Compositions and methods for detecting head and neck cancer

Abstract

Disclosed are compositions and methods for detecting biomarkers for proteins, typically associated with head and neck cancer, or more particularly head and neck squamous cell carcinoma (HNSCC). Such markers may be used to allow an individual susceptible to HNSCC to manage their lifestyle and / or drug therapy to avoid further development of disease.

Description

[0001] This application is a divisional application of CN201880082894.X.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 608,296 filed on December 20, 2017. The disclosure of U.S. Provisional Patent Application No. 62 / 608,296 is incorporated herein by reference in its entirety. Background Art

[0004] Head and neck cancer is a common disease. Histologically, most head and neck cancers are of the squamous cell type and are therefore classified as head and neck squamous cell carcinoma (HNSCC). HNSCC is the sixth most common cancer worldwide and the third most common cancer in developing countries.

[0005] The biological mechanisms behind HNSCC are unclear, and few biomarkers provide reliable indications of this condition. Still, it would be helpful for individuals susceptible to HNSCC to adjust their lifestyle to avoid triggering symptom onset and / or promoting further progression of the disease. Therefore, there is a need to develop and evaluate biomarkers for HNSCC. Summary of the invention

[0006] The present disclosure can be embodied in many ways.

[0007] In one embodiment, a method for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual is disclosed, the method comprising the steps of obtaining a sample from the individual; and measuring the amount of expression of at least one gene in Table 4 and / or Table 6 in the sample. In one embodiment, a method for detecting a biomarker associated with HNSCC in an individual is disclosed, comprising the steps of obtaining a sample from the individual; and measuring the amount of expression 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 for detecting a biomarker associated with HNSCC in an individual is disclosed, comprising the steps of obtaining a sample from the individual; and measuring the amount of expression of at least one of the human papillomavirus (HPV) E6 or E7 genes. Additionally and / or alternatively, the method may include measuring at least one normalized gene (e.g., a housekeeping gene). In one embodiment, the normalized gene may be KHDRBS1. In one embodiment, the normalizing gene can be RPL30 or another normalizing gene. Alternatively, the expression of various combinations of these genes can be measured.

[0008] In one embodiment, a plurality of disclosed biomarker groups are 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, the composition comprising a quantitative reagent for 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. In addition and / or alternatively, the composition may include at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1. In one embodiment, the standardized gene may be RPL30 or another standardized gene. In certain embodiments, the composition may include primers and / or probes for any of these genes, wherein the primers and / or probes are labeled with a detectable portion as described herein.

[0009] Other embodiments include systems for performing the methods disclosed herein and / or using the compositions disclosed herein.

[0010] Other features, objects and advantages of the present disclosure are apparent in the accompanying detailed description, drawings and claims. However, it should be understood that the detailed description, drawings and claims, although indicating the embodiments of the disclosed methods, compositions and systems, are for illustrative purposes only and not for limiting purposes. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention may be better understood with reference to the following non-limiting drawings.

[0013] Figure 1 Shown are the results of an experiment using the TCGA dataset and random forest analysis to identify differentially expressed genes in HNSCC according to an embodiment of the present disclosure.

[0014] Figure 2 It is shown that according to various embodiments of the present disclosure, evaluation of a genome comprising multiple markers can improve assay performance.

[0015] Figure 3 The gene expression of four markers of the present disclosure (SH3BGRL2, CAB39L, NRG2 and ADAM12) at various locations in normal and HNSCC tissues is shown. Figure 3For each graph, the 3 datasets on the left end of the x-axis (larynx, oral cavity, and oropharynx) are from normal tissues, and the 4 datasets on the right end of the x-axis (hypopharynx, larynx, oral cavity, and oropharynx) are from HNSCC tissues.

[0016] Figure 4 Gene expression of four additional markers of the present disclosure (LOXL2, COL13A1, HSD17B6, and GRIN2D) at various locations in normal and HNSCC tissues according to various embodiments of the present disclosure is shown. Figure 4 For each graph, the 3 datasets on the left end of the x-axis (larynx, oral cavity, and oropharynx) are from normal tissues, and the 3 datasets on the right end of the x-axis (hypopharynx, larynx, oral cavity, and oropharynx) are from HNSCC tissues.

[0017] Figure 5 Shown is a comparison of all HNSCC markers in HNSCC samples versus normal samples for all data, and a comparison of all HNSCC markers in the oral cavity (OC) or oropharynx (OP) in HNSCC versus normal samples, according to embodiments of the present disclosure.

[0018] Figure 6, Panel A (i.e. Fig. 6A ) shows the differential expression of the TCGA genome according to an embodiment of the present disclosure. Figure 6B ) shows the median ranks of 36 genes (darker symbols in the figure) from random forest analysis according to various embodiments of the present disclosure. For increased gene expression in HNSCC, the cutoff values ​​were the 5th percentile for HNSCC and the 95th percentile for normal values ​​(e.g., Figure 6B , GRIN2D illustration).

[0019] Figure 7 Differentially expressed markers identified from a literature search according to various embodiments of the present disclosure are shown (darker symbols in the figure).

[0020] Figure 8 Normalized markers from literature search according to various embodiments of the present disclosure are shown.

[0021] Figure 9, Figure A (i.e. Fig.9A) shows the identification of normalized genes using the TCGA dataset. The left figure shows the entire dataset; the middle figure shows those genes whose median fold change in gene expression between normal and cancer is less than 2 (positive or negative) and whose interquartile range (IQR) is less than 2, where IQR = 75th percentile expression / 25th percentile expression; the right figure shows the expression levels in normal relative HNSCC of genes from the TCGA database according to various embodiments of the present disclosure to identify genes with median expression levels similar to the target group. Figure B (i.e. Fig. 9B ) shows a KHDRBS1 differential map according to various embodiments of the present disclosure.

[0022] Fig.10 A comparison of droplet digital PCR (ddPCR) data relative to TCGA RNASeq data of gene expression levels of potential marker genes ADAM12 and SH3BGRL2 in cancer tissue (i.e., tongue squamous cell carcinoma) compared to normal tissue (i.e., buccal mucosa) according to various embodiments of the present disclosure is shown.

[0023] Fig.11 Additional ddPCR data are shown for three formalin-fixed, paraffin-embedded patient samples (DA1081983; DR1041686; DA0063595) and one URNA control sample (cancer tissue derived from cell culture) using ddPCR; replicate or triplicate sampling was performed according to various embodiments of the present disclosure.

[0024] Fig.12 Shown is the concentration dependence of SH3BGRL2 (potential cancer marker) (·) expression compared to KHDRBS1 (x) (potential normalizing gene) expression in three different patient samples (RNA 1, 3 and 5) and URNA control according to various embodiments of the present disclosure, showing a relatively constant ratio (dashed line) until the assay limit of one copy / μL.

[0025] Fig.13 Shown are the expression of the potential cancer marker SH3BGRL2 in formalin-fixed paraffin-embedded (FFPE) samples compared with URNA controls according to various embodiments of the present disclosure (left graph); the ratio of ddPCR products of SH3BGRL2 / KHDRBS1 in cancer tissues relative to normal tissues (middle graph); the distribution of reporter gene expression of these two markers in the TCGA database (right graph); in the figure, x is a sample from a cancer patient and the circle (hollow or solid) is a normal tissue sample.

[0026] Fig.14Shown are the analyses of various patient samples for SH3BGRL2 using either a singleplex assay format (Single) (ie comprising only SH3BGRL2 primers) or a duplex assay format (Duplex) (comprising SH3BGRL2 and KHDRBS1 primers) according to various embodiments of the present disclosure.

[0027] Fig.15 Shown is the expression of five biomarkers (SH3BGRL2, KRT4, EMP1, LOXL2, and ADAM12) and the housekeeping gene KHDRBS1 using duplex ddPCR from 22 benign (circles) and 8 cancer (x) FFPE samples according to various embodiments of the present disclosure.

[0028] Fig.16 The expression of 5 biomarkers from 22 benign and 8 cancer FFPE samples normalized to the housekeeping gene KHDRBS1 by ddPCR according to various embodiments of the present disclosure is shown (left panel), compared to RNASeq data of HNSCC TCGA for the same biomarkers and housekeeping genes (right panel), and the median fold change of expression of each biomarker is summarized (table). In this figure, N = normal tissue, C = cancer tissue.

[0029] Fig.17 Results of the ddPCR scoring algorithm for normalized ddPCR expression for distinguishing cancer from normal FFPE samples according to various embodiments of the present disclosure are shown (left panel), and receiver operating characteristic (ROC) analysis is shown (right panel).

[0030] Fig.18 Shown is the correlation between E6 and E7 HPV16 expression determined by ddPCR in p16-positive FFPE HNSCC samples (upper panel) and p16-negative FFPE HNSCC samples (lower panel) according to various embodiments of the present disclosure; and the normalized ddPCR expression levels of E6 and E7 from p16-positive samples (right panel).

[0031] Fig.19 Shown are tabular forms (left table) and box-and-whisker plots (right graph) of RNA yields (μg RNA / 2 mL saliva) and A260 / A280 ratios from 15 saliva samples according to various embodiments of the present disclosure.

[0032] Fig. 20Shown are the expression of five biomarkers (LOXL2, SH3BGRL2, CRISP3, EMP1, and KRT4) and the housekeeping gene RPL30 measured using double-stranded ddPCR from 15 saliva samples according to various embodiments of the present disclosure (left panel), and the expression of the housekeeping gene RPL30 from five double-stranded ddPCR reactions (right panel).

[0033] Fig.21 The expression of five biomarkers from 15 saliva samples determined by ddPCR after normalization to the housekeeping gene RPL30 according to various embodiments of the present disclosure is shown (left panel), compared to RNASeq data of the same biomarkers from HNSCCTCGA (right panel), where the median fold increase in expression relative to LOXL2 for each biomarker is summarized (table). Detailed Description

[0035] Terms and Definitions

[0036] In order to make the present disclosure more easily understood, certain terms are first defined. The following terms and additional definitions of other terms are set forth throughout the specification.

[0037] Although the numerical ranges and parameters describing the broad scope of the present invention are approximate, the numerical values ​​described in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors, which are necessarily caused by the standard deviation found in their respective test measurements. In addition, all ranges disclosed herein are understood to cover any and all subranges contained therein. For example, the specified range "1 to 10" should be deemed to include any and all subranges between a minimum value of 1 and a maximum value of 10 (including the end values); that is, all subranges start with a minimum value of 1 or a larger value (e.g., 1 to 6.1) and end with a maximum value of 10 or a smaller value (e.g., 5.5 to 10). In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0038] It should also be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "and / or" is generally used to refer to at least one or the other. In some cases, the term "and / or" is used interchangeably with the term "or." The term "including" is used herein to mean the phrase "including but not limited to," and can be used interchangeably with the phrase "including but not limited to." The term "for example" is used herein to mean and is used interchangeably with the phrase "for example, but not limited to."

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Practitioners are specifically referred to Current Protocols in Molecular Biology (Ausubel) for definitions and terminology of the art.

[0040] Antibody: As used herein, the term "antibody" refers to a polypeptide consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are usually classified as κ or λ. Heavy chains are usually classified as γ, μ, α, δ or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. It is known that a typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kD) and a "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light chains and heavy chains, respectively. Antibodies can be specific for specific antigens. Antibodies or their antigens can be analytes or binding partners. Antibodies exist in the form of complete immunoglobulins or in the form of many well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin disulfide-digests antibodies in the hinge region to produce F(ab)'2 (dimer of Fab), which itself is a light chain connected to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a partial hinge region (see, Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993) for a more detailed description of other antibody fragments). Although various antibody fragments are defined based on the digestion of complete antibodies, it will be understood by those of ordinary skill in the art that such Fab' fragments can be synthesized de novo by chemical methods or using recombinant DNA methods. Therefore, the term "antibody" used herein also includes antibody fragments synthesized de novo by modifying complete antibodies or using recombinant DNA methods. In some embodiments, the antibody is a single-chain antibody, such as a single-chain Fv (scFv) antibody, in which the variable heavy chain and the variable light chain are linked together (directly or through a peptide linker) to form a continuous polypeptide. A single-chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer that can be expressed from a nucleic acid comprising VH and VL coding sequences that are directly linked or linked through 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). There are many structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site.See, e.g., U.S. Patents 5,091,513 and 5,132,405 and 4,956,778.

[0041] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies and chimeric antibodies produced, for example, by combinatorial mutagenesis and phage display. The term "antibody" also includes mimetics or peptidomimetics of antibodies. Peptidomimetics are compounds based on or derived from peptides and proteins. Peptidomimetics of the present disclosure can generally be obtained by structurally modifying known peptide sequences using non-natural amino acids, conformational restrictions, isotopic substitutions, etc.

[0042] Allele: As used herein, the term "allele" refers to different forms of the nucleotide sequence of the same genetic locus (eg, gene).

[0043] Allele-specific primer extension (ASPE): As used herein, the term "allele-specific primer extension (ASPE)" refers to a mutation detection method utilizing a primer that hybridizes to a corresponding DNA sequence and is extended depending on the successful hybridization of the 3' terminal nucleotide of such primer. Typically, 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 may be incorporated into the extension product, such nucleotides effectively labeling the extension product for detection purposes. Alternatively, the extension primer may alternatively comprise 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.

[0044] Amplification: As used herein, the term "amplification" refers to any method known in the art for replicating a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. The target nucleic acid can be DNA or RNA. Typically, the sequence amplified in this manner forms an "amplicon". Amplification can be accomplished in a variety of ways, including but not limited to polymerase chain reaction ("PCR"), transcription-based amplification, isothermal amplification, rolling circle amplification, etc. Amplification can be performed using a relatively similar amount of each primer of a primer pair to produce a double-stranded amplicon. However, as is well known in the art, asymmetric PCR can be used to amplify single-stranded products primarily or exclusively (e.g., Poddar, Molec. And Cell. Probes 14: 25-32 (2000)). This can be achieved by using each pair of primers to significantly reduce the concentration of another primer relative to one primer in the primer pair (e.g., differing by 100 times). Amplification by asymmetric PCR is generally linear. Those skilled in the art will appreciate that different amplification methods can be used together.

[0045] 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, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, 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 can be transgenic animals, genetically modified animals, and / or clones.

[0046] Approximately: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​within a range of 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 than or less than) of the reference value, unless otherwise specified or obvious from the context (except that the number will exceed 100% of the possible value). Therefore, the term "approximately" is used to indicate that a value includes variations in the inherent errors of the device, the method for determining the value, or variations that exist between samples.

[0047] Associating with a syndrome or disease of interest: As used herein, "associating with a syndrome or disease of interest" means that the variant is found more frequently in patients with the syndrome or disease of interest than in non-syndrome or non-disease controls. Typically, the statistical significance of such an association can be determined by analyzing multiple patients.

[0048] Biological sample: As used herein, the term "biological sample" or "sample" encompasses any sample obtained from a biological source. As non-limiting examples, biological samples may include blood, amniotic fluid, serum, plasma, liquid or tissue biopsy, urine, feces, epidermal samples, skin samples, cheek swabs, sperm, amniotic fluid, cultured cells, bone marrow samples, and / or chorionic villi. Convenient biological samples can be obtained by, for example, scraping cells from the buccal cavity surface. The term biological sample includes samples that have been treated to release or otherwise make nucleic acids or proteins available for detection as described herein. The term biological sample also includes cell-free nucleic acids that may be present in a sample (e.g., plasma or amniotic fluid). For example, a biological sample may include cDNA obtained by reverse transcribing RNA from cells in a biological sample. Biological samples may be obtained from life stages such as fetuses, young people, adults, etc. Fixed or frozen tissues may also be used.

[0049] Biomarker: As used herein, the term "biomarker" or "marker" refers to one or more nucleic acids, polypeptides, and / or other biological molecules (e.g., cholesterol, lipids) that can be used to diagnose or aid in the diagnosis or prognosis of a disease or syndrome of interest (alone or in combination with other biomarkers); to monitor the progression of a disease or syndrome of interest; and / or to monitor the effectiveness of a treatment for a syndrome or disease of interest.

[0050] Binder: As used herein, the term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction can be non-covalent, for example as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently bound) to a solid support.

[0051] Carrier: The term "carrier" refers to a person who is asymptomatic but carries a mutation that can be passed on to his / her children. Typically, for autosomal recessive disorders, a carrier has one allele that contains the disease-causing mutation and another allele that is normal or unrelated to the disease.

[0052] Coding sequence and non-coding sequence: As used herein, the term "coding sequence" refers to a nucleic acid sequence or its complementary sequence or a portion thereof that can be transcribed and / or translated to produce a polypeptide or fragment thereof and / or an mRNA for a polypeptide or fragment thereof. Coding sequences include exons in genomic DNA or immature primary RNA transcripts, which are linked together by the biochemical machinery of the cell to provide mature mRNA. The antisense strand is the complementary sequence of this nucleic acid, and the coding sequence can be deduced therefrom. As used herein, the term "non-coding sequence" refers to a nucleic acid sequence or its complementary sequence or a portion thereof that is not transcribed into an amino acid in vivo or that tRNA does not interact with to place or attempt to place an amino acid. Non-coding sequences include intron sequences of genomic DNA, or immature primary RNA transcripts, and sequences associated with genes, such as promoters, enhancers, silencers, etc.

[0053] Complementary sequence: 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 an RNA sequence that is complementary to a DNA sequence. Certain bases that are not common 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 is not necessarily perfect; stable duplexes may contain unmatched base pairs, denatured or unmatched bases. A person skilled in the art of nucleic acid technology can determine duplex stability empirically taking into account multiple variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the ionic strength and the incidence of mismatched base pairs.

[0054] Conserved: As used herein, the term "conserved residues" refers to amino acids that are identical in multiple proteins with the same structure and / or function. Regions of conserved residues may be important for protein structure or function. Thus, consecutive conserved residues identified in a three-dimensional protein may be important for protein structure or function. In order to find conserved residues or conserved regions of 3-D structure, sequences of the same or similar proteins from different species or individuals of the same species may be compared.

[0055] Control: As used herein, the term "control" has its meaning as understood in the art, i.e., it is a standard with which the result is compared. Typically, the control increases the integrity of the experiment by isolating the variable, so as to draw a conclusion about such variable. In some embodiments, the control is a reaction or determination carried out simultaneously with the test reaction or determination to provide a comparator. In an experiment, (i.e., the variable being tested) is applied to the "test". In a second experiment, the variable being tested is not applied to the "control". In some embodiments, the control is a historical control (i.e., a previously performed test or determination or a previously known amount or result). In some embodiments, the control is or includes a printed or otherwise preserved record. The control can be a positive control or a negative control.

[0056] A "control" or "predetermined standard" for a biomarker refers to the expression level of the biomarker in a healthy subject or the expression level of the biomarker in a non-diseased or asymptomatic tissue from the same subject. The control or predetermined standard expression level or amount of protein for a given biomarker can be determined by prospective and / or retrospective statistical studies using only routine experiments. Such predetermined standard expression levels and / or protein levels (amounts) can be determined by one of ordinary skill in the art using well-known methods. A positive control is a sample (or reagent) that provides a predetermined amount of the signal to be measured.

[0057] Crude: As used herein, the term "crude" when used in conjunction with a biological sample refers to a sample that is substantially in an unrefined state. For example, a crude sample can be a cell lysate or a biopsy tissue sample. A crude sample may be in the form of a solution or in the form of a dry preparation.

[0058] Deletion: As used herein, the term "deletion" encompasses a mutation that removes one or more nucleotides from a naturally occurring nucleic acid.

[0059] Disease or syndrome of interest: As used herein, the disease or syndrome of interest is head and neck cancer, and in some embodiments, more specifically HNSCC.

[0060] Detect: As used herein, the terms "detect," "detected," or "detecting" include "measure," "measured," or "measuring" and vice versa.

[0061] Detectable moiety: As used herein, the term "detectable moiety" or "detectable biomolecule" or "reporter molecule" refers to a molecule that can be measured in a quantitative assay. For example, the detectable moiety can comprise an enzyme that can be used to convert a substrate into a measurable product (e.g., a visible product). Alternatively, the detectable moiety can be a radioisotope that can be quantified. Alternatively, the detectable moiety can be a fluorophore. Alternatively, the detectable moiety can be a luminescent molecule. Alternatively, other detectable molecules can be used.

[0062] Epigenetic: As used herein, epigenetic elements can alter gene expression by mechanisms other than changes in the underlying DNA sequence. Such elements can include those that regulate paramutation, imprinting, gene silencing, X chromosome inactivation, position effects, reprogramming, translocation, maternal effects, histone modifications, and heterochromatin.

[0063] 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 specific antibody or antibody-like protein.

[0064] 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 called introns) have been removed by RNA splicing. Thus, an exon sequence is often part of or encodes a protein. An intron is a portion of an RNA that is removed from the surrounding exon sequences by RNA splicing.

[0065] Expression and expressed RNA: As used herein, expressed RNA is RNA that encodes a protein or polypeptide ("coding RNA"), as well as any other RNA that is transcribed but not translated ("non-coding RNA"). As used herein, the term "expression" refers to the process by which a polypeptide is produced from DNA. The process involves transcription of a gene into mRNA and translation of the mRNA into a polypeptide. Depending on the context of use, "expression" can refer to the production of RNA, protein, or both.

[0066] The amount of protein of the present disclosure and / or the measurement of the expression of biomarkers can be assessed by any of a variety of well-known methods for detecting the expression of transcription molecules or their corresponding proteins. Non-limiting examples of such methods include immunological methods, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods for detecting secreted proteins. In certain embodiments, the expression of marker genes is assessed using antibodies (e.g., radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibodies) that specifically bind to proteins corresponding to marker genes (e.g., proteins encoded by open reading frames corresponding to marker genes or such proteins that have undergone all or part of their normal post-translational modifications), antibody derivatives (e.g., antibodies conjugated to proteins or ligands of substrates or protein-ligand pairs (e.g., biotin-streptavidin)), or antibody fragments (e.g., single-chain antibodies, isolated antibody hypervariable domains, etc.). In certain embodiments, reagents can be directly or indirectly labeled with detectable substances. The detectable substance may, for example, be selected from radioisotopes, fluorescent compounds, enzymes and enzyme cofactors.Methods for labeling antibodies are well known in the art.

[0067] In another embodiment, the expression of the marker gene is assessed by preparing mRNA / cDNA (i.e., transcribed polynucleotides) from cells in the sample and hybridizing the mRNA / cDNA with a reference polynucleotide and a fragment thereof that is a complementary sequence of a polynucleotide comprising the marker gene. Prior to hybridization with the reference polynucleotide, the cDNA may optionally be amplified using any of a variety of polymerase chain reaction methods; preferably, it is not amplified.

[0068] Family history: As used herein, the term "family history" generally refers to the occurrence of events (e.g., disease-related conditions or mutation carriers) associated with an individual's immediate family members, including parents and siblings. Family history may also include grandparents and other relatives.

[0069] Flanking: As used herein, the term "flanking" refers to the hybridization of a primer to a target nucleic acid adjacent to a region of interest to be amplified on a target. It will be appreciated by those skilled in the art that a preferred primer is a primer pair that hybridizes at the 5' (upstream) of the region of interest on each strand of the target double-stranded DNA molecule, so that nucleotides can be added to the 3' end of the primer by a suitable DNA polymerase. For example, the primer of the flanking mutant sequence does not actually anneal to the mutant sequence, but anneals to the sequence adjacent to the mutant sequence. In some cases, the primer of the flanking exon is usually designed not to anneal to the exon sequence, but to anneal to the sequence (e.g., intron sequence) adjacent to the exon. However, in some cases, an amplification primer can be designed to anneal to the exon sequence.

[0070] Gene: As used herein, a gene is a genetic unit. Typically, a gene is a portion of DNA that encodes a protein or functional RNA. A gene is a locatable region of a genomic sequence that corresponds to a genetic unit. A gene may be associated with a regulatory region, a transcription region, and / or other functional sequence regions.

[0071] 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" of an individual or DNA sample refers to the property of identifying the two alleles an individual possesses at a known polymorphic site based on nucleotide bases.

[0072] Gene regulatory element: As used herein, a gene regulatory element or regulatory sequence is a segment of DNA where regulatory proteins such as transcription factors bind to regulate gene expression. Such regulatory regions are usually upstream of the gene being regulated.

[0073] Healthy individual: As used herein, the term "healthy individual" or "control" refers to a subject who has not been diagnosed with the syndrome and / or disease of interest.

[0074] Heterozygote: As used herein, the term "heterozygote" or "HET" refers to an individual with two different alleles of the same gene. As used herein, the term "heterozygote" encompasses a "compound heterozygote" or "compound heterozygous mutant". As used herein, the term "compound heterozygote" refers to an individual with two different alleles. As used herein, the term "compound heterozygous mutant" refers to an individual with two different copies of an allele, such alleles being characterized as mutant forms of a gene.

[0075] Homozygous: As used herein, the term "homozygous" refers to an individual having two copies of the same allele. As used herein, the term "homozygous mutant" refers to an individual having two copies of the same allele, such alleles being characterized as mutant forms of a gene.

[0076] Housekeeping or normalizing genes: As used herein, "housekeeping genes" are those genes that are usually constitutively expressed in all cells because they provide basic functions required to maintain all cell types. Housekeeping genes or "normalizing genes" are measured simultaneously with the gene of interest to account for differences due to differences between samples. Differences between such samples may reflect differences in experimental variables (such as, but not limited to, RNA isolation and reverse transcription and PCR efficiency). Normalization involves reporting the ratio of the gene of interest to the housekeeping gene. See, for example, Bustin, SA et al. 2009. The MIQE guidelines: minimuminformation for publication of quantitative real-time PCR experiments. Clin Chem, Apr; 55 (4): 611-622.

[0077] Hybridization: As used herein, the term "hybridization" or "hybridized" refers to the process in which two complementary nucleic acid chains anneal to each other under appropriately stringent conditions. The length of the oligonucleotide or probe suitable for hybridization is generally 10-100 nucleotides (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 understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the 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 Biology. John Wiley & Sons, Secaucus, NJ.

[0078] 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. The percent identity can be determined by comparing two sequences and refers to the number of identical residues (i.e., amino acids or nucleotides) at positions common to the compared sequences. Sequence alignment and comparison can be performed using algorithms standard 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 by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI) (publicly available as BLAST and FASTA). In addition, ENTREZ available through the National Institutes of Health (Bethesda MD) can be used for sequence comparison. In other cases, commercial software (e.g., GenomeQuest) can be used to determine the identity percentage. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., BLASTN; available on the Internet website of the National Center for Biotechnology Information) can be used. In one embodiment, the identity percentage of two sequences can be determined using GCG with a gap weight of 1, so that each amino acid gap is weighted, as if it is a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0) can be used, which is a part of the GCG (Accelrys, San Diego, CA) sequence alignment software package.

[0079] As used herein, the term "at least 90% identical thereto" includes sequences having 90 to 100% identity to the indicated sequence, and includes 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% identical to the indicated sequence. Similarly, the term "at least 70% identical thereto" includes sequences that are 70 to 100% identical, including all ranges therebetween. Percent identity is determined using the algorithm described herein.

[0080] Insertion or addition: As used herein, the term "insertion" or "addition" refers to a change in an amino acid or nucleotide sequence which results in the addition of one or more amino acid residues or nucleotides, respectively, compared to the naturally occurring molecule.

[0081] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0082] In vivo: As used herein, the term "in vivo" refers to events that occur in a multicellular organism, such as a human or non-human animal.

[0083] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity may be 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% separated from other components with which they were 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 components. As used herein, the term "isolated cell" refers to a cell that is not contained within a multicellular organism.

[0084] 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 by, for example, detecting the label (e.g., visualization, detecting radioactivity, etc.) after binding to another entity (e.g., a nucleic acid, a polypeptide, etc.). The detectable agent or moiety can be selected so that it produces a signal that can be measured and whose intensity is related to (e.g., proportional to) the amount of the bound entity. Various systems for labeling and / or detecting proteins and peptides are known in the art. Labeled proteins and peptides can be prepared by incorporating or conjugating a label that can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemistry or other means. The label or labeling moiety can be detected directly (i.e., it does not require any further reaction or manipulation to detect, e.g., a fluorophore can be detected directly) or it can be detected indirectly (i.e., by reacting or binding to another detectable entity to make it detectable) (e.g., a hapten can be detected by immunostaining after reacting with an appropriate antibody containing a reporter molecule (e.g., 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.

[0085] MicroRNA: As used herein, microRNA is such microRNA (miRNA), which is a short (20-24 nucleotides) non-coding RNA that participates in post-transcriptional regulation of gene expression. MicroRNA can affect the stability and translation of mRNA. For example, microRNA can bind to the complementary sequence in the 3'UTR of the target mRNA and cause gene silencing. MiRNA is transcribed by RNA polymerase II as part of a capped and polyadenylated primary transcript (pri-miRNA), which can be protein-encoding or non-coding. The primary transcript can be cleaved by the Drosha ribonuclease III enzyme to produce a stem-loop precursor miRNA (pre-miRNA) of about 70 nucleotides, which can then be cleaved by the cytoplasmic Dicer ribonuclease to generate mature miRNA and antisense miRNA star (miRNA*) products. Mature miRNA can be incorporated into the RNA-induced silencing complex (RISC), which can recognize the target mRNA by imperfect base pairing with the miRNA, and most commonly leads to translational inhibition or instability of the target mRNA.

[0086] Multiplex PCR: As used herein, the term "multiplex PCR" refers to the simultaneous amplification of two or more regions, each region being primed using a different primer pair.

[0087] Multiplex ASPE: As used herein, the term "multiplex ASPE" refers to an assay that combines multiplex PCR and allele-specific primer extension (ASPE) to detect polymorphisms. Typically, multiplex PCR is first used to amplify the DNA region that will serve as the target sequence for the ASPE primers. See definition of allele-specific primer extension.

[0088] Mutation and / or variant: As used herein, the terms mutation and variant are used interchangeably to describe changes in nucleic acid or protein sequences. As used herein, the term "mutant" refers to a mutated or potentially non-functional form of a gene.

[0089] Nucleic acid: As used herein, "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.

[0090] Obtain or Obtaining: As used herein, the terms "obtain" or "obtaining" include receiving directly or indirectly (ie, from a third party).

[0091] Polypeptide or protein: As used herein, the terms "polypeptide" and / or "protein" refer to a polymer of amino acids, rather than a specific length. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide and / or protein. "Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may include partial or full-length proteins. Unless the context indicates otherwise, the term "peptide" is used to refer to less than full-length proteins or very short proteins.

[0092] As known in the art, "proteins", "peptides", "polypeptides" and "oligopeptides" are chains of amino acids (usually L-amino acids) whose alpha carbons are linked by a peptide bond formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. Typically, the amino acids that make up a protein are numbered sequentially, starting with the amino terminal residue and increasing toward the carboxyl terminal residue of the protein. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0093] As used herein, a "domain" of a polypeptide or protein includes a region along a polypeptide or protein that contains independent units. Domains can be defined based on 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 of the rest of the protein. Domains can be identified using domain databases such as, but not limited to, PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

[0094] Primer: As used herein, the term "primer" refers to a short single-stranded oligonucleotide that can hybridize with a complementary sequence in a nucleic acid sample. Typically, primers are used as the starting point for template-dependent DNA synthesis. Deoxyribonucleotides can be added to primers by DNA polymerase. In some embodiments, the addition of such deoxyribonucleotides to primers is also referred to 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, thiophosphate-modified primers, labeled primers, etc. A "primer pair" or "primer set" for PCR reaction generally refers to a primer set that generally 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.

[0095] Polymorphism: As used herein, the term "polymorphism" refers to the coexistence of more than one form of a gene or part thereof.

[0096] Portion and fragment: As used herein, the terms "portion" and "fragment" are used interchangeably to refer to a portion of a polypeptide, nucleic acid or other molecular construct.

[0097] Sample: As used herein, the term "sample" refers to material obtained from an individual or subject or patient. The sample can be from any biological source, including all body fluids (e.g., whole blood, plasma, serum, saliva, lens fluid, sweat, urine, milk, etc.), tissues or extracts, cells, cell-free nucleic acids, formalin-fixed paraffin-embedded (FFPE) tissues, etc.

[0098] Sense strand and antisense strand: 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 such a strand of dsDNA that is the reverse complementary sequence of the sense strand.

[0099] Significant difference: As used herein, the term "significant difference" is well within the knowledge of those skilled in the art and will be determined empirically with reference to each specific biomarker. For example, a significant difference in biomarker expression in subjects with a disease or syndrome of interest compared to healthy subjects is any statistically significant difference in the amount of protein.

[0100] Similar or homologous: As used herein, the term "similar" or "homologous" refers to a polypeptide having a certain degree of homology or identity with a wild-type amino acid sequence when referring to an amino acid or nucleotide sequence. Homology comparisons can be performed by eye, or more generally, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology percentage 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 considered to include amino acid sequences that are at least 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 97% identical, or completely identical 98% to each other in alternative embodiments.

[0101] Specificity: As used herein, the term "specificity" when used in conjunction with an oligonucleotide primer refers to an oligonucleotide or primer that is capable of hybridizing to a target of interest and substantially not hybridizing to nucleic acids of no interest under appropriate hybridization or washing conditions. Higher levels of sequence identity are preferred and include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity. In some embodiments, when comparing oligonucleotides and nucleic acids, a specific oligonucleotide or primer comprises a sequence identity of 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 to be hybridized or amplified.

[0102] As known in the art, the conditions under which nucleic acid sequences hybridize to each other can be described as from low to high stringency. Typically, highly stringent hybridization conditions refer to washing the hybrid in a low-salt buffer at high temperatures. Hybridization can be performed using standard hybridization solutions in the art (e.g., 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS) at 65°C, and then in 0.25 M NaHPO 4, 3.5% SDS, and then 0.1x SSC / 0.1% SDS at a temperature ranging from room temperature to 68°C depending on the length of the probe (see, e.g., Ausubel, FM et al., Short Protocols in Molecular Biology, 4 th Ed., Chapter 2, John Wiley & Sons, NY). For example, high stringency washing includes washing a 14-base oligonucleotide probe with 6x SSC / 0.05% sodium pyrophosphate at 37°C, or washing a 17-base oligonucleotide probe at 48°C, or washing a 20-base oligonucleotide probe at 55°C, or washing a 25-base oligonucleotide probe at 60°C, or washing a nucleotide probe of about 250 nucleotides at 65°C. Nucleic acid probes can be prepared by, for example, washing with [γ- 32 P]ATP can be terminally labeled with radioactive nucleotides, or radioactively labeled nucleotides such as [α- 32 Alternatively, the probe can be labeled by incorporating biotinylated or fluorescein-labeled nucleotides and detected using streptavidin or anti-fluorescein antibodies.

[0103] siRNA: As used herein, siRNA (small inhibitory RNA) is essentially a double-stranded RNA molecule consisting of about 20 complementary nucleotides. siRNA is produced by the decomposition of a larger double-stranded (ds) RNA molecule. siRNA can inhibit gene expression by intrinsically splitting its corresponding mRNA into two parts through the interaction of siRNA with mRNA, thereby causing mRNA degradation. siRNA can also interact with DNA to promote chromatin silencing and amplification of heterochromatin.

[0104] 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 person who presents to a medical provider for diagnosis or treatment of a disease. Humans include prenatal and postnatal forms. Likewise, 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 subject is a subject with an increased risk of developing HNSCC.

[0105] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, reach completeness and / or proceed until complete or an absolute result is achieved or avoided. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0106] Substantially complementary: As used herein, the term "substantially complementary" refers to two sequences that can hybridize under stringent hybridization conditions. Those skilled in the art will understand that substantially complementary sequences do not need to hybridize along their entire length. In some embodiments, "stringent hybridization conditions" refer to hybridization conditions that are at least as stringent as: in 50% formamide, 5X SSC, 50 mM NaHCO 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5X Denhart solution at 42°C overnight; washed with 2X SSC, 0.1% SDS at 45°C; and washed 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 two bases over a stretch of 20 consecutive nucleotides.

[0107] Substitution: As used herein, the term "substitution" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the native molecule.

[0108] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0109] Susceptible to: An individual who is "susceptible" to a disease, disorder, and / or condition has not yet 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 exhibit 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.

[0110] Solid support: The term "solid support" refers to a structure that provides a substrate on which biomolecules can be bound. For example, the solid support can be an assay well (e.g., a microtiter plate), or the solid support can be a location on an array, or can be a mobile support, such as a bead.

[0111] Upstream and downstream: As used herein, the term "upstream" refers to the N-terminal of the second residue when the molecule is a protein or the residue 5' of the second residue when the molecule is a nucleic acid. Also as used herein, the term "downstream" refers to the C-terminal of the second residue when the molecule is a protein or the residue 3' of the second residue when the molecule is a nucleic acid. The protein, polypeptide and peptide sequences disclosed herein are all listed from the N-terminal amino acid to the C-terminal acid, and the nucleic acid sequences disclosed herein are all listed from the 5' end of the molecule to the 3' end of the molecule.

[0112] Overview

[0113] The disclosure herein provides novel mutations identified in certain genes that are associated with diseases and / or syndromes of interest and that can be used to more accurately diagnose diseases associated with the genes and / or syndromes of interest.

[0114] In some embodiments, the sample comprises nucleic acid. In some embodiments, the testing step comprises nucleic acid sequencing. In some embodiments, the testing step comprises hybridization. In some embodiments, hybridization is performed using one or more oligonucleotide probes specific to the region in the biomarker of interest. In some embodiments, in order to detect mutations, hybridization is performed under conditions that are sufficiently strict to not allow single nucleotide mismatches. In some embodiments, hybridization is performed with a microarray. In some embodiments, the testing step comprises restriction enzyme digestion. In some embodiments, the testing step comprises PCR amplification. In some embodiments, the testing step comprises reverse transcriptase PCR (rtPCR). In some embodiments, PCR amplification is digital PCR amplification. In some embodiments, the testing step comprises primer extension. In some embodiments, primer extension is single base primer extension. In some embodiments, the testing step comprises performing multiple allele-specific primer extension (ASPE).

[0115] In some embodiments, the sample comprises protein. In some embodiments, the testing step comprises amino acid sequencing. In some embodiments, the testing step comprises using one or more antibodies that specifically recognize the biomarker of interest to perform an immunoassay. In some embodiments, the testing step comprises protease digestion (e.g., trypsin digestion). In some embodiments, the testing step also comprises performing 2D gel electrophoresis.

[0116] In some embodiments, the testing step comprises determining the presence of one or more biomarkers using mass spectrometry. In some embodiments, the mass spectrometry modality is selected from matrix assisted laser desorption / ionization, time of flight (MALDI-TOF), electrospray (ES), IR-MALDI, ion cyclotron resonance (ICR), Fourier transform, and combinations thereof.

[0117] In some embodiments, the sample is obtained from cells, tissues (e.g., FFPE tissues), whole blood, mouthwash, plasma, serum, urine, feces, saliva, cord blood, chorionic villus samples, chorionic villus sample cultures, amniotic fluid, amniotic fluid cultures, transcervical lavage fluid, or a combination thereof. In some cases, the sample can be a liquid or tissue biopsy. In some embodiments, the sample comprises cell-free nucleic acids (e.g., DNA) that may be present in a biological sample such as blood, plasma, serum, or amniotic fluid.

[0118] In some embodiments, the testing step comprises determining the identity of the nucleotide and / or amino acid at a predetermined position in the biomarker. In some embodiments, the presence of a mutation is determined by comparing the identity of the nucleotide and / or amino acid at the predetermined position to a control.

[0119] In embodiments, methods may include performing an assay (eg, nucleic acid sequencing) in multiple individuals to determine the statistical significance of the association.

[0120] On the other hand, the present disclosure provides reagents for detecting biomarkers of interest, such as, but not limited to, nucleic acid probes that specifically bind to biomarkers (e.g., mutations in DNA sequences, mRNAs, proteins) or arrays comprising one or more probes that specifically bind to biomarkers. In some embodiments, the present disclosure provides antibodies that specifically bind to biomarkers. In some embodiments, the present disclosure provides kits for containing one or more such reagents. In some embodiments, one or more reagents are provided in the form of microarrays. In some embodiments, the kit further comprises reagents for primer extension. In some embodiments, the kit also comprises controls indicating healthy individuals. In some embodiments, the kit further comprises instructions on how to determine whether an individual suffers from a syndrome or disease of interest based on a biomarker of interest.

[0121] In certain cases, in certain embodiments, the amount of one or more biomarkers can be detected by: (a) detecting the amount of a polypeptide or protein regulated by the one or more biomarkers; (b) detecting the amount of a polypeptide or protein that regulates the biomarker; or (c) detecting the amount of a metabolite of the biomarker.

[0122] In another aspect, the disclosure herein provides a computer-readable medium encoding information corresponding to the detection of a biomarker.

[0123] Methods and compositions for diagnosing HNSCC

[0124] Embodiments of the present disclosure include compositions and methods for diagnosing the presence of HNSCC or an increased risk of developing HNSCC. The methods and compositions of the present disclosure can be used to obtain or provide genetic information from a subject in order to objectively diagnose the presence of HNSCC or an increased risk of developing HNSCC in the subject or other subjects. The methods and compositions can be embodied in a variety of ways.

[0125] In one embodiment, a method for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual is disclosed, the method comprising the steps of obtaining a sample from the individual; and measuring the amount of expression of at least one gene in Table 4 and / or Table 6 in the sample. In one embodiment, a method for detecting a biomarker associated with HNSCC in an individual is disclosed, comprising the steps of obtaining a sample from the individual; and measuring the amount of expression 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 for detecting a biomarker associated with HNSCC in an individual is disclosed, comprising the steps of obtaining a sample from the individual; and measuring the amount of expression of at least one of the human papillomavirus (HPV) E6 or E7 genes. Alternatively, various combinations of genes may be evaluated. In certain embodiments, the measured expression of any of these genes may be compared to a control value. In various embodiments, the difference between the gene expression in an individual and the control value indicates that the individual may have (i.e., diagnosed with the presence of HNSCC) or be susceptible to developing (i.e., an increased risk of developing HNSCC) HNSCC. The control value can be from one or more samples of normal (non-cancerous tissue) or from a normal (non-cancerous) population. Additionally and / or alternatively, the method can include measuring at least one normalizing gene (e.g., a housekeeping gene). In one embodiment, the normalizing gene can be KHDRBS1. In other embodiments, RPL30 or other normalizing genes can be used.

[0126] 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; and measuring the amount of an expression product of a gene from at least one of the genes 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 with a control value of expression. In various embodiments, the difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., the presence of HNSCC is diagnosed) or be susceptible to developing (i.e., an increased risk of developing HNSCC) HNSCC. The control value may be from one or more samples of normal (non-cancerous tissue) or from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include measuring at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1. In other embodiments, RPL30 or other standardized genes may be used.

[0127] Additionally and / or alternatively, a method of detecting susceptibility to HNSCC in an individual is disclosed, the method comprising: obtaining a sample from the individual; measuring the amount of at least one expression product from at least one gene comprising at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6 in the sample; and comparing the expression of at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6 in the sample to a control value of 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, the method comprising: obtaining a sample from the individual; measuring the amount of at least one expression product of a gene comprising at least one of HPV E6 and / or HPV E7 in the sample; and comparing the expression of at least one HPV E6 and / or HPV E7 expression product in the sample with a control value of the expression of each of HPV E6 and / or HPV E7. In various embodiments, the difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., the presence of HNSCC is diagnosed) or be susceptible to developing (i.e., an increased risk of HNSCC) HNSCC. The control value may be from one or more samples of normal (non-cancerous tissue) or from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include measuring at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1. In other embodiments, RPL30 or other standardized genes may be used.

[0128] In certain embodiments, the measuring comprises measuring RNA (eg, mRNA). Alternatively, the measuring may comprise an immunoassay.

[0129] In some cases, increasing the number of biomarkers can improve the statistical power of the method. For example, in certain embodiments, the method can include measuring the expression of at least two, three, four, five or more biomarkers. In some cases, at least four biomarkers are measured.

[0130] A variety of samples can be analyzed. In certain embodiments, the sample comprises serum, plasma, saliva, or tissue (eg, FFPE tissue).

[0131] The disclosed method also includes a method for identifying a marker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, the method comprising: identifying at least one marker that is expressed increased or decreased in HNSCC but not in HNSCC disease compared to a normal control. As disclosed herein, such a method may include a statistical evaluation of a marker that shows differential expression in HNSCC compared to normal tissue. Alternatively, the method may include a biomarker that distinguishes HNSCC from normal people based on other biological criteria (e.g., mutated genes, copy number differences and translocations, DNA methylation and / or microRNAs). Alternatively, other biological aspects of biomarkers may be evaluated.

[0132] As disclosed herein, various methods can be used to measure biomarkers of interest. In one embodiment, the measurement comprises measurement of mRNA. In one embodiment, the measurement comprises measurement of peptide or polypeptide biomarkers. For example, in one embodiment, the measurement comprises immunoassay. Alternatively, the measurement can comprise flow cytometry. Alternatively, as discussed in detail herein, nucleic acid methods can be used.

[0133] In other embodiments, a method for treating HNSCC is disclosed. The treatment method may include: obtaining a sample from an individual; and measuring the amount of an expression product of a gene from 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; when the difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., diagnose the presence of HNSCC) or be prone to developing (i.e., increase the risk of suffering from HNSCC) HNSCC, the individual is treated for HNSCC. The control value may be from one or more samples of normal (non-cancerous tissue) or from a normal (non-cancerous) population. Additionally and / or alternatively, the method may include measuring at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1. In other embodiments, RPL30 or other standardized genes may be used.

[0134] For example, in certain embodiments, the method of treatment may include: obtaining a sample from an individual; measuring the amount of at least one expression product from at least one gene comprising at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6 in the sample; 4. The expression of at least one of EMP1 or HSD17B6 is compared with a control value of the expression of each of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6; when the difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., the presence of HNSCC is diagnosed) or is susceptible to developing (i.e., the risk of developing HNSCC is increased) HNSCC, the individual is treated for HNSCC.

[0135] The treatment method may further include measuring the amount of at least one expression product from a gene comprising at least one of HPV E6 and / or HPV E7 in the sample; and comparing the expression of at least one HPV E6 and / or HPV E7 expression product in the sample with a control value for the expression of each of HPV E6 and / or HPV E7; when the difference between the gene expression in the individual and the control value indicates that the individual may have (i.e., the presence of HNSCC is diagnosed) or is susceptible to developing (i.e., an increased risk of developing HNSCC) HNSCC, treating the individual for HNSCC.

[0136] In various embodiments of the method of treatment, the control value can be from one or more samples of normal (non-cancerous tissue) or derived from a normal (non-cancerous) population. Additionally and / or alternatively, the method can include measuring at least one normalizing gene (e.g., a housekeeping gene). In one embodiment, the normalizing gene can be KHDRBS1. In other embodiments, RPL30 or other normalizing genes can be used.

[0137] As described above, other embodiments include compositions for detecting biomarkers associated with HNSCC in individuals. In certain embodiments, the composition comprises 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 comprise a reagent for measuring mRNA. Or the composition may comprise a reagent for measuring a peptide or polypeptide biomarker. In one embodiment, the composition comprises a reagent for performing an immunoassay. Alternatively, the composition may comprise a reagent for performing flow cytometry. Alternatively, as discussed in detail herein, the composition may comprise a reagent for determining the presence of a specific sequence and / or the expression level of a nucleic acid. As described in detail herein, the reagent may be labeled with a detectable moiety.

[0138] Thus, other aspects of the disclosure include compositions for detecting biomarkers associated with HNSCC in individuals, the compositions comprising reagents for quantifying the expression level of at least one of the genes of Table 4 and / or Table 6. Aspects of the disclosure include compositions for detecting biomarkers associated with HNSCC in individuals, the compositions comprising reagents 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, aspects of the disclosure include compositions for detecting biomarkers associated with HNSCC in individuals, the compositions comprising reagents for quantifying the expression level of at least one of HPV E6 and / or HPV E7. Additionally and / or alternatively, the composition may include reagents for detecting at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1. In other embodiments, RPL30 or other standardized genes may be used. For example, in some embodiments, the reagent detects mRNA. Alternatively, the reagent may detect protein.

[0139] Therefore, in certain embodiments, compositions can include primers (e.g., primer pairs) and / or probes for any of these genes, wherein the primers and / or probes are labeled with detectable moieties as described herein. Additionally and / or alternatively, primers and / or probes can also include arrays in which primers and / or probes are fixed on surfaces. In other embodiments, reagents can include reagents for measuring peptides and / or proteins expressed from disclosed genes. For example, compositions can include reagents for performing immunoassays. In some embodiments, these reagents can include arrays as described in detail herein. As described in detail herein, reagents can be labeled with detectable moieties.

[0140] Other embodiments include systems for performing the methods disclosed herein and / or using the compositions described herein. For example, other aspects of the present disclosure include compositions containing the present disclosure or kits for performing the methods of the present disclosure. For example, other embodiments include systems, such as kits, containing at least some compositions disclosed herein and / or reagents for performing the methods disclosed herein. Such systems or kits may include computer-readable media containing instructions and / or other information for performing the methods of the present disclosure and / or using the compositions of the present disclosure.

[0141] A variety of sample types can be used for any method, composition or system disclosed herein. In certain embodiments, the sample comprises serum, plasma, saliva or tissue (e.g., FFPE tissue). Alternatively, other sample types can be used.

[0142] Peptide, polypeptide and protein assays

[0143] 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 its fragment can be analyzed by amino acid sequencing methods or by immunoassays using one or more antibodies that specifically recognize one or more epitopes present on the biomarker of interest or in some cases have specificity for the mutation of interest. Proteins can also be analyzed by protease digestion (e.g., trypsin digestion), and in some embodiments, the digested protein products can be further analyzed by 2D gel electrophoresis.

[0144] Antibody-based detection methods

[0145] Specific antibodies that recognize a biomarker of interest can be used in any of a variety of methods known in the art. Antibodies to specific epitopes, polypeptides, and / or proteins can be produced using any of a variety of methods known in the art. For example, an epitope, polypeptide, or protein of a desired antibody 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 produced by producing hybridomas with immortal cell lines that express the antibody of interest.

[0146] In some embodiments, the antibody is labeled with a detectable moiety described herein.

[0147] 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 execution in an array format.

[0148] For example, in some embodiments, a first antibody (or antibody fragment) that specifically recognizes a biomarker is used to detect the biomarker of interest. The antibody can be labeled with a detectable portion (e.g., a chemiluminescent molecule), an enzyme, or a second binding agent (e.g., streptavidin). Alternatively, a second antibody can be used to detect the first antibody, as is known in the art.

[0149] In certain embodiments, the method may further include adding a capture support, which includes at least one recognition and binding biomarker to fix the biomarker on the capture support binding agent. In certain embodiments, the method may further include adding a second binding agent, which can specifically recognize and bind at least some of the multiple binding agent molecules and / or biomarkers on the capture support. In one embodiment, the binding agent that can specifically recognize and bind at least some of the biomarkers on the multiple binding agent molecules and / or capture support is a soluble binding agent (e.g., a second antibody). The second binding agent can be labeled (e.g., with an enzyme), so as to measure the combination of the biomarker of interest by adding the substrate of the enzyme and the amount of the product formed quantitatively.

[0150] In one embodiment, the capture solid support can be an assay well (ie, for example, a microtiter plate). Alternatively, the capture solid support can be a location on an array, or a removable support, such as a bead. Alternatively, the capture support can be a filter.

[0151] In some cases, the biomarker can be complexed with a first binding agent (e.g., a first antibody specific for the biomarker and labeled with a detectable moiety) and a second binding agent (e.g., a second antibody that recognizes the first antibody or a second first antibody), wherein the second binding agent is complexed with a third binding agent (e.g., biotin), which can then interact with a capture support (e.g., magnetic beads) having an agent that recognizes the third binding agent (e.g., streptavidin) attached to the capture support. The complex (labeled first antibody: biomarker: second first antibody-biotin: streptavidin-beads) can then be captured using a magnet (e.g., a magnetic probe) to measure the amount of the complex.

[0152] Various binding agents can be used in the methods of the present disclosure. For example, the binding agent or second antibody attached to the capture support can be an antibody or antibody fragment that recognizes a biomarker. Alternatively, the binding agent can include a protein that binds to a non-protein target (i.e., such as a protein that specifically binds to a small molecule biomarker or a receptor that binds to a protein).

[0153] In certain embodiments, the solid support can be treated with a passivating agent. For example, in certain embodiments, the biomarker of interest can be captured on a passivated surface (i.e., a surface that has been treated to reduce non-specific binding). One such passivating agent is BSA. Additionally and / or alternatively, in the case where the binding agent used is an antibody, the solid support can be coated with protein A, protein G, protein A / G, protein L, or another reagent that binds to the binding agent (e.g., antibody) with high affinity. These proteins bind to the Fc domain of the antibody, so the binding of the antibody of one or more proteins of interest can be directed to be recognized.

[0154] Nucleic acid assay

[0155] In certain embodiments, the biomarkers disclosed herein are detected at the nucleic acid level. In one embodiment, the disclosure includes methods for diagnosing the presence of a syndrome or disease of interest (e.g., HNSCC) or an increased risk of developing a syndrome or disease of interest (e.g., HNSCC) in a subject.

[0156] The method may include the following steps: obtaining nucleic acid from a tissue or body fluid sample of a subject and performing an assay to identify whether there is overexpression of a gene of interest. For example, overexpression of certain gene products can be quantified using reverse transcriptase PCR (RT-PCR). Alternatively, droplet digital PCR (ddPCR) can be used.

[0157] Alternatively, the method may include the following steps: obtaining nucleic acid from a tissue or body fluid sample of a subject, and determining whether there is a variant sequence (i.e., mutation) in the nucleic acid of the subject. In certain embodiments, the method may include comparing the variation with a known variation associated with a syndrome or disease of interest, and determining whether the variation is a variation previously identified as being associated with a syndrome or disease of interest. Alternatively, the method may include identifying the variation as a new, previously uncharacterized variation. If the variation is a new variation, the method may further include analyzing to determine whether the mutation is expected to be harmful to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using a variation profile (i.e., a compilation of mutations identified in a subject) to diagnose the presence of a syndrome or disease of interest or the increased risk of developing a syndrome or disease of interest.

[0158] Nucleic acid analysis can be performed on genomic DNA, messenger RNA and / or cDNA. Similarly, in various embodiments, nucleic acids include genes, RNA, exons, introns, gene regulatory elements, expressed RNA, siRNA or epigenetic elements. In addition, regulatory elements can be evaluated for mutations (i.e., variants) including splicing sites, transcription factor binding, AI editing sites, microRNA binding sites and functional RNA structural sites. Therefore, for each method and composition of the present disclosure, the variant may include a nucleic acid sequence covering at least one of the following: (1) A to I editing sites; (2) splicing sites; (3) conserved functional RNA structures; (4) verified transcription factor binding sites (TFBS); (5) microRNA (miRNA) binding sites; (6) polyadenylation sites; (7) known regulatory elements; (8) miRNA genes; (9) small nucleolar RNA genes encoded in ROIs; and / or (10) ultra-conserved elements in placental mammals.

[0159] In many embodiments, nucleic acid is extracted from biological sample. In some embodiments, nucleic acid is analyzed without amplification. In some embodiments, nucleic acid is amplified using techniques known in the art (e.g., cDNA amplified using polymerase chain reaction (PCR) is produced), and the amplified nucleic acid is used for subsequent analysis. Multiple PCR can be used, wherein multiple groups of primers are used to amplify several amplicons (e.g., from different genomic regions) at one time. For example, nucleic acid can be analyzed by sequencing, hybridization, PCR amplification, restriction enzyme digestion, primer extension (e.g., single base primer extension or multiple allele-specific primer extension (ASPE)) or DNA sequencing. In some embodiments, nucleic acid is amplified in a manner that makes the amplified product of wild-type allele different from the amplified product of mutant allele in size. Therefore, it is possible to determine whether there is a specific mutant allele by, for example, detecting the size difference of the amplified product on an electrophoresis gel. For example, the deletion or insertion of a gene region may be particularly suitable for using a method based on size.

[0160] Certain exemplary nucleic acid analysis methods are described in detail below.

[0161] mRNA analysis

[0162] 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 products generated in each cycle of PCR, which are proportional to the number of template nucleic acids before the start of PCR. The information obtained, such as an amplification curve, can be used to determine the presence of target nucleic acids and / or the initial amount of quantitative target nucleic acid sequences. The term "real-time PCR" is used to represent a subset of PCR techniques that allows detection of PCR products in real time or throughout the PCR reaction.

[0163] In some instances, rPCR is real-time reverse transcriptase (RT) PCR (rRT-PCR). Alternatively, droplet digital PCR can be used. When the starting material is RNA and / or mRNA, reverse transcriptase PCR is used. RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase. In rRT-PCR, cDNA is then used as a template for qPCR reactions. rRT-PCR can be performed in a one-step method, which combines reverse transcription and PCR in one test tube and buffer, using reverse transcriptase and DNA polymerase simultaneously. In one-step rRT-PCR, RNA and DNA targets are amplified using sequence-specific targets. The term "quantitative PCR" encompasses all PCR-based techniques that allow quantitative or semi-quantitative determination of the target nucleic acid sequence initially present.

[0164] The principles of real-time PCR (rPCR) are generally described in, for example, Held et al. "Real Time Quantitative PCR" Genome Research 6:986-994 (1996). Typically, rPCR measures a signal in each amplification cycle. Some rPCR techniques rely on fluorophores that emit a signal when each amplification cycle is completed. Examples of such fluorophores are fluorescent dyes such as SYBR Green that emit fluorescence at a defined wavelength when bound to double-stranded DNA. Therefore, as each PCR product accumulates, the increase in double-stranded DNA results in an increase in fluorescence intensity. Another example of a fluorophore for rPCR detection is a sequence-specific fluorescent reporter probe, which is described elsewhere herein. Examples of such probes are Probes. The use of sequence-specific reporter probes provides detection of target sequences with high specificity and can be quantified even in the presence of nonspecific DNA amplification. Fluorescent probes can also be used for multiplex determinations (for detecting multiple genes in the same reaction) based on specific probes labeled with different colors. For example, multiplex determinations can use several sequence-specific probes labeled with multiple fluorophores (including but not limited to FAM, JA270, CY5.5 and HEX) in the same PCR reaction mixture.

[0165] rPCR relies on the detection of a measurable parameter (e.g., fluorescence) during the PCR reaction. The amount of the measurable parameter is proportional to the amount of PCR product, which makes it possible to observe the increase of PCR product in "real time." Some rPCR methods allow the quantification of the input DNA template based on the observable progress of the PCR reaction. The analysis and processing of the data are discussed below. In nucleic acid amplification assays, a "growth curve" or "amplification curve" is a graph of a function in which the independent variable is the number of amplification cycles and the dependent variable is a measurable parameter of amplification dependence measured at each amplification cycle, such as the fluorescence emitted by a fluorophore. As described above, a fluorophore-labeled probe can be used to detect the amount of amplified target nucleic acid. Typically, the measurable parameter of amplification dependence is the amount of fluorescence emitted by the probe during hybridization or after the probe is hydrolyzed 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 equation dR is used. n =R n+ -R n- To calculate the change in fluorescence (dR n ), where R n+ is the fluorescence emission of the product at each time point, and R n- is the baseline fluorescence emission. n The values ​​are plotted against the cycle number to produce an amplification plot. In a typical polymerase chain reaction, the growth curve consists of a period of exponential growth followed by a plateau, which when a linear scale is used results in an S-shaped amplification curve. The growth curve is characterized by the "crossing point" value or "C p " value, also known as the "threshold" or "cycle threshold" (C t ), which is the number of cycles at which a predetermined magnitude of a measurable parameter is achieved. For example, when using a fluorophore-labeled probe, the threshold (C t ) is the fluorescence emission (dR n ) exceeds a selected threshold, which is typically 10 times the standard deviation of the baseline (however, this threshold level can be changed as desired). The lower C t A higher C value indicates faster completion of amplification, while a higher C t A lower C value indicates a slower completion of the amplification. t The values ​​reflect the higher starting amount of target nucleic acid, while the higher C t The value reflects the lower starting amount of target nucleic acid. If a control nucleic acid of known concentration is used to generate a "standard curve" or a set of "control" C at various known concentrations of control nucleic acid, t The C value of the target nucleic acid and the control nucleic acid can be compared. t The absolute amount of target nucleic acid in the sample was determined by quantifying the target nucleic acid in the sample.

[0166] Allele-specific amplification

[0167] In some embodiments, for example, in the case where the biomarker for the disease and / or syndrome of interest is a mutation, the biomarker is detected using an allele-specific amplification assay. The method is variously referred to as PCR amplification (PASA) of specific alleles (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 retardation mutation system (ARMS) (Newton et al., 1989 Nucleic Acids Res. 17: 2503-2516). The entire contents of each of these references are incorporated herein. The method is applicable to single-base substitutions and microdeletions / insertions.

[0168] For example, for amplification methods based on PCR, amplification primers can be designed so that they can distinguish different alleles (e.g., between wild-type alleles and mutant alleles). Therefore, the presence or absence of an amplified product can be used to determine whether there is a gene mutation in a given nucleic acid sample. In some embodiments, allele-specific primers can be designed so that the presence of an amplified product indicates a gene mutation. In some embodiments, allele-specific primers can be designed so that the absence of an amplified product indicates a gene mutation.

[0169] In some embodiments, two complementary reactions are used.One reaction adopts a primer ("wild-type specific reaction") having specificity to wild-type alleles, and the other reaction uses a primer ("mutant specific reaction") having specificity to mutant alleles. These two reactions can use a common second primer. Specific PCR primers for specific alleles (such as wild-type alleles or mutant alleles) are usually fully matched to an allele variant of the target, but with other allele variants (such as mutant alleles or wild-type alleles) mismatch. Mismatch may be located at the 3' end / near the primer, thereby causing the preferential amplification of the allele of perfect match. Whether amplified production can be detected from one or two reactions to indicate the presence or absence of mutant alleles. Only detecting amplified production from wild-type specific reactions indicates that there is only wild-type allele (such as, the homozygosity of wild-type alleles). Only detecting amplified production in mutant specific reactions indicates that there is only mutant allele (such as, the homozygosity of mutant alleles). Detection of amplification products from both reactions indicates (eg, heterozygosity). As used herein, this method will be referred to as "allele-specific amplification (ASA)".

[0170] Allele-specific amplification can also be used to detect duplications, insertions or inversions by using primers that partially hybridize at the junction. The degree of junction overlap can be varied to allow specific amplification.

[0171] Amplification products can be examined by methods known in the art, including by visualizing (eg, with one or more dyes) nucleic acid bands that have migrated through a gel (eg, by electrophoresis) to separate nucleic acids by size.

[0172] Allele-specific primer extension

[0173] In some embodiments, allele-specific primer extension (ASPE) method is used to detect gene mutation.ASPE uses allele-specific primers that can distinguish alleles (e.g., mutant alleles and wild-type alleles) in extension reactions, so that extension products are obtained only in the presence of specific alleles (e.g., mutant alleles or wild-type alleles).Extension products can be detectable or, for example, can become detectable by using labeled deoxynucleotides in extension reactions.Any of various labels can be used for these methods, including but not limited to radioactive labels, fluorescent labels, chemiluminescent labels, enzyme labels, etc.In some embodiments, nucleotides are labeled with entities (e.g., biotin molecules that can be combined with fluorescent dyes that can be conjugated to streptavidin) that can be subsequently detected.In some embodiments, the reaction is carried out in a multiplex manner, for example, in the same extension reaction, many allele-specific primers are used.

[0174] In some embodiments, the extension products are hybridized to a solid or semi-solid support such as a bead, matrix, gel, etc. For example, the extension products can be labeled with a specific nucleic acid sequence (e.g., included as part of an allele-specific primer), and the solid support can be attached to an "anti-tag" (e.g., a nucleic acid sequence complementary to the tag in the extension product). The extension products can be captured and detected on the solid support. For example, beads can be sorted and detected.

[0175] Single nucleotide primer extension

[0176] In some embodiments, a single nucleotide primer extension (SNuPE) assay is used, in which the primer is designed to extend only one nucleotide. In this method, the identity of the nucleotide immediately downstream of the 3' end of the primer is known and is different in the mutant allele compared to the wild-type allele. SNuPE can be performed using an extension reaction in which only one special type of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP, labeled dGTP, or labeled dTTP). Therefore, the presence of a detectable extension product can be used as an indication of the identity of a nucleotide at a position of interest (e.g., a position immediately downstream of the 3' end of the primer), and can therefore be used as an indication of the presence or absence of a mutation at that position. SNuPE can be as described in U.S. Pat. No. 5,888,819; U.S. Pat. No. 5,846,710; U.S. Pat. No. 6,280,947; U.S. Pat. No. 6,482,595; U.S. Pat. No. 6,503,718; U.S. Pat. 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"), the entire contents of each of which are incorporated herein by reference.

[0177] In some embodiments, primer extension can be combined with mass spectrometry to accurately and quickly detect the presence or absence of mutations. See U.S. Pat. No. 5,885,775 (analysis of single nucleotide polymorphism analysis by mass spectrometry) by Haff et al.; U.S. Pat. No. 7,501,251 (DNA diagnosis based on mass spectrometry) by Koster; 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.

[0178] Oligonucleotide ligation assay

[0179] In some embodiments, an oligonucleotide ligation assay ("OLA" or "OL") is used. OLA uses two oligonucleotides that are designed to hybridize to adjacent sequences of a single strand of a target molecule. Typically, one of the oligonucleotides is biotinylated and the other is detectably labeled, for example, using a fluorescent moiety conjugated to streptavidin. If the exact complementary sequence is found in the target molecule, the oligonucleotides will hybridize so that their ends are adjacent and produce 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. Pat. No. 4,998,617, the entire contents of which are incorporated herein by reference in their entirety.

[0180] Hybridization method

[0181] In some embodiments, nucleic acids are analyzed by hybridization using one or more oligonucleotide probes specific for the biomarker of interest and under sufficiently stringent conditions to not allow single nucleotide mismatches. In certain embodiments, suitable nucleic acid probes can distinguish between normal genes and mutant genes. Thus, for example, one of ordinary skill in the art can use the probes of the present invention to determine whether an individual is homozygous or heterozygous for a particular allele.

[0182] Nucleic acid hybridization techniques are well known in the art. One skilled in the art understands 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 Biology. John Wiley & Sons, Secaucus, NJ.

[0183] In some embodiments, probe molecules that hybridize to mutant or wild-type sequences can be used to detect such sequences in amplified products by solution phase or, more preferably, solid phase hybridization.Solid phase hybridization can be achieved, for example, by attaching the probe to a microchip.

[0184] Nucleic acid probe can comprise ribonucleic acid and / or deoxyribonucleic acid.In some embodiments, the nucleic acid probe provided is an oligonucleotide (i.e. " oligonucleotide probe "). Generally speaking, oligonucleotide probe is long enough to specifically bind with the homologous region of gene interested, but is short enough to make the difference of a nucleotide between probe and the tested nucleic acid sample destroy hybridization.Usually, the size of oligonucleotide probe is about 10 to 100 nucleotide.In some embodiments, the length of oligonucleotide probe is 15 to 90,15 to 80,15 to 70,15 to 60,15 to 50,15 to 40,15 to 35,15 to 30,18 to 30 or 18 to 26 nucleotide.As understood by those of ordinary skill in the art, the optimal length of oligonucleotide probe can depend on the ad hoc method and / or the condition that oligonucleotide probe can be used.

[0185] In some embodiments, nucleic acid probes can be used as primers, for example, for nucleic acid amplification and / or extension reactions. For example, in certain embodiments, the gene sequence evaluated for variation comprises an exon sequence. In certain embodiments, exon sequences and other flanking sequences (for example, 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, potential harmful mutations in intron sequences or other non-coding regions can be assessed. Alternatively, a portion of these sequences can be used. Such variant gene sequences may include sequences having at least one mutation as described herein.

[0186] Other embodiments of the present disclosure provide isolated gene sequences, which include mutations associated with syndromes and / or diseases of interest. Such gene sequences can be used to objectively diagnose the presence of HNSCC in a subject or the increased risk of developing HNSCC. In certain embodiments, the isolated nucleic acid can include any non-variant sequence or variant sequence or a combination thereof. For example, in certain embodiments, the gene sequence includes an exon sequence. In certain embodiments, exon sequences and other 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 used. Alternatively, a portion of these sequences can be used. In certain embodiments, the gene sequence includes an exon sequence from at least one biomarker gene disclosed herein.

[0187] In some embodiments, the nucleic acid probe is labeled with a detectable moiety as described herein.

[0188] Array

[0189] The various methods mentioned herein can be suitable for use as an array allowing the analysis and / or detection of a set of biomarkers in a single experiment. For example, multiple mutations comprising 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 being adapted to array-based platforms (e.g., microarrays). In some embodiments, the array comprises one or more probes specifically for detecting mutations in a biomarker of interest.

[0190] In one embodiment, a plurality of disclosed biomarker groups are 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, the composition 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. In addition and / or alternatively, the composition may include at least one standardized gene (e.g., a housekeeping gene). In one embodiment, the standardized gene may be KHDRBS1 and / or RPL30 or other standardized genes. In certain embodiments, the composition may include primers and / or probes for any of these genes, wherein the primers and / or probes are labeled with a detectable moiety as described herein.

[0191] DNA Sequencing

[0192] In certain embodiments, diagnosis of a biomarker of interest is performed by nucleic acid sequencing to detect variations in the sequence, genomic location or arrangement, and / or genomic copy number of a nucleic acid or a group of nucleic acids.

[0193] In some embodiments, the method may include obtaining nucleic acid from a tissue or body fluid sample of 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 variation with a variation known to be associated with HNSCC, and determining whether the variation is a variation previously identified as being associated with HNSCC. Alternatively, the method may include identifying the variation as a new, previously uncharacterized variation. If the variation is a new variation, or in some cases a previously characterized (i.e., identified) variation, the method may further include analyzing to determine whether the mutation is expected to be harmful to the expression of the gene and / or the function of the protein encoded by the gene. The method may further include using a variation profile (i.e., a compilation of mutations identified in a subject) to diagnose the presence of HNSCC or the increased risk of developing HNSCC.

[0194] For example, in certain embodiments, the next generation (massive parallel sequencing) can be used. Alternatively, Sanger sequencing can be used. Alternatively, the next generation (massive parallel sequencing) and Sanger sequencing can be used in combination. Additionally and / or alternatively, sequencing includes at least one of single molecule synthesis sequencing. Therefore, in certain embodiments, multiple DNA samples in the analysis library are used to identify samples that show variation. Additionally or alternatively, in certain embodiments, multiple DNA samples in multiple libraries are analyzed to identify individual samples that show the same variation in at least two libraries.

[0195] One conventional method for sequencing is by chain termination and gel separation, as described by Sanger et al., 1977, Proc Natl Acad Sci USA, 74:5463-67. Another conventional sequencing method involves chemical degradation of nucleic acid fragments. See Maxam et al., 1977, Proc. Natl. Acad. Sci., 74:560-564. In addition, methods have been developed based on hybridization sequencing. See, for example, Harris et al., U.S. Patent Application Publication No. 20090156412. Each of these references is incorporated herein by reference in its entirety.

[0196] In other embodiments, sequencing of nucleic acids is accomplished by massively parallel sequencing (also referred to as "next generation sequencing") of a single molecule or by amplification of substantially identical groups of molecules derived from a single molecule by methods such as PCR. Massively parallel sequencing is shown in, for example, Lapidus et al., U.S. Pat. No. 7,169,560, Quake et al., U.S. Pat. No. 6,818,395, Harris U.S. Pat. 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.

[0197] In next generation sequencing, PCR or whole genome amplification can be performed on the nucleic acid to obtain a sufficient amount of nucleic acid for analysis. In some forms of next generation sequencing, amplification is not required because the method can evaluate the DNA sequence from unamplified DNA. After determination, 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 derived from one or more individuals who were not known to have HNSCC when their samples were obtained. All differences 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 are considered variations.

[0198] In the next generation (massive parallel sequencing), all regions of interest are sequenced together, and the source of each sequence sequencing fragment is determined by comparing (alignment) with a reference sequence. The regions of interest can be enriched together in a reaction, or they can be enriched separately and then merged before sequencing. In certain embodiments, and as described in more detail in the embodiments herein, the DNA sequence of the coding exon derived from the gene included in the assay is enriched by a large amount of hybridization of the genomic DNA of random fragmentation and specific RNA probes. The same adapter sequence is attached to the end of all fragments, thereby allowing the fragments captured by a pair of primers to be enriched in a reaction by PCR. The region with lower efficiency is captured by hybridization by PCR amplification with specific primers. In addition, PCR using specific primers can be used to amplify exons ("pseudo-exons") that have similar sequences elsewhere in the genome.

[0199] In certain embodiments using massive parallel sequencing, PCR products are connected in series to form long DNA segments, which are sheared into short fragments (e.g., by acoustic energy). This step ensures that the fragment ends are distributed throughout the region of interest. Subsequently, a section of dA nucleotides is added to the 3' end of each fragment, which enables these fragments to be bound to a flat surface ("flow cell") coated with an oligo (dT) primer. Each fragment can then be sequenced by extending the oligo (dT) primer with fluorescently labeled nucleotides. In each sequencing cycle, only one type of nucleotide (A, G, T or C) is added, and only one nucleotide is allowed to be incorporated by using a chain termination nucleotide. For example, in the first sequencing cycle, fluorescently labeled dCTP can be added. The nucleotide will only be incorporated into those growing complementary DNA chains that need C as the next nucleotide. After each sequencing cycle, an image of the flow cell is obtained to determine which fragment is extended. The DNA chain that has been incorporated with C will emit light, while the DNA chain that has not been incorporated with C will appear dark. The chain termination is reversed so that the growing DNA chain can be extended again, and the process is repeated for a total of 120 cycles.

[0200] The images are converted into strings of bases commonly called "sequencing fragments," which summarize the 25 to 60 bases at the 3' end of each fragment. The sequenced fragments are then compared to a reference sequence of the DNA being analyzed. Since any given string of 25 bases typically occurs only once in the human genome, most sequenced fragments can be "aligned" to a specific location in the human genome. Finally, a consensus sequence for each genomic region can be established from the available sequenced fragments and compared to the exact reference sequence at that location. Any differences between the consensus sequence and the reference are called sequence variants.

[0201] Detectable part

[0202] In certain embodiments, certain molecules (e.g., nucleic acid probes, antibodies, etc.) according to and / or provided by the present invention comprise one or more detectable entities or moieties, i.e., such molecules are "labeled" with such entities or moieties.

[0203] Any of a variety of detectable agents may be used in the practice of the present disclosure. Suitable detectable agents include, but are not limited to, various ligands, radionucleotides; fluorescent dyes; chemiluminescent agents (e.g., acridinium esters, stable 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; digoxin; haptens; and proteins for which antisera or monoclonal antibodies are available.

[0204] In some embodiments, the detectable moiety is biotin. Biotin can be bound to avidin (e.g., streptavidin), which is typically conjugated (directly or indirectly) to other moieties that are themselves detectable (e.g., fluorescent moieties).

[0205] Some non-limiting examples of detectable moieties that can be used are described below.

[0206] Fluorescent dyes

[0207] In certain embodiments, the detectable portion is a fluorescent dye. A variety of known fluorescent dyes with a wide variety of chemical structures and physical properties are suitable for the practice of the present disclosure. The fluorescent detectable portion can be excited 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, which records its intensity.

[0208] Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthylfluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), hexachlorofluorescein (HEX), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine amine (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.), ALEXAFLUOR dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXAFLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY530 / 550, BODIPIP 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY630 / 650, BODIPY 650 / 665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), etc. For more examples of suitable fluorescent dyes and methods of coupling fluorescent dyes to other chemical entities (e.g., proteins and peptides), see, for example, "The Handbook of Fluorescent Probes and Research Products", 9th Ed., Molecular Probes, Inc., Eugene, OR. Advantageous properties of fluorescent labeling agents include high molar absorption coefficients, high fluorescence quantum yields, and photostability. In some embodiments, the labeled fluorophore exhibits absorption and emission wavelengths in the visible light of the spectrum (i.e., 400 to 750 nm) rather than in the ultraviolet range of the spectrum (i.e., below 400 nm).

[0209] Detectable part can include more than one chemical entity, such as in fluorescence resonance energy transfer (FRET). Resonance transfer leads to an overall enhancement of emission intensity. For example, see Ju et al. (1995) Proc.Nat'l Acad.Sci.(USA)92:4347, the entire contents of which are incorporated herein by reference. In order to achieve resonance energy transfer, the first fluorescent molecule (" donor "fluorescence) absorbs light and transfers it to the second fluorescent molecule (" acceptor "fluorescence) by the resonance of the excited electron. In one method, the donor and acceptor dyes can be linked together and attached to the oligonucleotide primer. The method of connecting the donor and acceptor dyes to nucleic acids has been described in, for example, U.S. Patent No. 5,945,526 of Lee et al., the entire contents of which are incorporated herein by reference. The donor / acceptor pair of dyes that can be used includes, for example, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL and fluorescein / QSY 7 dyes. See, e.g., U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes are also commercially available, e.g., 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.

[0210] Enzymes

[0211] In certain embodiments, the detectable moiety is an enzyme. Examples of suitable enzymes include, but are not limited to, those used in ELISA, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, etc. Other examples include beta-glucuronidase, beta-D-glucosidase, urease, glucose oxidase, etc. The enzyme can be conjugated to the molecule using a linking group such as carbodiimide, diisocyanate, glutaraldehyde, etc.

[0212] radioactive isotopes

[0213] In certain embodiments, the detectable moiety is a radioactive isotope. For example, the molecule can be isotopically labeled (i.e., can contain one or more atoms that have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature) or an isotope can be attached to the molecule. Non-limiting examples of isotopes that can be incorporated into the molecule 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).

[0214] Dendrimers

[0215] 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 commercially available from Genisphere (Montvale, NJ). These can be chemically coupled to oligonucleotide primers by methods known in the art.

[0216] system

[0217] In certain embodiments, the disclosure provides systems for performing the methods disclosed herein and / or using the compositions described herein. In certain embodiments, the system may include a kit. Alternatively, the system may include computerized instructions and / or reagents for performing the methods disclosed herein.

[0218] Reagent test kit

[0219] In certain embodiments, the disclosure provides a kit for use according to the methods and compositions disclosed herein. Typically, the kit comprises one or more reagents for detecting a biomarker 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 comprise a reagent for a positive control of a biomarker of interest (i.e., a gene).

[0220] In one embodiment, a group of multiple biomarkers disclosed 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, the kit comprising a reagent for quantitatively measuring 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. In addition and / or alternatively, the kit may include at least one standardized (e.g., housekeeping) gene and / or a reagent for detecting such a housekeeping gene. In one embodiment, the standardized gene may be KHDRBS1 and / or RPL30 or other standardized genes. In some embodiments, the kit may include a positive control for any disclosed biomarker and / or standardized gene.

[0221] In some embodiments, provided kits further comprise reagents for performing various detection methods described herein (e.g., RT-PCR, sequencing, hybridization, primer extension, multiplex ASPE, immunoassays, etc.). For example, the kits may optionally comprise buffers, enzymes, and / or reagents for the methods described herein (e.g., for amplifying nucleic acids by RT-PCR, primer-directed amplification, for performing ELISA experiments, etc.). In certain embodiments, the kits may comprise primers and / or probes for any of these genes, wherein the primers and / or probes are labeled with a detectable moiety as described herein.

[0222] In some embodiments, the kit provided further comprises a control indicating a healthy individual, e.g., a nucleic acid and / or protein sample from an individual that does not have the disease and / or syndrome of interest. Alternatively, the kit may comprise a positive control comprising a known amount of one or more measured biomarker genes. The kit may also comprise instructions on how to determine whether an individual suffers from a disease and / or syndrome of interest or is at risk of developing a disease and / or syndrome of interest.

[0223] In some embodiments, a computer readable medium is provided that encodes information corresponding to a biomarker of interest. Such a computer readable medium can be included in a kit of the present invention.

[0224] Methods for identifying HNSCC markers

[0225] Data Mining

[0226] In certain embodiments of the present disclosure, data mining methods are used to identify biomarkers. 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 with a disease (directly or indirectly) and / or differentially expressed in cancer compared to normal tissue. Such genes can then be evaluated as biomarkers.

[0227] molecular

[0228] In certain embodiments, the disclosure includes methods for identifying biomarkers of syndromes or diseases of interest (i.e., variants in nucleic acid sequences that are statistically significant and associated with HNSCC). For example, genes of interest and potential normalizing genes can be identified by evaluating gene expression in tissue samples isolated from patients with head and neck cancer using random forest analysis (see, e.g., L. Breiman, "Random Forests" Machine Learning, 2001, 45: 5-32) and discussed in detail herein. In this method, a random forest is a combination of tree predictors, so each tree depends on the value of an independently sampled random vector and has the same distribution for all trees in the forest.

[0229] Alternatively, the genes and / or genomic regions assayed for new markers can be selected based on their importance in biochemical pathways that show genetic associations and / or biological causal relationships with syndromes and / or diseases of interest. Alternatively, the genes and / or genomic regions assayed for markers can be selected based on genetic associations with DNA regions that are associated with the inheritance of HNSCC in a family. Alternatively, the genes and / or genomic regions assayed for markers can be systematically evaluated to cover certain regions of the chromosome that have not yet been evaluated.

[0230] In other embodiments, the gene or genomic region evaluated for the new marker can be a part of a biochemical pathway associated with the development of a syndrome and / or disease of interest (e.g., HNSCC). The clinical significance of a variation and / or a combination of variations can be assessed based on one or more of the following methods. If a report or known variation or a combination of variations occurs more frequently in a subject with a syndrome and / or disease of interest than in a nucleic acid in a subject without a syndrome and / or disease of interest, it is considered that it is at least potentially susceptible to the syndrome and / or disease of interest. If a report or known variation or a combination of variations is propagated exclusively or preferentially to an individual with a syndrome and / or disease of interest, it is considered that it is at least potentially susceptible to the syndrome and / or disease of interest. On the contrary, if a variation is found with a similar frequency in two populations, the variation is unlikely to be associated with the development of a related syndrome and / or disease.

[0231] If a variant or combination of variants is reported or known to have an overall deleterious effect on the function of a protein or biological system in an experimental model system suitable for measuring the function of the protein or biological system, and if the variant or combination of variants affects one or more genes known to be associated with the syndrome and / or disease of interest, it is considered to be at least potentially susceptible to the syndrome and / or disease of interest. For example, if a variant or combination of variants is predicted to have an overall deleterious effect on protein or gene expression (i.e., causing a nonsense mutation, a frameshift mutation, or a splice site mutation, or even a missense mutation) based on the predicted effect on the sequence and / or structure of the protein or nucleic acid, and if the variant or combination of variants affects one or more genes known to be associated with the syndrome and / or disease of interest, it is considered to be at least potentially susceptible to the syndrome and / or disease of interest.

[0232] Likewise, in certain embodiments, the total number of variants may be important. If one or several variants are detected in a test sample that are assessed, alone or in combination, as at least potentially associated with a syndrome and / or disease of interest, then the individual in whose genetic material the variant or variants are detected may be diagnosed as having or being at high risk of developing the syndrome and / or disease of interest.

[0233] For example, the disclosure herein provides a method for diagnosing the presence of HNSCC in a subject or an increased risk of developing HNSCC. Such a method may include obtaining nucleic acids from a sample of tissue or body fluid. The method may include determining the expression of at least one gene in normal tissue and cancerous tissue to identify potential biomarkers of interest. The method may further include sequencing the nucleic acid or determining the genomic arrangement or copy number of the nucleic acid to detect whether there are one or more variants in the nucleic acid sequence or genomic arrangement or copy number. The method may further include a step of evaluating the clinical significance of one or more variants. Such analysis may include evaluating the degree of association of variant sequences in an affected population (i.e., a subject suffering from the disease). Such an analysis may also include analyzing the extent to which mutations may affect gene expression and / or protein function. The method may also include an increased risk of diagnosing the presence of HNSCC or developing HNSCC based on an assessment. DETAILED DESCRIPTION

[0234] Example

[0235] The following examples are provided to illustrate certain aspects of the present disclosure. These examples are in no way limiting.

[0236] Example 1 - Literature-based identification of potential HNSCC markers

[0237] A preliminary literature search was performed to identify markers associated with HNSCC. Table 1 shows the type of markers and the number of markers found, and Table 2 shows the potential biomarkers identified.

[0238] Table 1

[0239] HNSCC markers serial number Mutant gene 9 Copy number and translocations 14 Methylation 71 Gene expression 29 MicroRNA 46 Normalizing genes 15 total 184

[0240] Table 2

[0241]

[0242]

[0243] Based on this initial search, it was decided to seek markers associated with differential gene expression

[0244] Example 2 - Identification of biomarkers using the TCGA database

[0245] Data from the Cancer Genome Atlas (TCGA) database were mined to identify markers that showed differential expression in HNSCC. The TCGA database (RNASeqV2) contains data on 18,379 genes that can be used for differential expression. The data includes information on: clinical information (e.g., age, smoking, stage, treatment, and survival); copy number; methylation; gene expression; mutations, microRNA expression.

[0246] The HNSCC data included 530 samples from 4 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 tissues. Of the 530 samples, 70 were human papillomavirus (HPV) positive, 279 were HPV negative, and 181 had unknown or uncertain HPV status.

[0247] Random forest analysis was performed to identify genes that can strongly predict HNSCC classification from normal samples. In this analysis, genes with reportable data and expression changes less than 2 times (Wilcox test, adjusted p value <0.001) were discarded for 50% of the samples. For each round of analysis, 75% of the samples were used as training sets and 25% of the samples were used as test sets. The data were optimized for κ and 10-fold cross validation (repeated 10 times and averaged performance) was performed. The 20 strongest predictions were identified, and the entire process was repeated 4 times. The gene list obtained for each run is shown in Table 3, and the 36 unique genes combined are shown in Table 4. The data in Table 3 are shown in order from the highest level (20) to the lowest level (1).

[0248] Table 3

[0249]

[0250] Table 4

[0251]

[0252]

[0253] The top 4 genes from each of the 4 analyses were then selected to provide an initial candidate list of 8 unique genes listed in Table 5.

[0254] Table 5

[0255] Gene name Gene product CAB39L Calcium Binding Protein ADAM12 Metalloproteinases involved in the shedding of the EGFR ligand HBEGF SH3BGRL2 SH3 domain-binding glutamate-rich protein-like 2 NRG2 Neuregulin 2 (ligand for HER3 receptor) COL13A1 Type XIII collagen α1 chain GRIN2D Glutamate receptor subunit 2D LOXL2 Lysyl oxidase homolog 2 HSD17B6 Hydroxysteroid 17-β dehydrogenase 6

[0256] Figure 1 Also shown are the statistical analysis results (ie, accuracy, kappa, sensitivity, and specificity) for several individual genes, listed in order of specificity.

[0257] Example 3 - Gene Panel

[0258] Analyses were performed to determine whether the use of the gene panel was expected to improve assay performance. Figure 2 As shown, using a 4 or 5 gene panel can significantly improve gene performance. Panel construction was performed by adding the most informative marker (CAB39L) to the second most informative marker (ADAM12) to form a 2-marker panel, and then adding the next most informative marker (NRG2) to form a 3-marker panel. Each of the other six markers was then added and the performance of the predictions was evaluated ( Figure 2 , top table). The results showed that the four-marker panel of CAB39L, ADAM12, NRG2, and GRIN2D provided the highest levels of accuracy, kappa value, sensitivity, and specificity. The results of the 5-gene panel are shown in the table below ( Figure 2 ). Find (e.g. Figure 2 ) There is only minimal improvement when more than 4-5 genes are added. Such panels are still useful if one of the markers identified as one of the top 4-5 markers is technically challenging.

[0259] Example 4 - Gene expression at tumor site

[0260] Most HNSCCs are found in the oral cavity (mouth) or oropharynx (throat). Analyses were 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 for eight markers are shown in Figure 3 and 4Middle: CAB39L, ADAM12, SH3BGRL2, NRG2 ( Figure 3 ); and COL13A1, GRIN2D, LOXL2, and HSD17B6 ( Figure 4 ); using the TCGA dataset with oral cavity and oropharynx as the majority of TCGA samples, among which HNSCC oral cavity (320) and normal oropharynx (82) = 402 out of a total of 530 samples (402 / 530 = 76%) and normal oral cavity (30) and oropharynx (3) = 33 out of a total of 44 samples (33 / 44 = 75%).

[0261] It was found that for both sites as well as the larynx, the markers had very different expression levels in normal versus cancerous tissue. Figure 3 and Figure 4 In the figure, the 3 leftmost data sets on the x-axis (larynx, oral cavity, and oropharynx) are the expression levels of normal tissues, and the 4 rightmost data sets on the x-axis (hypopharynx, larynx, oral cavity, and oropharynx) are the expression levels of cancer tissues; the data of hypopharynx are merged. It can be seen that there are similar distributions in normal and HNSCC sites (i.e., the levels in normal are similar regardless of tissue, and the levels in HNSCC are similar regardless of tissue). For example, it can be seen that for the larynx, oral cavity, and oropharynx, the expression levels of CAB39L in HNSCC are significantly lower than those in normal tissues, respectively, while the expression level of ADAM12 in HNSCC is significantly higher than that in normal tissues. Figure 5 A statistical compilation of the data is shown in , which shows the results for markers for all HNSCC samples compared to samples from the oral cavity and oropharynx. Accuracy, sensitivity, and specificity were only minimally altered when all samples were compared to the oral cavity and oropharynx. For all 8 markers, the sample set was reduced by approximately 25%; median gene expression levels were only minimally altered; and the distributions were significantly different (HNSCC vs. normal). In both sets, the Mann-Whitney p value was less than 0.0001.

[0262] Example 5 - Analysis of differential expression of TCGA gene sets for median fold expression versus expression overlap percentage.

[0263] Fig. 6AThe figure above shows the number of times the markers from Table 4 originally selected by random forest analysis were identified from four random forest analysis replicates compared to the median rank (discriminating power) of the markers. It can be seen that as the number of times the markers were repeatedly identified from the random forest analysis increases, the median rank tends to increase. The four tables below the figure list the markers and median ranks grouped according to the number of times the markers were repeatedly identified from the random forest analysis. The figure and the table together provide a measure that allows the 36 genes of Table 4 identified from the random forest analysis to be prioritized; first by the number of replicates, then by the median rank.

[0264] Figure 6B The left figure shows an analysis of the 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 of expression as an increase in gene expression (data pointing to the right of 0) or a decrease in gene expression (data pointing to the left of 0). The y-axis shows the overlap percentage of gene expression in HNSCC relative to normal tissue. It can be seen that the markers disclosed in Table 4 (N=36) show a substantial increase or decrease in gene expression, with a very low overlap percentage compared to other genes in the database. The entire TCGA HNSCC gene set is RNASeq data, using genes representing more than 80% of the samples (i.e., 16,161 (88%) of 18,379 genes can be reported for HNSCC and normal samples). The x-axis shows the median fold change (=HNSCC / normal). 8,352 genes (52%) were found to be increased in HNSCC compared with normal, while 7,809 genes (48%) were decreased, of which 1,387 genes (8.6%) were increased > 2-fold, while 1,701 genes (10.5%) were decreased > 2-fold. For increased gene expression in HNSCC, the cutoff value was the 5th percentile in HNSCC and the 95th percentile in normal (e.g., Figure 6B , GRIN2D illustration).

[0265] Figure 6B The dashed line on the graph in shows that nine markers from the random forest analysis repeated four times had less than 20% overlap in expression (range 0 to 19%) (i.e., these markers are below the dashed line). Figure 6B The dashed line on the graph in shows that 23 of the 36 unique markers in Table 4, or 23 / 36 = 64%, have less than 20% overlap in expression because they are clustered below the dashed line. Table 6 lists an additional 45 genes that were not identified by the random forest analysis and whose expression has < 20% overlap. < 20% of the overlapping genes, e.g. Figure 6BGLT25D1, identified in the literature, may be considered as an additional biomarker to help classify HNSCC from normal samples.

[0266] Table 6

[0267]

[0268]

[0269] You can Figure 6B The data in Figure 7 and Figure 8 Compare the data in . Figure 7 A similar analysis of median fold expression versus percent expression overlap from TCGA HNSCC RNASeq data is shown, and this example shows tissue and saliva markers identified by literature search highlighted on the graph. The upper and lower graphs show results for markers identified in both tissue (upper graph) and saliva (lower graph). Figure 7 Some of the literature markers in showed some evidence of differential expression, but only a few markers showed high levels of differential expression with low overlap percentages. Based on this analysis, the markers MAL, MMP1, CEP55, CENPA, AURKA, and FOXM1 appear to be the most useful additional biomarkers and can be included in the disclosed methods and compositions.

[0270] Figure 8 A similar analysis of the analysis of median expression fold relative expression overlap percentage from TCGA HNSCC RNASeq data is shown, and this example shows the standardized markers used in tissues (upper figure) and saliva (lower figure) identified by literature search. It can be seen that these markers have little variation in expression and have significant overlap in normal versus HNSCC. An ideal standardized marker should have minimal variation and expression levels similar to the gene panel of interest.

[0271] Example 6 - Identification of potential normalizing or housekeeping genes

[0272] The TGCA database was analyzed using three criteria to identify potential normalizing genes: (1) minimum median change in expression between HNSCC and normal tissues; (2) minimum interquartile range (IQR) in HNSCC and normal, where IQR is defined as gene expression in the 75th quartile / gene expression in the 25th quartile; and (3) expression levels close to the median of the gene panel of interest to facilitate experimental comparisons between the expression of potential candidate genes and normalizing genes.

[0273] The analysis is summarized in Fig.9AIn. The left figure shows a graph of the median fold change of gene expression in HNSCC relative to normal (x-axis) relative to the average IQR in normal and cancer cells (= [HNSCC IQR + normal IQR] / 2) (Y-axis). In this figure, the positive number on the x-axis corresponds to an increase in gene expression in cancer cells compared to normal, and the negative number corresponds to a decrease in gene expression in cancer cells compared to normal. Data from a total of 16,161 genes were analyzed (left figure). This corresponds to all genes with data for normal and HNSCC in the TCGA database, and therefore corresponds to 88% (16,151 / 18,379) of the total number of genes in the TCGA database. It was found again that compared with normal, 8,352 genes (52%) increased in HNSCC, while 7,809 genes (48%) decreased, and 1,387 genes (8.6%) increased>2 times, while 1,701 genes (10.5%) decreased>2 times.

[0274] The most interesting potential normalized genes are those with a fold change (x-axis) of 0 and an average IQR of 1 (area circled in the figure). The middle figure shows the data of those genes with a median fold change of less than 2 and an average IQR of less than 2 (n=7,949 genes). The right figure shows the gene expression data of 7949 candidate normalized genes. Those genes with median expression are considered to be the most interesting. Based on this analysis, KHDRBS1 (containing KH domain, RNA binding, signal transduction related protein 1) was identified as a normalized gene of interest. Some other more common normalized genes, such as RPLPO, RPL10, RPL30 and GAPDH, were identified in the middle figure. Data from the TCGA database for KHDRBS1 are shown in Table 6 below. Fig. 9B showed that KHDRBS1 exhibited similar characteristics (low expression fold change and low IQR) in many cancer types.

[0275] The average expression level of 11.60 to 11.90 is higher than the proposed panel markers, which range from 1.52 (NRG2 in HNSCC) to 11.44 (SH3BGRL2 in normal). This is still within the range of cancer-specific markers and should therefore be a good normalizing gene. It is noted that for FFPE samples that may contain a lot of degraded RNA, an amplicon length of <100 bp is preferred.

[0276] Table 7

[0277]

[0278] These data can be compared with the data of the well-known housekeeping gene (normalization gene) GAPDH (Table 7). The median expression of 16.3-16.50 is about 30 times higher than the gene (SH3BGRL2) showing the highest expression level in the above candidate panel. Therefore, GAPDH may not be very useful as a marker for the above disclosed HNSCC panel.

[0279] Example 7 - Evaluation of expression assays

[0280] 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 Fig.10 , which presents data showing the reproducibility of ddPCR analysis of ADAM12 and SH3BGRL2 in tongue squamous cell carcinoma (SCC) and normal tissue (buccal mucosa) ( Fig.10 Also shown are data from the ddPCR data (lower table) relative to the TCGA RNASeq data (middle table) for gene expression levels of ADAM12 and SH3BGRL2 in cancer compared to normal tissue. It can be seen that ADAM12 expression is significantly increased in cancer compared to normal, while SH3BGRL2 expression is significantly decreased in cancer compared to normal, as measured by both methods. In these experiments, two aliquots from the same sample were analyzed. For the cheek sample, the expression level in one of the samples was too low to be accurately measured. Although the ddPCR values ​​were generally lower than the TCGA RNASeq data, the trends were the same for both markers (see Fig.10 ). Reproducibility was excellent down to 1 copy / μL.

[0281] Fig.11 Additional digital PCR data are shown for three formalin-fixed, paraffin-embedded patient samples (DA1081983; DR1041686; DA0063595) and one URNA control sample from a cell cultured cancer tissue. URNA is a universal human reference RNA available from Agilent (Catalog No. 740000). It contains 10 somatic cancer cell lines that can be used as a consistent control for comparison to standard data sets. According to an embodiment of the present disclosure, repeated or triplicate sampling was performed. Again, it was found that SH3BGRL2 expression was significantly reduced in cancer compared to normal. It can be seen that the number of copies per uL from the URNA control is much greater than the cross-linked and possibly fragmented RNA from the FFPE sample, which is most likely due to the intact nature of the isolated RNA.

[0282] Fig.12RNA titration experiments using ddPCR and markers SH3BGRL2 (labeled with FAM) and KHDRBS1 (labeled with HEX) are shown. In this experiment, RNA was isolated from FFPE samples (or URNA was used as a positive control) and diluted 2-fold or 10-fold. cDNA was generated using standard techniques, 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). The results of three samples (#1, #3, or #5) are shown. It can be seen that, except for very dilute samples (i.e., close to or below one copy per μL), there is a good correlation between the ratios of markers and housekeeping genes at different concentrations, indicating that a good range of sample concentrations can be measured using this assay.

[0283] Fig.13 The relative abundance of the biomarker SH3BGRL2 and housekeeping RNA KHDRBS1 in FFPE samples compared to the positive control URNA (left panel); the ratio of the biomarker SH3BGRL2 to KHDRBS1 in cancer cells compared to normal cells and URNA (middle panel); the relative abundance of SH3BGRL2 to KHDRBS1 in cancer cells and normal cells as measured using RNASeq (right panel). Again, consistent patterns can be seen for the biomarkers regardless of how they are measured (although the absolute values ​​may vary).

[0284] Fig.14 Results are shown for SH3BGRL2 measured by a singleplex assay (ie only SH3BGRL2-FAM generated by PCR, compared to a duplex reaction measuring both SH3BGRL2 and KHDRBS1 in samples of normal or cancer origin). Results were generally very similar.

[0285] Example 8 - Differential expression in FFPE tissue samples measured by ddPCR

[0286] Fig.15Shows the measurement results of 5 biomarkers and the housekeeping gene KHDRBS1 from 22 benign and 8 head and neck cancer FFPE samples. RNA was extracted from FFPE tissues using the Roche High Pure FFPET RNA isolation kit essentially according to the manufacturer's protocol. Five graphs show the copy number / μL of duplex ddPCR of 5 biomarkers (SH3BGRL2, KRT4, EMP1, LOXL2, and ADAM12) and the housekeeping gene KHDRBS1 from 22 benign and 8 head and neck cancer FFPE samples. KHDRBS1 showed a very similar distribution pattern and copy number / μL in all samples and all assays. In contrast, the biomarkers showed >3-log 10 different distributions in copies / μL. One HNSCC sample in all assays resulted in "no response" for both the biomarkers and KHDRBS1.

[0287] Fig.16 The left graph shows the expression of the five biomarkers normalized to KHDRBS1. The biomarker copy number / μL was divided by the KHDRBS1 copy number / μL in the duplex ddPCR reaction to obtain the biomarker normalization or ratio for each sample. Fig.16 The right graph is the RNASeq expression data of the same biomarkers from the HNSCC TCGA dataset. The table shows the median fold change in the expression of each biomarker from the ddPCR experiment compared to the TCGA data. Both datasets showed the same genes (SH3BGRL2, KRT4, and EMP1) downregulated in cancer, and the same genes (LOXL2 and ADAM12) upregulated in cancer. The ddPCR results were consistent with the TCGA dataset, but the magnitude of the change did vary.

[0288] In Fig.17 , a ddPCR score was developed to separate cancer from normal samples by determining the difference of (total log of upregulated genes) - (total log of downregulated genes). After adding the biomarkers, the equation became (logLOXL2 + logADAM12) - (logSH3BGRL2 + logEMP1 + logKRT4). The left graph shows the ddPCR scores of cancer samples, alongside the ddPCR scores of normal samples. At a ddPCR cut-off score >0.24, the assay specificity was 95.4% and the sensitivity was 85.7%. The right graph shows the receiver operating characteristic (ROC) analysis with an AUC of 0.961 and p = 0.0003. The specificity of ddPCR was similar to the TCGA dataset (see Figure 2 ), while the sensitivity of ddPCR < TCGA, possibly due to differences in sample size and / or gene selection.

[0289] Example 9 - HPV16 E6 and E7 expression in FFPE HNSCC tissue samples

[0290] Fig.18 The correlation between E6 and E7 HPV16 expression and p16 from FFPE HNSCC tissue samples is shown. The top table shows the HPV16 E6, E7, and housekeeping gene KHDRBS1 ddPCR copy numbers / uL obtained from 4 p16-positive samples. The bottom table shows the results from 10 p16-negative samples, as well as the HPV16 E6, E7, and housekeeping gene KHDRBS1 ddPCR copy numbers / uL. The right figure shows the standardized ddPCR ratio of the biomarkers of the four p16-positive samples divided by the housekeeping gene KHDRBS1. The two samples in which the HPV16 E6 and E7 reportable copy numbers / uL were greater than no response (No Call) showed that the standardized ddPCR expression of E7 was approximately 5 times that of E6. In addition, in preparations and replicates, all p16-negative samples were negative (no response) for E6 and E7 expression measured by ddPCR. There was good overall agreement between p16 measured by IHC and E6 or E7 expression measured by ddPCR (Cohen's κ=0.81).

[0291] Example 10 - Isolation of RNA and Gene Expression in Saliva Samples

[0292] In some cases, saliva can be used as a biological sample. Fig.19 The yield of RNA in saliva is shown. Saliva samples were collected using a DNAGenotek CP-190 human RNA collection device. After sample collection, each sample was thoroughly mixed and incubated at 50 degrees Celsius for 2 hours, and then the samples were stored at -20 degrees Celsius until processing. For each saliva aliquot to be processed, 1 / 10 sample volume of DNA Genotek neutralization solution (catalog number RELONN-5) was added. RNA was purified using the Roche HighPure RNA Paraffin kit (catalog number 03270289001). The table on the left shows 15 saliva RNA samples, as well as the μg RNA / 2mL saliva calculated from 250μL saliva sample preparation solution and the A260 / A280 ratio of each saliva sample. The median μg RNA isolated was 5.8μg, and the median A260 / A280 ratio was 2.05. The scatter plot on the right shows the same data in a box-and-whisker plot format, with the whiskers at the maximum and minimum, the boxes around the 75th and 25th percentiles, and a line through the median.

[0293] Fig. 20 Shows the Fig.19Measurement results of 5 biomarkers and the housekeeping (HK) gene RPL30 in 15 saliva RNA samples. The left figure shows the copy number / μL of the dual ddPCR of 5 biomarkers (LOXL2, SH3BGRL2, CRISP3, EMP1 and KRT4) and the housekeeping gene RPL30 in 15 saliva samples. The biomarker distribution range is 0.38 to 650 copies / μL. The right figure should be the housekeeping gene in each dual ddPCR. In 5 dual ddPCR reactions, the housekeeping gene (RPL30) averaged 8 to 170 copies / μL and the %CV was 6 to 27%. One sample averaged 1.3 copies / μL and the %CV was 57%.

[0294] Fig.21 Normalized ddPCR from saliva compared to TCGA RNASeq is shown. The left panel shows the expression of biomarkers normalized to the housekeeping gene RPL30. The biomarker normalization or ratio for each sample was obtained by dividing the biomarker copy number / μL by the housekeeping gene RPL30 copy number / μL in the dual ddPCR reaction. One sample with an average HK gene of 1.3 copies / uL and samples with a biomarker of "no response" or <1 copy / uL were excluded. The normalized ddPCR expression range was 0.018 to 9.7 = 540 times. Fig.21 The right panel is RNASeq expression data for the same biomarkers from "normal" in the HNSCC dataset. The table shows the median fold increase in expression of each biomarker from the ddPCR experiment relative to LOXL2 compared to the TCGA data. The median fold increase in expression from saliva measured by ddPCR tends to be similar to the TCGA (tissue) dataset, but the magnitude of change is different.

[0295] Example 11 - Implementation

[0296] The present disclosure includes, but is not limited to, the following embodiments.

[0297] A.1 A method for detecting biomarkers associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising the following steps:

[0298] Obtaining a sample from an individual; and

[0299] The amount of expression product from a gene comprising at least one gene in Table 4 and / or Table 6 is measured.

[0300] A.2 The method of any of the preceding paragraphs, wherein the gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6.

[0301] A.3 The method of any of the preceding paragraphs, wherein the genes comprise at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, and HSD17B6.

[0302] 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.

[0303] A.5 The method of any of the preceding paragraphs, wherein the genes consist of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6 and an expression product from at least one of the HPV E6 and / or HPV E7 genes.

[0304] A.6 The method of any of the preceding paragraphs, further comprising measuring the amount of a normalizing gene such as KHDRBS1 or RPL30 or another normalizing gene.

[0305] A.7 The method of any of the preceding paragraphs, wherein the measuring comprises measuring mRNA.

[0306] A.8 The method of any of the preceding paragraphs, wherein the measuring comprises an immunoassay.

[0307] A.9 The method of any of the preceding paragraphs, comprising measuring the expression of at least four genes.

[0308] A.10 The method of any of the preceding paragraphs, wherein the sample comprises serum, tissue, FFPE, saliva, or plasma.

[0309] A.11 The method of any of the preceding paragraphs, comprising comparing the expression level to a control value from a normal population.

[0310] A.12 The method of any of the preceding paragraphs, wherein a difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC.

[0311] B.1 A method for identifying a marker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, comprising: identifying at least one marker whose expression is increased or decreased in HNSCC but not in HNSCC disease compared to a normal control.

[0312] The method of B.2B.1, wherein the gene comprises at least one of the genes of Table 4 and / or Table 6, and / or at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0313] The method described in any of B.3B.1-B.2, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0314] B.4 The method of any of B.1-B.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 and / or HPV E7 genes.

[0315] The method described in any of B.5B.1-B.4, wherein the genes are composed of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6 and expression products from at least one of the HPV E6 and / or HPV E7 genes.

[0316] B.6 The method of any of B.1-B.5, further comprising measuring the amount of a normalizing gene such as KHDRBS1 or RPL30 or another normalizing gene.

[0317] B.7 The method of any of B.1-B.6, wherein the measuring comprises measuring mRNA.

[0318] B.8 The method of any of B.1-B.7, wherein the measuring comprises an immunoassay.

[0319] B.9 The method of any of B.1-B.8, comprising measuring the expression of at least four genes.

[0320] B.10 The method of any of B.1-B.9, wherein the sample comprises serum, tissue, FFPE, saliva, or plasma.

[0321] B.11 The method of any one of B.1-B.10, wherein a difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC.

[0322] C.1 A method for determining susceptibility of an individual to head and neck squamous cell carcinoma (HNSCC), comprising:

[0323] Obtaining a sample from an individual; and

[0324] measuring the amount of at least one expression product of at least one gene from Table 4 and / or Table 6; and

[0325] The expression of at least one gene in Table 4 and / or Table 6 in the sample is compared to a control value of the expression product of the gene.

[0326] The method of C.2C.1, wherein the gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0327] C.3 The method of any one of C.1-C.2, wherein the genes comprise at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, and HSD17B6.

[0328] C.4 The method of any of C.1-C.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 and / or HPV E7 genes.

[0329] C.5 The method of any one of C.1-C.4, wherein the genes consist of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6 and expression products from at least one of the HPV E6 and / or HPV E7 genes.

[0330] C.6 The method of any of C.1-C.5, further comprising measuring the amount of a normalizing gene such as KHDRBS1 or RPL30 or another normalizing gene.

[0331] C.7 The method of any of C.1-C.6, wherein the measuring comprises measuring mRNA.

[0332] C.8 The method of any of C.1-C.7, wherein the measuring comprises an immunoassay.

[0333] C.9 The method of any of C.1-C.8, comprising measuring the expression of at least four genes.

[0334] C.10 The method of any of C.1-C.9, wherein the sample comprises serum, tissue, FFPE, saliva, or plasma.

[0335] C.11 The method of any one of C.1-C.10, wherein a difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC.

[0336] D.1 A composition for detecting a biomarker associated with head and neck squamous cell carcinoma (HNSCC) in an individual, the composition comprising a reagent for quantifying the expression level of at least one gene in Table 4 and / or Table 6.

[0337] D.2 The composition of any of D.1, wherein the at least one gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6.

[0338] D.3 The composition of D.1-D.2, wherein at least one gene comprises at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0339] D.4 The composition of any of D.1-D.3, further comprising at least one reagent for quantifying the expression level of at least one of HPV E6 and / or E7.

[0340] D.5 The composition of any one of D.1-D.4, ​​wherein the at least one gene consists of 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.

[0341] D.6 The composition of any of D.1-D.5, further comprising at least one reagent for measuring at least one normalizing gene, such as KHDRBS1 or RPL30 or another normalizing gene.

[0342] D.7 The composition of any of D.1-D.6, wherein the reagent detects mRNA.

[0343] D.8 The composition of any of D.1-D.7, wherein the reagent detects a protein.

[0344] D.9 The composition of any one of D.1-D.8, wherein the reagent comprises at least one primer and / or probe for any one of these genes, wherein the at least one primer and / or probe is labeled with a detectable portion.

[0345] D.10 The composition of any one of D.1-D.9, wherein a difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC.

[0346] E.1 A kit comprising the compositions of the preceding paragraphs.

[0347] E.2 The kit of E.1, further comprising instructions for measuring the at least one gene and / or determining whether the value is different from a control value.

[0348] E.3 The kit of any of E.1-E.2, comprising at least one of the positive controls for at least one normalizing gene, such as KHDRBS1 or RPL30 or another normalizing gene.

[0349] E.4 The kit of any one of E.1-E.3, further comprising at least one of the positive controls for any one of the genes in Table 4 and / or Table 6.

[0350] E.5 The kit described in any one of E.1-E.4, wherein the at least one gene includes at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0351] E.6 The kit described in any of E.1-E.5, wherein the at least one gene comprises at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 or HSD17B6.

[0352] E.7 The kit of any one of E.1-E.6, wherein the at least one gene comprises at least one of HPV E6 and / or E7.

[0353] E.8 The kit described in any of E.1-E.7, wherein the at least one gene consists of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6 and at least one of HPV E6 and / or HPV E7.

[0354] E.9 The kit of any one of E.1-E.8, wherein the reagents comprise at least one primer and / or probe for any one of these genes, wherein the at least one primer and / or probe is labeled with a detectable moiety.

[0355] E.10 The kit of any of E.1-E.9, wherein a difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC.

[0356] F.1 A method of treating HNSCC comprising:

[0357] obtaining a sample from an individual;

[0358] Measuring the amount of an expression product from a gene comprising at least one gene in Table 4 and / or Table 6 in the sample;

[0359] Comparing the expression of at least one gene in Table 4 and / or Table 6 in the sample to a control value of expression; and

[0360] When the difference between the gene expression in the individual and the control value indicates that the individual may be suffering from (ie, diagnosed with) HNSCC or is susceptible to developing (ie, at risk of developing) HNSCC, the individual is treated for HNSCC.

[0361] F.2F.1, wherein the gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6.

[0362] F.3 The method of F.1-F.2, wherein the genes include at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6.

[0363] F.4 The method of any of F.1-F.3, further comprising measuring the amount of an expression product from at least one of the HPV E6 and / or HPV E7 genes.

[0364] F.5 The method of any one of F.1-F.4, wherein the genes consist of at least four of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1 and HSD17B6 and the expression products of at least one of the HPV E6 and / or HPV E7 genes.

[0365] F.6 The method of any of F.1-F.5, further comprising measuring the amount of a normalizing gene such as KHDRBS1 or RPL30 or another normalizing gene.

[0366] F.7 The method of any of F.1-F.6, wherein the measuring comprises measuring mRNA.

[0367] F.8 The method of any of F.1-F.7, wherein the measuring comprises an immunoassay.

[0368] F.9 The method of any of F.1-F.8, comprising measuring the expression of at least four genes.

[0369] F.10 The method of any of F.1-F.9, wherein the sample comprises serum, tissue, FFPE, saliva, or plasma.

[0370] F.11 The method of any of F.1-F.10, comprising comparing the expression level to a control value from a normal population.

[0371] Throughout this disclosure, other documents, such as patents, patent applications, patent publications, periodicals, books, papers, web page content, have been referenced and cited. For all purposes, all of these documents are incorporated herein by reference in their entirety. According to the full content of this article, including the science and patent literature cited herein, various modifications and equivalents to those described herein will become apparent to those skilled in the art. The subject matter of this article includes information, examples and guidance that can be adjusted to be applicable to practicing this disclosure in its various embodiments and equivalent forms thereof.

Claims

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

2. The method of claim 1, wherein the gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6.

3. 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. The method according to claim 1 , further comprising measuring the amount of a normalizing gene.

5. The method of claim 1, wherein the measuring comprises measuring mRNA. The method of claim 1 , wherein the measuring comprises an immunoassay.

7. The method of claim 1, comprising measuring the expression of at least four of said genes.

8. The method of claim 1, wherein the sample comprises serum, tissue, FFPE, saliva, or plasma.

9. A method for detecting susceptibility of an individual to head and neck squamous cell carcinoma (HNSCC), comprising: obtaining a sample from an individual; and Measuring the amount of at least one expression product of at least one gene from Table 4 and / or Table 6; and The expression of at least one gene in Table 4 and / or Table 6 in the sample is compared to a control value of the expression product of the gene, wherein a difference between the gene expression in the individual and the control value indicates that the individual may have or be susceptible to HNSCC (i.e., have an increased risk of developing HNSCC).

10. The method of claim 9, wherein the gene comprises at least one of CAB39L, ADAM12, SH3BGRL2, NRG2, COL13A1, GRIN2D, LOXL2, KRT4, EMP1, or HSD17B6.

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