing complementary DNA strand that requires a C as the next nucleotide. After each sequencing cycle, an image of the flow cell is taken to determine which fragments have been extended. DNA strands that have incorporated a C will emit light, while those that have not incorporated a C will appear dark. Chain termination is reversed to allow the growing DNA strand to extend again, and the process is repeated for a total of 120 cycles. The image is converted into a string of bases, commonly called a "read", which reproduces the 25-60 bases at the 3' end of each fragment. The reads are then compared to a reference sequence for the DNA analyzed. Because any string of 25 bases typically occurs only once in the human genome, most reads can be "aligned" to one specific location in the human genome. Finally, a consensus sequence for each genomic region can be constructed from the available reads and compared to the exact sequence of the reference at that location. Differences between the consensus sequence and the reference are called sequence variants.

[0182] Detectable Part In certain embodiments, certain molecules used in accordance with and / or provided by the present invention (e.g., nucleic acid probes, antibodies, etc.) comprise one or more detectable entities or moieties, i.e., such molecules are "labeled" with such entities or moieties.

[0183] Any of a wide variety of detectable agents can be used in the practice of the present disclosure. Suitable detectable agents include, but are not limited to: various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (e.g., dyes, colloidal gold, etc.); biotin; dioxygenin; haptens; and proteins for which antisera or monoclonal antibodies are available.

[0184] In some embodiments, the detectable moiety is biotin, which can be bound to avidin (such as streptavidin), which is generally bound (directly or indirectly) to other moieties that are themselves detectable (e.g., fluorescent moieties).

[0185] Below are some non-limiting examples of some detectable moieties that can be used.

[0186] Fluorescent dyes In certain embodiments, the detectable moiety is a fluorescent dye. A large number of known fluorescent dyes with a wide variety of chemical structures and physical properties are suitable for use in the practice of the present disclosure. The fluorescent detectable moiety is stimulated by a laser and the emitted light is captured by a detector. The detector can be a charge-coupled device (CCD) or a confocal microscope that records its intensity.

[0187] Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), hexachlorofluorescein (HEX), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Q-DOTS. Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, CY-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc.), the BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), the IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), etc. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, "The Handbook See, "A Guide to the Study of Fluorescent Probes and Research Products," 9th Edition, Molecular Probes, Inc., Eugene, OR. Favorable properties of fluorescent labeling agents include high molar absorption coefficients, high fluorescence quantum yields, and photostability. In some embodiments, the labeling fluorophores exhibit absorption and emission wavelengths in the visible range (i.e., between 400-750 nm) rather than the ultraviolet range (i.e., below 400 nm) of the spectrum.

[0188] The detectable moiety may include multiple chemical entities, such as those in fluorescence resonance energy transfer (FRET). Resonance transfer results in an overall increase in emission intensity. See, for example, Ju et al. (1995) Proc. Nat'l, the entire contents of which are incorporated herein by reference. See Acad. Sci. (USA) 92:4347. To achieve resonance energy transfer, a first fluorescent molecule (the "donor" fluorophore) absorbs light and transfers it to a second fluorescent molecule (the "acceptor" fluorophore) through the resonance of excited electrons. In one approach, both donor and acceptor dyes can be linked together and attached to an oligo primer. Methods for linking donor and acceptor dyes to nucleic acids are described, for example, in U.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which are incorporated herein by reference. Donor / acceptor dye pairs that can be used include, for example, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, and fluorescein / QSY7 dye. See, for example, U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes are also commercially available, for example from Molecular Probes Inc. (Eugene, Oreg.) Suitable donor fluorophores include 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and the like.

[0189] enzyme In certain embodiments, the detectable moiety is an enzyme. Examples of suitable enzymes include, but are not limited to, enzymes used in ELISA, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, etc. Other examples include β-glucuronidase, β-D-glucosidase, urease, glucose oxidase, etc. Enzymes can be attached to molecules using linker groups such as carbodiimides, diisocyanates, glutaraldehyde, etc.

[0190] radioactive isotope In certain embodiments, the detectable moiety is a radioisotope. For example, the molecule may be isotopically labeled (i.e., may contain one or more atoms replaced with an atom having an atomic mass or mass number different from that normally found in nature), or the isotope may be attached to the molecule. Non-limiting examples of isotopes that can be incorporated into the molecules include isotopes of hydrogen, carbon, fluorine, phosphorus, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I, 135I, 186Re, 187Re, 201T1, 212Bi, 213Bi, 211At, 153Sm, 177Lu).

[0191] Dendrimer In some embodiments, signal amplification is achieved using labeled dendrimers as detectable moieties (see, e.g., Physiol Genomics 3:93-99, 2000), the entire contents of which are incorporated herein by reference in their entirety. Fluorescently labeled dendrimers are available from Genisphere (Montvale, NJ). These can be chemically attached to oligonucleotide primers by methods known in the art.

[0192] system In certain embodiments, the present disclosure provides a system for carrying out the methods disclosed herein and / or for using the compositions described herein. In certain embodiments, the system may include a kit. Or, the system may include computerized instructions and / or reagents for carrying out the methods disclosed herein.

[0193] kit In certain embodiments, the present disclosure provides a kit for use according to the methods and compositions disclosed herein. In general, the kit includes one or more reagents for detecting biomarkers of interest. Suitable reagents may include nucleic acid probes and / or antibodies or fragments thereof. In some embodiments, suitable reagents are provided in the form of arrays such as microarrays or mutation panels. The kit may further include a reagent that serves as a positive control for the biomarkers (i.e., genes) of interest.

[0194] In one embodiment, a panel of the disclosed biomarkers is used. In one embodiment, the present disclosure includes a kit for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising a reagent for quantifying the expression level of at least one gene in Table 4 and / or Table 6, and / or at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6, and / or at least one of HPV E6 and E7 genes. Additionally and / or alternatively, the kit may include at least one normalization (e.g., housekeeping) gene and / or a reagent for detecting such a housekeeping gene. In one embodiment, the normalization gene may be KHDRBS1 and / or RPL30 or other normalization genes. In some embodiments, the kit may include a positive control of any of the disclosed biomarkers and / or normalization genes.

[0195] In some embodiments, the kit provided further comprises reagents for carrying out various detection methods described herein (e.g., RT-PCR, sequencing, hybridization, primer extension, multiplex ASPE, immunoassays, etc.). For example, the kit may optionally comprise buffers, enzymes, and / or reagents for use in the methods described herein, e.g., for amplifying nucleic acids via RT-PCR, primer-directed amplification, for carrying out ELISA experiments, etc. The kit may, in certain embodiments, comprise a primer and / or probe for any one of these genes, the primer and / or probe being labeled with a detectable moiety as described herein.

[0196] In some embodiments, the kit provided further comprises a control representative of a healthy individual, e.g., a nucleic acid and / or protein sample from an individual not having the disease and / or condition of interest. Alternatively, the kit may comprise a positive control comprising a known amount of one (or more) biomarker genes to be measured. The kit may also comprise instructions on how to determine whether an individual has the disease and / or condition of interest or is at risk of developing the disease and / or condition of interest.

[0197] In some embodiments, computer readable media is provided that encodes information corresponding to a biomarker of interest. Such computer readable media may be included in a kit of the invention.

[0198] How to Identify HNSCC Markers Data Mining In certain embodiments of the present disclosure, biomarkers are identified using data mining approaches.For example, in some cases public databases (e.g., PubMed, The Cancer Genome Atlas (TCGA)) can be searched for genes that have been shown to be associated (directly or indirectly) with a particular disease and / or genes that are differentially expressed in cancer compared to normal tissue.Such genes can then be evaluated as biomarkers.

[0199] molecule In certain embodiments, the present disclosure includes a method for identifying a biomarker (i.e., a variant in a nucleic acid sequence that is associated with HNSCC in a statistically significant manner) of a symptom cluster or disease of interest. For example, a gene of interest and a potential normalizing gene can be identified by evaluating gene expression in tissue samples isolated from patients with head and neck cancer using random forest analysis (see, for example, L. Breiman, "Random Forests" Machine Learning, 2001, 45:5-32), as discussed in detail herein. In this approach, a random forest is a combination of tree predictors, where each tree depends on the value of an independently sampled random vector, with the same distribution for all trees in the forest.

[0200] Alternatively, the genes and / or genomic regions that are assayed for new markers can be selected based on their importance in biochemical pathways that show genetic linkage and / or biological causation to symptoms and / or diseases of interest.Or, the genes and / or genomic regions that are assayed for markers can be selected based on genetic linkage with DNA regions that are genetically related to the inheritance of HNSCC in families.Or, the genes and / or genomic regions that are assayed for markers can be systematically evaluated to cover specific regions of chromosomes that have not yet been evaluated.

[0201] In other embodiments, the gene or genomic region evaluated for new markers may be part of a biochemical pathway that may be related to the onset of the condition and / or disease of interest (e.g., HNSCC). Variants and / or combinations of variants may be evaluated for their clinical significance based on one or more of the following methods. If a variant and / or combination of variants is reported or known to occur more frequently in the nucleic acid from subjects with the condition and / or disease of interest than subjects without it, it is considered to be at least potentially susceptible to the condition and / or disease of interest. If a variant and / or combination of variants is reported or known to be exclusively or preferentially transmitted to individuals with the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest. Conversely, if a variant is found at similar frequency in both populations, it is unlikely to be related to the onset of the condition and / or disease of interest.

[0202] If a variant or combination of variants is reported or known to have a global detrimental effect on the function of this protein or biological system in an experimental model system suitable for measuring the function of the protein or biological system, and if this variant or combination of variants affects one or more genes known to be related to the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest.For example, if a variant or combination of variants is predicted based on the predicted effect on the sequence and / or structure of protein or nucleic acid to have a global detrimental effect on protein or gene expression (i.e., to cause nonsense mutation, frameshift mutation, or splice site mutation, or even missense mutation), and if this variant or combination of variants affects one or more genes known to be related to the condition and / or disease of interest, it is considered to be at least potentially susceptible to the condition and / or disease of interest.

[0203] Also, in certain embodiments, the total number of variants may be important.When one or several variants are detected in test sample, which are evaluated as possibly being related to at least symptoms and / or disease of interest, either individually or in combination, the individual who has this variant or these variants detected in their genetic material can be diagnosed as suffering from or at high risk of developing symptoms and / or disease of interest.

[0204] For example, the disclosure herein provides a method for diagnosing the presence of or increased risk of developing HNSCC in a subject. Such a method may include obtaining nucleic acid from a tissue or bodily fluid sample. The method may include determining the expression of at least one gene in both normal and cancer tissues to identify potential biomarkers of interest. The method may further include sequencing the nucleic acid or determining the genomic location or copy number of the nucleic acid to detect whether there are one or more variants in the nucleic acid sequence or genomic location or copy number. The method may further include evaluating the clinical significance of the one or more variants. Such analysis may include evaluating the degree of association of the variant sequence in an affected population (i.e., subjects with the disease). Such analysis may also include analyzing the degree of effect that a mutation may have on gene expression and / or protein function. The method may also include diagnosing the presence of or increased risk of developing HNSCC based on the evaluation. EXAMPLES

[0205] The following examples serve to illustrate certain aspects of the present disclosure and are not intended to be limiting in any way.

[0206] Example 1 - Literature-based identification of potential HNSCC markers A preliminary literature search was performed to identify markers associated with HNSCC. Table 1 shows the type and number of markers found, and Table 2 shows the potential biomarkers identified.

[0207] [Table 1]

[0208] [Table 2-1] [Table 2-2] [Table 2-3]

[0209] Based on this initial search, it was decided to pursue markers associated with differential gene expression.

[0210] Example 2 - Biomarker identification using the TCGA database Data from The Cancer Genome Atlas (TCGA) database was used to identify markers that show differential expression in HNSCC. The TCGA database (RNASeqV2) contains data on 18,379 genes that are available for differential expression. The data includes information on clinical information (e.g., age, smoking, stage, treatment, and survival); copy number; methylation; gene expression; mutations, and microRNA expression.

[0211] The HNSCC data consisted of 530 samples from four tumor sites: oral cavity n=320; 60.4%), oropharynx (n=82; 15.5%), larynx (n=117; 22.1%) and hypopharynx (n=10; 1.5%). An additional 44 samples were from adjacent normal tissue. Of the total 530 samples, 70 were human papillomavirus (HPV) positive, 279 were HPV negative and 181 had unknown or undetermined HPV status.

[0212] Random forest analysis was performed to identify genes that were strong predictors for classifying HNSCC from normal samples. In this analysis, genes with 50% of samples with reportable data and less than 2-fold change in expression (Wilcox test, adjusted p-value <0.001) were discarded. In each round of analysis, 75% of the samples were used as the training set and 25% of the samples were used as the test set. The data was kappa optimized and 10-fold cross-validation was performed (repeated 10 times and performance averaged). The top 20 strong predictors were identified and the entire process was repeated 4 times. The gene lists obtained from each run are shown in Table 3 and the combination of 36 unique genes is shown in Table 4. The data in Table 3 are presented in order of highest rank (20) to lowest (1).

[0213] [Table 3]

[0214] [Table 4-1] [Table 4-2]

[0215] The top four genes from each of the four analyses were then selected to obtain an initial candidate list of eight unique genes, listed in Table 5 .

[0216] [Table 5]

[0217] The results of several statistical analyses of the individual genes (ie, accuracy, kappa, sensitivity and specificity) are also shown in Figure 1, listed in order of specificity.

[0218] Example 3 - Gene Panel Analysis was performed to determine whether the use of a gene panel would be expected to improve assay performance. As shown in Figure 2, the use of a four or five gene panel should significantly improve gene performance. Panels were constructed by adding the most informative marker (CAB39L) to the next least informative marker (ADAM12) to form a two-marker panel, followed by the next least informative marker (NRG2) to form a three-marker panel. The other six markers were then added, respectively, and the predicted performance was assessed (Figure 2, top table). Results showed that a four marker panel of CAB39L, ADAM12, NRG2, and GRIN2D provided the highest levels of accuracy, kappa value, sensitivity, and specificity. Results for the five-gene panel are shown in the table below (Figure 2). The addition of more than four to five genes was found to provide minimal improvement (e.g., graph in Figure 2). Nevertheless, such panels may be useful when there are technical challenges with one of the markers identified to be one of the top four to five markers.

[0219] Example 4 - Gene expression by tumor location Most HNSCCs are found in either the oral cavity (mouth) or oropharynx (throat). An analysis was performed to determine whether the same gene panel developed using the entire HNSCC dataset could also be used to distinguish HNSCC of the oral cavity or oropharynx from normal tissue. Results are shown in Figures 3 and 4 for eight markers, CAB39L, ADAM12, SH3BGRL2, NRG2 (Figure 3); and COL13A1, GRIN2D, LOXL2, and HSD17B6 (Figure 4); using the TCGA dataset including oral cavity and oropharynx as the majority of TCGA samples (HNSCC oral cavity (320) and normal oropharynx (82) = 402 (402 / 530 = 76%) out of a total of 530 samples, and normal oral cavity (30) and oropharynx (3) = 33 (33 / 44 = 75%) out of a total of 44 samples).

[0220] For both sites, and for the larynx, the markers were found to show very different levels of expression in normal and cancer tissues. In Figures 3 and 4, the three datasets on the far left of the x-axis (larynx, oral cavity, oropharynx) are expression levels in normal tissues, while the four datasets on the far right of the x-axis (hypopharynx, larynx, oral cavity, oropharynx) are expression levels in cancer tissues combined with the hypopharynx data. It can be seen that there was a similar distribution in normal and HNSCC sites (i.e., normal levels were similar regardless of tissue, and HNSCC levels were similar regardless of tissue). For example, CAB39L is seen to be expressed at significantly lower levels in HNSCC than normal tissues for the larynx, oral cavity, and oropharynx, respectively, whereas ADAM12 is seen to be expressed at significantly higher levels in HNSCC than normal tissues. A statistical compilation of data showing the results with the markers for all HNSCC samples compared to samples from the oral cavity and oropharynx is shown in Figure 5. There was minimal change in accuracy, sensitivity, and specificity when comparing all samples to the oral cavity and oropharynx. For all eight markers, the sample sets were reduced by approximately 25%, with minimal changes in median gene expression levels and significantly different distributions (HNSCC vs. normal), with Mann-Whitney p-values ​​less than 0.0001 for both sets.

[0221] Example 5 - Analysis of differential expression of TCGA gene sets in terms of median fold expression compared to overlap in expression. The top graph in Figure 6A shows the number of times markers from the 36 genes in Table 4 initially selected by random forest analysis were identified from four repeats of random forest analysis compared to the median rank (discriminating ability) of the markers. It can be seen that the median rank tends to increase with increasing number of times the markers were repeatedly identified from the random forest analysis. The four tables below the graph list the markers and median ranks grouped by the number of times the markers were repeatedly identified from the random forest analysis. Together, the graphs and tables provide a measure that allows prioritization of the 36 genes in Table 4 identified from the random forest analysis, first by number of repeats and then by median rank.

[0222] The graph on the left of FIG. 6B shows an analysis of differential expression of certain selected HNSCC markers of the present disclosure compared to the entire TCGA HNSCC gene set. The x-axis shows the median fold change in expression as either increased gene expression (data points to the right of 0) or decreased gene expression (data points to the left of 0). The y-axis shows the overlap in gene expression in HNSCC and normal tissues. It can be seen that the disclosed markers in Table 4 (n=36) show either a significant increase or decrease in gene expression, showing a very low overlap rate compared to other genes in the database. The entire TCGA HNSCC gene set is RNASeq data with genes from over 80% of the samples represented (i.e., 16,161 (88%) of the 18,379 genes were reportable for HNSCC and normal samples). The x-axis shows the median fold change (=HNSCC / Normal). We found that 8,352 genes (52%) were increased, 7,809 genes (48%) were decreased in HNSCC compared to normal, 1,387 genes (8.6%) were increased more than 2-fold, and 1,701 genes (10.5%) were decreased more than 2-fold. For increased gene expression in HNSCC, the cutoffs were the 5th percentile for HNSCC and the 95th percentile for normal (e.g., Figure 6B, inset for GRIN2D).

[0223] The dotted line across the graph in Figure 6B indicates that the nine markers repeated four times from the random forest analysis had less than 20% overlap in expression (range 0-19%) (i.e., these markers are below the dotted line). Additionally, the dotted line across the graph in Figure 6B indicates that 23 of the 36 unique markers in Table 4, i.e., 23 / 36=64%, have less than 20% overlap in expression because the markers cluster below the dotted line. Table 6 lists 45 additional genes with less than 20% overlap in expression that are not identified by the random forest analysis. Genes with less than 20% overlap in expression, such as GLT25D1 identified in Figure 6B, can be considered as additional biomarkers that are useful for classifying HNSCC from normal samples.

[0224] [Table 6-1] [Table 6-2]

[0225] The data in Figure 6B can be compared to those in Figures 7 and 8. Figure 7 shows a similar analysis of median fold expression and percent overlap in expression from TCGA HNSCC RNASeq data, with the example showing tissue and salivary markers identified by the literature search highlighted on the graph. The top and bottom panels show results for markers identified in both tissue (top panel) and saliva (bottom panel). While certain literature markers in Figure 7 show some evidence of differential expression, only a few markers show high levels of differential expression with low percent overlap. Based on this analysis, markers MAL, MMP1, CEP55, CENPA, AURKA and FOXM1 appear to be the most informative additional biomarkers and may be included in the disclosed methods and compositions.

[0226] Figure 8 shows a similar analysis of median fold expression and percent overlap in expression from TCGA HNSCC RNASeq data, with this example showing normalization markers used in tissue (top panel) and saliva (bottom panel) identified by literature search. It can be seen that these markers show little variation in expression, with significant overlap between HNSCC and normal. An ideal normalization marker should have minimal variation and similar expression levels as the gene panel of interest.

[0227] Example 6 - Identification of potential normalization or housekeeping genes The TGCA database was analyzed to identify potential normalization genes using three criteria: (1) the minimum median fold change in expression between HNSCC and normal tissues; (2) the minimum interquartile range (IQR) in both HNSCC and normal (IQR is defined as the 75th quartile of gene expression / 25th quartile of gene expression); and (3) a median expression level close to the panel of genes of interest to facilitate experimental comparison between potential candidate gene expression and normalization genes.

[0228] The analysis is summarized in Figure 9A. The left panel shows a plot of the median fold change of gene expression in HNSCC and normal (x-axis) against the mean IQR (=[HNSCC IQR+Normal IQR] / 2) for both normal and cancer cells (y-axis). In this figure, positive numbers on the x-axis correspond to increased gene expression in cancer cells compared to normal, and negative numbers correspond to decreased gene expression in cancer cells compared to normal. Data from a total of 16,161 genes were analyzed (left panel). This matches all genes with both normal and HNSCC data in the TCGA database, and thus matches 88% (16,151 / 18,379) of the total number of genes in the TCGA database. Again, 8,352 genes (52%) were found to be increased, 7,809 genes (48%) were decreased, 1,387 genes (8.6%) were increased more than two-fold, and 1,701 genes (10.5%) were decreased more than two-fold in HNSCC compared to normal.

[0229] Potential normalization genes of most interest are those with a fold change (x-axis) of 0 and a mean IQR of 1 (circled area on the plot). The middle plot shows data for genes with a median fold change of less than 2 and a mean IQR of less than 2 (n=7,949 genes). The right panel shows gene expression data for 7,949 candidate normalization genes. Genes with median expression were considered of most interest. Based on this analysis, KHDRBS1 (KH domain-containing, RNA-binding, signal transduction-related protein 1) was identified as a normalization gene of interest. Several other more common normalization genes, such as RPLPO, RPL10, RPL30, GAPDH, etc., are identified in the middle panel. TCGA database data for KHDRBS1 is shown in Table 6 below. Figure 9B shows that KHDRBS1 showed similar characteristics (low fold change expression and low IQR) across many cancer types.

[0230] The mean level of expression, 11.60-11.90, is higher than the proposed panel markers, which range from 1.52 (NRG2 in HNSCC) to 11.44 (SH3BGRL2 in normal). Nevertheless, this is within the range of the cancer-specific markers and should therefore be a good normalization gene. Note that amplicon lengths of less than 100 bp are preferred for FFPE samples, which may contain substantially degraded RNA.

[0231] [Table 7]

[0232] These data can be compared to those of the well-known housekeeping (normalizing) gene GAPDH (Table 7). The median expression of 16.3-16.50 is about 30-fold higher than the gene (SH3BGRL2) with the highest level of expression in the candidate panel discussed above. Thus, GAPDH may be less useful as a marker in the HNSCC panel of the present disclosure discussed above.

[0233] Example 7 - Evaluation of expression assays Experiments were performed to compare expression levels determined from data in the TCGA database (RNASeq assessment of gene expression) with expression levels measured using droplet digital PCR (ddPCR). The results are shown in Figure 10. The figure shows data demonstrating the reproducibility of ddPCR analysis of ADAM12 and SH3BGRL2 by ddPCR in tongue squamous cell carcinoma (SCC) and normal tissues (oral mucosa) (top table in Figure 10). A comparison of ddPCR data (bottom table) and TCGA RNASeq data (middle table) for gene expression levels of ADAM12 and SH3BGRL2 in cancer compared to normal tissues is also shown. As measured by both techniques, it can be seen that there is a significant increase in the expression of ADAM12 in cancer compared to normal, and a significant decrease in the expression of SH3BGRL2 in cancer compared to normal. In these experiments, two aliquots from the same sample were analyzed. Regarding the buccal samples, one of the samples had expression levels that were too low to be measured accurately. Although ddPCR values ​​were generally lower than the TCGA RNASeq data, the trend was the same for both markers (see Figure 10). Reproducibility was good down to 1 copy / μL.

[0234] FIG. 11 shows further digital PCR data for three formalin-fixed paraffin-embedded patient samples (DA1081983; DR1041686; DA0063595) and one URNA control sample derived from cell culture cancer tissue. URNA is a universal human reference RNA available from Agilent (cat. no. 740000). It is comprised of 10 human cancer cell lines that serve as a consistent control for standard data set comparison. Either duplicate or triplicate sampling was performed according to embodiments of the present disclosure. Again, it was found that there was a substantial reduction in SH3BGRL2 expression in cancer compared to normal. It can be seen that the copy number per uL from the URNA control is much greater compared to the cross-linked and possibly fragmented RNA from the FFPE sample, likely due to the intact nature of the isolated RNA.

[0235] Figure 12 shows an RNA titration experiment using ddPCR and the markers SH3BGRL2 (labeled with FAM) and KHDRBS1 (labeled with HEX). In this experiment, RNA was isolated from FFPE samples (or URNA was used as a positive control) and diluted 2- or 10-fold. Standard techniques were used to generate cDNA, and ddPCR was used to detect the presence of the biomarker SH3BGRL2 (amplified sequence labeled with FAM) or the housekeeping gene KHDRBS1 (labeled with HEX). Results for three samples (#1, #3, or #5) are shown. It can be seen that there is a good correlation between the ratios of the marker and the housekeeping gene at various concentrations, except for very dilute samples (i.e., approaching or below 1 copy / μL). This indicates that the range of sample concentrations that can be measured using this assay technique is adequate.

[0236] Figure 13 shows the relative abundance of biomarkers SH3BGRL2 and housekeeping RNA KHDRBS1 in FFPE samples compared to positive control URNA (left panel), the ratio of biomarkers SH3BGRL2 / KHDRBS1 in cancer cells compared to normal cells and URNA (middle panel), and the relative abundance of KHDRBS1 to SH3BGRL2 in cancer and normal cells measured using RNASeq (right panel). Again, consistent patterns of biomarkers are seen regardless of how they are measured (absolute values ​​may vary).

[0237] Figure 14 shows the measurement of SH3BGRL2 measured as a singleplex assay (i.e. only SH3BGRL2-FAM generated by PCR was measured in either normal or cancer derived samples compared to a duplex reaction where both SH3BGRL2 and KHDRBS1 were measured). The results are overall very similar.

[0238] Example 8 - Differential expression in FFPE tissue samples by ddPCR Figure 15 shows the measurement of five biomarkers and the housekeeping gene KHDRBS1 from 22 benign and eight head and neck carcinoma FFPE samples. RNA was extracted from FFPE tissues using the Roche High Pure FFPET RNA Isolation Kit essentially according to the manufacture's protocol. The five panels show the copy numbers / μL from duplex ddPCR of the five biomarkers (SH3BGRL2, KRT4, EMP1, LOXL2 and ADAM12) and the housekeeping gene KHDRBS1 from 22 benign, eight head and neck carcinoma FFPE samples. KHDRBS1 showed a very similar distribution pattern and copy numbers / μL across all samples and all assays. In contrast, the biomarkers were >3-log 10 There was a variable distribution of copies / μL. One HNSCC sample had a "no call" for both biomarkers and KHDRBS1 across all assays.

[0239] The left graph in Figure 16 shows the expression of the five biomarkers normalized to KHDRBS1. Dividing the biomarker copy number / uL by the copy number / uL of the housekeeping gene KHDRBS1 from the duplex ddPCR reaction gives the normalization or ratio of the biomarkers for each sample. The right graph in Figure 16 is the RNASeq expression data for the same biomarkers from the HNSCC TCGA dataset. The table shows the median fold change in expression of each biomarker from the ddPCR experiments compared to the TCGA data. Both datasets show the same genes downregulated in cancer (SH3BGRL2, KRT4 and EMP1) and the same genes upregulated in cancer (LOXL2 and ADAM12). The ddPCR results are consistent with the TCGA dataset, but the magnitude of change is different.

[0240] Figure 17, ddPCR score was developed to separate cancer from normal samples by taking the difference of (log sum of upregulated genes)-(log sum of downregulated genes), and when biomarkers are added, the formula becomes (logLOXL2+logADAM12)-(logSH3BGRL2+logEMP1+logKRT4). The left panel shows the ddPCR scores of cancer samples plotted next to the ddPCR scores of normal samples. When the ddPCR cutoff score is >0.24, the specificity of the assay is 95.4% and the sensitivity is 85.7%. The right plot shows the receiver operator characteristic (ROC) analysis, with an AUC of 0.961, p=0.0003. The specificity from ddPCR is similar to the TCGA dataset (see Figure 2), but the sensitivity is lower from ddPCR than TCGA, likely due to differences in sample size and gene selection.

[0241] Example 9 - HPV16 E6 and E7 Expression in FFPE HNSCC Tissue Samples Figure 18 shows the correlation between E6 and E7 HPV16 expression and p16 from FFPE HNSCC tissue samples. The top table shows the HPV16 expression from four p16 positive samples. The table below shows the ddPCR copy numbers / uL for E6, E7 and housekeeping gene KHDRBS1. The table below shows the results for 10 p16 negative samples and the ddPCR copy numbers / uL for HPV16 E6, E7 and housekeeping gene KHDRBS1. The plot on the right shows the normalized ddPCR ratios of the biomarkers divided by the housekeeping gene KHDRBS1 for the four p16 positive samples. Two of the samples with reportable copies / uL for both HPV16 E6 and E7 greater than "no call" showed normalized ddPCR expression of E7 that was approximately 5-fold greater than E6. In addition, all p16 negative samples were also negative (no call) for E6 and E7 expression by ddPCR in both preparations and replicates. There is very good overall agreement between p16 by IHC and E6 or E7 expression by ddPCR (Cohen's Kappa = 0.81).

[0242] Example 10 - RNA isolation and gene expression in saliva samples In some cases, saliva can be used as a biological sample. Figure 19 shows the yield of RNA from saliva. Saliva samples were collected using a DNA Genotek CP-190 human RNA collection device. After sample collection, each sample was mixed thoroughly and incubated at 50°C for 2 hours, and the samples were stored at -20°C until processing. DNA Genotek Neutralizer Solution (catalog number RELONN-5) was added at 1 / 10 of the sample volume to each aliquot of saliva to be processed. RNA was purified using the Roche High Pure RNA Paraffin Kit (catalog number 03 270 289 001). The table on the left shows 15 saliva RNA samples and the μg RNA / 2mL saliva calculated from 250 μL of saliva sample preparation and the A260 / A280 ratio from each saliva sample. The median μg of RNA isolated was 5.8 μg, and the median A260 / A280 ratio was 2.05. The scatter plot on the right shows the same data in box-and-whisker format, with whiskers denoted by the maximum and minimum, boxes around the 75th and 25th percentiles and a line through the median.

[0243] Figure 20 shows the measurement of the five biomarkers and the housekeeping (HK) gene RPL30 from the 15 saliva RNA samples in Figure 19. The left panel shows the copy numbers / μL from duplex ddPCR of the five biomarkers (LOXL2, SH3BGRL2, CRISP3, EMP1, and KRT4) and the housekeeping gene RPL30 from the 15 saliva samples. The distribution of the biomarkers ranged from 0.38 to 650 copies / μL. The right panel should be the housekeeping gene for each duplex ddPCR. The housekeeping gene (RPL30) averaged 8-170 copies / μL, with a CV% of 6-27% across the five duplex ddPCR reactions. One sample averaged 1.3 copies / μL, with a CV% of 57%.

[0244] Figure 21 shows normalized ddPCR from saliva compared to TCGA RNASeq. The graph on the left shows the expression of the biomarkers normalized to the housekeeping gene RPL30. Dividing the number of copies / μL of the biomarker by the number of copies / μL of the housekeeping gene RPL30 from the duplex ddPCR reaction gives the normalization or ratio of the biomarker for each sample. One sample with an average of 1.3 copies / uL of the HK gene and samples with a "no call" or less than 1 copy / uL of the biomarker are excluded. The range of normalized ddPCR expression was 0.018-9.7 = 540-fold. The graph on the right of Figure 21 is the RNASeq expression data of the same biomarkers from "normals" in the HNSCC dataset. The table shows the median fold increase in expression relative to LOXL2 for each biomarker from the ddPCR experiment compared to the TCGA data. The median fold increase in expression from saliva by ddPCR tends to be similar to the TCGA (tissue) dataset, but the magnitude of change varies.

[0245] Example 11 - Embodiment The present disclosure includes, but is not limited to, the following embodiments.

[0246] A.1 A method for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: obtaining a sample from an individual; measuring the amount of an expression product from a gene comprising at least one gene in Table 4 and / or Table 6; A method comprising:

[0247] A.2 The method of any of the preceding paragraphs, wherein the genes include at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0248] A.3 The method of any of the preceding paragraphs, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0249] A.4 The method of any of the preceding paragraphs, further comprising measuring the amount of an expression product from at least one of the HPV E6 and / or HPV E7 genes.

[0250] A.5 The method of any of the preceding paragraphs, wherein the genes consist of expression products from at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6, and at least one of the HPV E6 and / or HPV E7 genes.

[0251] A.6 The method of any of the preceding paragraphs, further comprising measuring the amount of a normalization gene, such as KHDRBS1, or RPL30, or another normalization gene.

[0252] A.7 The method of any of the preceding paragraphs, wherein the measuring step comprises measuring mRNA.

[0253] A.8 The method of any of the preceding paragraphs, wherein the measuring step comprises an immunoassay.

[0254] A.9 The method of any of the preceding paragraphs, comprising measuring the expression of at least four of the genes.

[0255] A.10 The method of any of the preceding paragraphs, wherein the sample comprises serum, tissue, FFPE, saliva or plasma.

[0256] A.11 The method of any of the preceding paragraphs, comprising comparing the expression level with a control value from a normal population.

[0257] A.12 The method of any of the preceding paragraphs, wherein a difference between gene expression in the individual and the control value indicates that the individual may have HNSCC (i.e., is diagnostic) or is predisposed to developing HNSCC (i.e., is at high risk).

[0258] B.1 A method of identifying markers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising identifying at least one marker that has increased or decreased expression in HNSCC but is not expressed in HNSCC disease compared to normal controls.

[0259] B.2 The method of B.1, wherein the genes include at least one of one of the genes in Table 4 and / or Table 6, and / or at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0260] B.3 A method according to any one of B.1 to B.2, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0261] B.4 The method according to any one of B.1 to B.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 gene and / or the HPV E7 gene.

[0262] B.5 A method according to any one of B.1 to B.4, wherein the genes consist of expression products from at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6, and at least one of the HPV E6 and / or HPV E7 genes.

[0263] B.6 The method according to any one of B.1 to B.5, further comprising measuring the amount of a normalization gene such as KHDRBS1, or RPL30, or another normalization gene.

[0264] B.7 A method according to any of B.1 to B.6, wherein the measuring step includes measuring mRNA.

[0265] B.8 The method of any of B.1 to B.7, wherein the measuring step comprises an immunoassay.

[0266] B.9 A method according to any of B.1 to B.8, comprising measuring the expression of at least four of the genes.

[0267] B.10 The method of any of B.1 to B.9, wherein the sample comprises serum, tissue, FFPE, saliva or plasma.

[0268] B.11 A method according to any of B.1 to B.10, wherein a difference between gene expression in the individual and the control value indicates that the individual may have HNSCC (i.e., is diagnostic) or is predisposed to developing HNSCC (i.e., is at high risk).

[0269] C.1 Obtaining a sample from an individual; measuring the amount of at least one expression product from at least one gene in Table 4 and / or Table 6; comparing the expression of at least one gene in Table 4 and / or Table 6 in the sample with a control value of the expression product of the gene; 1. A method for detecting susceptibility to head and neck squamous cell carcinoma (HNSCC) in an individual, comprising:

[0270] C.2 The method of C.1, wherein the genes include at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0271] C.3 A method according to any one of C.1 to C.2, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0272] C.4 The method according to any one of C.1 to C.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 gene and / or the HPV E7 gene.

[0273] C.5 A method according to any one of C.1 to C.4, wherein the genes consist of expression products from at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6, and at least one of the HPV E6 and / or HPV E7 genes.

[0274] C.6 The method according to any one of C.1 to C.5, further comprising measuring the amount of a normalization gene such as KHDRBS1, or RPL30, or another normalization gene.

[0275] C.7 A method according to any of C.1 to C.6, wherein the measuring step includes measuring mRNA.

[0276] C.8 A method according to any of C.1 to C.7, wherein the measuring step comprises an immunoassay.

[0277] C.9 A method according to any of C.1 to C.8, comprising measuring the expression of at least four of the genes.

[0278] C.10 The method of any of C.1 to C.9, wherein the sample comprises serum, tissue, FFPE, saliva or plasma.

[0279] C.11 The method of any of C.1-C.10, wherein a difference between gene expression in the individual and a control value indicates that the individual is likely to have HNSCC or is predisposed (i.e., at high risk) to developing HNSCC.

[0280] D.1 A composition for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual comprising a reagent that quantifies the expression level of at least one gene in Table 4 and / or Table 6.

[0281] D.2 The composition of any of D.1, wherein the at least one gene includes at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0282] D.3 A composition described in D.1 to D.2, wherein the at least one gene includes at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0283] D.4 A composition described in any of D.1 to D.3, further comprising at least one reagent for quantifying the expression level of at least one HPV E6 and / or E7.

[0284] D.5 A composition described in any of D.1 to D.4, wherein the at least one gene consists of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6, and at least one of HPV E6 and / or E7.

[0285] D.6 A composition described in any of D.1 to D.5, further comprising at least one reagent for measuring at least one normalization gene, such as KHDRBS1, or RPL30, or another normalization gene.

[0286] D.7 A composition described in any of D.1 to D.6, wherein the reagent detects mRNA.

[0287] D.8 A composition described in any of D.1 to D.7, wherein the reagent detects a protein.

[0288] D.9 A composition described in any of D.1-D.8, wherein the reagent comprises at least one primer and / or probe for any one of these genes, and at least one primer and / or probe is labeled with a detectable moiety.

[0289] D.10 A composition described in any of D.1-D.9, wherein a difference between gene expression in an individual and a control value indicates that the individual may have HNSCC (i.e., is diagnostic) or is predisposed to developing HNSCC (i.e., is at high risk).

[0290] E.1 A kit comprising a composition described in any of the preceding paragraphs.

[0291] E.2 The kit of E.1, further comprising instructions for measuring at least one gene and / or for determining whether the value differs from a control value.

[0292] E.3 A kit described in any of E.1 to E.2, comprising at least one normalization gene such as KHDRBS1, or RPL30, or at least one positive control of another normalization gene.

[0293] E.4. The kit of any of E.1 to E.3, further comprising at least one positive control for any one of the genes in Table 4 and / or Table 6.

[0294] E.5 A kit described in any of E.1 to E.4, wherein the at least one gene includes at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0295] E.6 A kit described in any of E.1 to E.5, wherein the at least one gene includes at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0296] E.7 A kit described in any of E.1 to E.6, wherein the at least one gene includes at least one of HPV E6 and / or E7.

[0297] E.8 A kit described in any of E.1 to E.7, wherein the at least one gene consists of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6, and at least one of HPV E6 and / or E7.

[0298] E.9 A kit described in any of E.1 to E.8, wherein the reagents comprise at least one primer and / or probe for any one of these genes, and at least one primer and / or probe is labeled with a detectable moiety.

[0299] E.10 A kit described in any of E.1 to E.9, wherein a difference between gene expression in an individual and a control value indicates that the individual is likely to have HNSCC or is predisposed (i.e., at high risk) to developing HNSCC.

[0300] F.1 Obtaining a sample from an individual; measuring in the sample the amount of an expression product from a gene comprising at least one gene in Table 4 and / or Table 6; comparing expression of at least one gene in Table 4 and / or Table 6 in the sample to a control value of expression; and treating the individual for HNSCC if a difference between the gene expression in the individual and the control value indicates that the individual may have HNSCC (i.e., is diagnostic of the presence) or is susceptible to developing HNSCC (i.e., is at high risk). 23. A method for treating HNSCC comprising:

[0301] F.2 The method of F.1, wherein the genes include at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0302] F.3 The method according to F.1 to F.2, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0303] F.4 A method according to any one of F.1 to F.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 gene and / or the HPV E7 gene.

[0304] F.5 A method according to any one of F.1 to F.4, wherein the genes consist of expression products from at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6, and at least one of the HPV E6 and / or HPV E7 genes.

[0305] F.6 The method according to any one of F.1 to F.5, further comprising measuring the amount of a normalization gene such as KHDRBS1, or RPL30, or another normalization gene.

[0306] F.7 A method according to any of F.1 to F.6, wherein the measuring step includes measuring mRNA.

[0307] F.8 A method according to any of F.1 to F.7, wherein the measuring step comprises an immunoassay.

[0308] F.9 A method according to any of F.1 to F.8, comprising measuring the expression of at least four of the genes.

[0309] F.10 A method according to any of F.1 to F.9, wherein the sample comprises serum, tissue, FFPE, saliva or plasma.

[0310] F.11 A method according to any of F.1 to F.10, comprising comparing the expression level with a control value from a normal population.

[0311] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. Various modifications and equivalents of what is described herein will be apparent to those skilled in the art from the entire contents of this document, including references to the scientific and patent literature cited herein. The subject matter of this specification, in its various embodiments and equivalents thereof, contains information, examples, and guidance that can be adapted to the practice of the present disclosure.

Claims

1. A method for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: measuring the amount of expression products from at least the ADAM12, CRISP3, MUC21 and MMP9 genes in a sample obtained from said individual. A method comprising:

2. The method of claim 1, further comprising the step of measuring a sample amount of an expression product from at least one additional gene from Table 4 and / or Table 6.

3. The method of claim 1, further comprising measuring a sample amount of an expression product from at least one of the HPV E6 gene and / or the HPV E7 gene in the sample.

4. The method of claim 3, further comprising the step of comparing the measured amount of expression product of at least one of the HPV E6 and / or HPV E7 genes with a corresponding control value of expression.

5. A method for using expression of at least one gene as an indicator for detecting susceptibility to head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: measuring the amount of expression products from at least the ADAM12, CRISP3, MUC21 and MMP9 genes in a sample obtained from said individual; comparing the measured amount of each expression product in the sample to a corresponding control value for each expression product; wherein a difference between the measured amount of each expression product in the sample and the corresponding control value indicates that the individual is likely to have or is susceptible to developing HNSCC.

6. The method further comprising: determining in said sample an amount of an expression product from at least one gene from Table 4 and / or Table 6; comparing said measured amount of each expression product from said at least one gene from Table 4 and / or Table 6 in said sample with a corresponding control value for each expression product from said at least one gene; The method of claim 5 further comprising:

7. The method of claim 1, further comprising: determining in said sample an amount of at least one expression product of at least one of the HPV E6 and / or E7 genes; comparing said amount of measured expression of said at least one expression product in said sample with a corresponding control value of expression; The method of claim 5 or 6, further comprising:

8. A method described in any one of claims 1 to 7, further comprising a step of measuring the amount of a standardization gene.

9. The method described in claim 8, wherein the standardization gene is KHDRBS1 or RPL30.

10. The method of claim 1, wherein the measuring step comprises measuring mRNA.

11. The method of claim 1, wherein the measuring step comprises measuring a protein.

12. The method of claim 1, wherein the measuring step comprises performing a peptide, polypeptide or protein assay.

13. The method of claim 12, wherein the peptide, polypeptide or protein assay is an immunoassay.

14. The method of claim 1, wherein the measuring step comprises performing a nucleic acid assay.

15. The method described in claim 14, wherein the nucleic acid assay is droplet digital PCR (ddPCR).

16. The method of claim 1, wherein the measuring step comprises using an array of expression products.

17. The method of any one of claims 1 to 16, wherein the sample comprises serum tissue, FFPE, saliva or plasma.

Images