TITLE OF THE INVENTION

"BlOMARKES OF DISEASE AMD THEIR USE: IN DISEASE DETECTION AND MANAGEMENT"

FIELD OF THE INVENTION

[0001] The present invention relates to methods and reagents for determining the likelihood of the presence or a bsence of head and neck cancer or susceptibility to head and neck cancer, including virus infection positive and virus infection negative head and neck cancer, and for monitoring, treatment, response to therapy and management of these conditions. The invention has practical use in early diagnosis of head and neck cancer and in enabling better treatment and management decisions to be made In clinically and sub-clinically affected subjects.

BACKGROUND OF THE INVENTION

[0002] Head and neck cancer refers to a group of biologically distinct cancers of the; upper aerodigestive tract (UADT), including the Up, oral cavity (mouth), nasal cavity (inside the nose), paranasal sinuses, pharynx, and larynx. Ninety percent of cancers associated with the UADT are squamous cell carcinomas (5CC), which are collectively referred to as head and neck squamous cell carcinoma (HNSCC). HNSCC is the sixth most common cancer in the world (Chin etaf., 2006. Anttemcerpier, 6:1111-1118; Weiss £t 3i>, 2013, Cell, Oncol. 36:213-224; Nakaofca etal, 2014. Cell. Oncol, 37:119- 129) and annually approximately 900,000 new cases are reported worldwide, including 300,000 deaths (Wingo er a/., 1995. Cancer J. Clin. 45:8-30). HNSCC is diverse in nature, both biologically and clinically. The primary risk factors for the development of HNSCC include tobacco use, alcohol consumption, human papilloma virus (HPV) infection (for oropharyngeal cancer) and Epstein-Barr virus (EBV) infection (for nasopharyngeal cancer), in the initial stages of the disease a patient may not show any clinical symptoms and, consequently, a significant number of patients present with metastatic disease at the time of diagnosis (regional nodal involvement in 43% and distant metastasis in 10%), leading to 5-years survival rates of less than 40% (Halier ef al. _f Cancer

Management Handbook, 12th Edition, (Cancer Network, 2013)).

[0003] The early diagnosis of HNSCC holds promise for an improved prognosis, but this option is currently impeded because current diagnosis relies heavily on the histological assessment of tissue biopsy samples, tumor size, anatomic location and the presence of lymph node metastases. Additionally, there is a lack of clinically validated biomarkers to reliably detect the disease at an early stage and the paucity of targeted molecular therapeutics (Pfaffe etal., 2011. Clin. Chem. 57:675-687).

[0004] Hence, there is a clear need for the development of new methods for the detection of head and neck cancer, includi HNSCC which could lead to earlier diagnosis and improvement in the survival rate of patients afflicted with this debilitating disease.

SUMMARY OF THE INVENTION

[0005] The present invention arises from the determination that certain microRNA (miRNA) biomarkers are differentially expressed between head and neck cancer subjects and healthy controls, between subjects with precancer of the head neck and healthy controls, between subjects with precancer of the head neck and HPV infection positive head and neck cancer subjects, between subjects with precancer of the head neck and HPV infection negative head and neck cancer subjects, and between HPV infection positive head and neck cancer subjects and HPV infection negative head and neck cancer subjects. It has also been determined that this differential expression can be conveniently detected using saliva as a sample. Based on these determinations methods and reagents are proposed that take advantage of salivary miRNA biomarkers to determine the likelihood that head and neck cancer (e.g., HNSCC), precancer of the head and neck, HPV infection positive head and neck cancer or HPV infection negative head and neck cancer is present or absent in a subject, to treat and manage these conditions, and to monitor the efficacy of treatment of those conditions, as described hereafter.

[0006] The present invention thus represents a significant advance over current technologies for the management of head and neck cancer (e.g., HNSCC), including precancer of the head and neck, as well as HPV infection positive or negative forms thereof. In certain advantageous embodiments, it relies upon measuring the level of miRNA biomarkers in saliva, in some embodiments where saliva is the subject of analysis, it is proposed that detection of the presence or absence of head and neck cancer, including HNSCC, will be feasible at very early stages of its development and/or progression.

[0007] Accordingly, in one aspect, the present invention provides methods for determining the likelihood of the presence or absence of a condition selected from a healthy condition (e.g., a normal condition or one in which head arid neck cancer is absent), precancer of the head and neck (pHN), head and neck cancer generally (HNC) (i.e., regard I ess of HPV infection status) including HPV infection positive HNC (HPV ⁺ HNC) or HPV infection negative HNC (HPV HNC). These methods generally comprise, consist or consist essentially of: (I) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from a healthy condition (also referred to herein as a "reference healthy salivary miRNA profile"), pHN (also referred to herein as a "reference pHN salivary miRNA profile"), HNC (also referred to herein as a "reference HNC salivary miRNA profile"), HPV ⁺ HNC (also referred to herein as a "reference HPV ^f HNC salivary miRNA profile") or HPV- HNC (also referred to herein as a "reference HPV- HNC salivary miRNA profile"), wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6 or 7 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, miR-134, miR-196a, miR-196b, miR-210, and miR-455; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3)

determining a likelihood of the subject having or not having the condition based on the sample salivary miRNA profile and the reference salivary miRNA profile. Suitably, the cancer is a squamous cell carcinoma (SCC). Suitably, the precancer is a squamous cell precarcinoma. In some embodiments, the reference salivary miRNA profile further evaluates one or both of miR-191 and miR-222. Individual reference salivary miRNA profiles are suitably obtained from a corresponding control subject or control population. Suitably, the miRNA biomarkers of the salivary miRNA profiles disclosed herein are selected from the group consisting of: hsa-miR-9-5p, hsa-miR-127-5p, hsa-miR-134, hsa^miR-l9l-5p, hsa-miR-196a, hsa-miR-196b, hsa-miR-210, hsa-miR-222-3p and hsa- miR-455.

[0008] In some embodiments, the methods determine the likelihood that pHN or a healthy condition is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from a healthy condition and pHN, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, miR-134, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or not haying the healthy condition or pHN based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0009] In other embodiments, the methods determine the likelihood that HNC generally (i.e., regardless of HPV infection status) or a healthy condition is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from a a healthy condition and HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, miR-134, miR-196a, miR-l96b, miR-210, rniR~45S, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or riot having the healthy condition or HNC based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0010] In some embodiments, the methods determine the likelihood that pHN or HNC generally (i.e., regardless of HPV infection status) is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition pHiSl and HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biorrtarker(s}} selected from the group consisting of; miR- 9, mi R- 127, miR-134, miR-196a, mi R- 196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a ewresponding salivary miRNA; and (3) determining a likelihood of the subject having or not having pHN or HNC based on the sample salivary miRNA profile and the reference sa I ivary miRNA profi le«

[0011] In other embodiments, the methods determine the likelihood that pHN or HPV ⁺ HNC is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from pHN and HPV ⁺ HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, mi R- 196a, miR- 196b, miR-210, mlR-455, mjR- 191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or not having pHN of HPV ⁺ HNC based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0012] In still other embodiments, the methods determine the likelihood that pHN or HPV HNC is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from pHN and HPV HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, mi R- 196a, miR- 196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or not having pHN or HPV HNC based Oh the sample salivary miRNA profile and the reference salivary miRNA profile.

[0013] In other embodiments, the methods determine the likelihood that HPV ⁺ HNC or HPV- HNC is present or absent in the subject, wherein the method comprises: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from HPV ^: HNC and HPV- HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA {e.g., 1, 2, 3, 4, % 6, 7, 8 or 9 salivary miRNA bjomarker(s)) selected from the group consisting of: miR-9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or not having HPV ^f HNC or HPV- HNC based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0014] In some embodiments, the methods comprise comparing the sample salivary miRNA profile with the reference salivary miRNA profile and determining a likelihood of the presence or absence of the i condition based on that comparison.

Suitably, the methods further comprise correlating the reference salivary miRNA profile with the presence or absence of a respective condition.

[0015] Evaluation of salivary miRNAs suitably includes determining the levels of individual salivary miRNAs, which correlate with the presence of a condition, as broadly described above and elsewhere herein.

[0016] In some embodiments, the methods of determining the likelihood of the presence or absence of a condition, as broadly described above and elsewhere herein, comprise comparing the level of a first salivary miRNA in the sample salivary miRNA with the level of a second salivary miRNA in the sample salivary miRNA profile to provide a ratio and determining a likelihood of the presence or absence of the condition based on that ratio, in illustrative examples of this type, the determination is carried out in the absence of comparing the level of the first or second salivary miRNA in the sample salivary miRNA profile to the level of a corresponding salivary miRNA in the reference salivary miRNA profile.

[0017] In a related aspect, the present invention provides kits for determining the likelihood of the presence or absence of a condition, as broadly described above and elsewhere herein. These kits generally comprise, consist or consist essentially of: one or more reagents and/or devices for use in performing the methods of determining the likelihood of the presence or absence of a condition as broadly described above and elsewhere herein. [0018] The present findings enable treatment regimens, which can be used to treat subjects with biomarker profiles that correlate with the presence of precancer (/.e, pHN), or HNC including specific forms of HNC {e.g., HPV ⁺ HNC or HPV- HNC). In some embodiments, these treatment regimens can be adopted or prescribed, particularly at an earlier stage in the progression towards HNC, with a view to treating HNC or preventing or delaying the onset of HNC in a subject

[0019] Yet another aspect of the present invention provides methods for treating, preventing or inhibiting the development or progression of a condition in a subject wherein the condition is selected from pHN, HNC, HPV ' HNC or HPV- HNC (also referred to herein as "treatment methods") « These methods generally comprise, consist or consist essentially of: exposing the subject to a treatment regimen for treating the conditon based on results obtained from any one or more of the methods of determining the likelihood of the presence or absence of the condition, as broadly described above and elsewhere herein (also referred to herein as "determination methods"), using a sample salivary miRNA profile from the subject for the determination, wherein the results indicate that the condition is present in the subject. In some embodiments, the treatments methods further comprise conducting a determination method as broadly described above and elsewhere herein on the subject prior to exposing the subject to the treatment regimen. In illustrative examples of this type* the determination method is performed by a person who exposes the subject to the treatment regimen. In other illustrative examples, a saliva sample from the subject or nucleic acid extract thereof is provided to another person (e.g., a person in a laboratory) who performs the

determination method and provides the results of the determination method to the person who exposes the subject to the treatment regimen.

[0020] In a related aspect, the present invention provides methods for treating a condition in a subject wherein the condition is selected from pHN, HNC, HPV ⁺ HNC or HPV- HNC (also referred to herein as "treatment methods"). These methods generally comprise, consist or consist essentially of: (a) sending a saliva sample or nucleic acid extract thereof from the subject to a laboratory to have a determination method as broadly described above and elsewhere herein conducted; (b) receiving the results of the determination method of step (a); and (c) exposing the subject to a treatment regimen for treating the condition.

[0021] In another related aspect, the present invention provides methods for treating, preventing or inhibiting the development or progression of pHN, HNC, HPV ⁺ HNC or HPV- HNC in a subject. These methods generally comprise, consist or consist essentially of: (1) providing a correlation of a reference salivary miRNA profile with the presence of a condition selected from a healthy condition, pHN, HNC, HPV ⁺ HNC or HPV- HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA {e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)) selected from the group consisting of: miR-9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; (3) determining a likelihood of the subject having or not having the condition based on the sample salivary miRNA profile and the reference salivary miRNA profile, and (4) administering to the subject, on the basis that the subject has an increased likelihood of having pHN an effective amount Of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of pHN, or

administering to the subject, on the basis that the subject has an increased ftke!thood of having HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of HNC, or administering to the subject, on the basis that the subject has an increased likelihood of having HPV ⁺ HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of HPV ⁺ HNC, or administering to the subject, on the basts that the subject has an increased likelihood of having HPV ^" HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of HPV ^" HNC.

[0022] The present findings also enable methods of monitoring the. efficacy of a treatment regimen for treating pHN, HNC, HPV ⁺ HNC or HPV ^" HNC, and determining a subject's response to such treatment (e.g., whether it is a positive or negative response to such treatment). Thus, in another aspect, methods are provided for monitoring the efficacy of a particular treatment regimen in a subject towards a desired health state (e.g., healthy condition). These methods generally comprise, consist or consist essentially of: (1) providing a correlation of a reference salivary miRNA profile with the likelihood of having a healthy condition, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (eg., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)} selected from the group consisting of: miR-9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; (2) obtaining a corresponding salivary miRNA profile of a subject having pHN, HNC, HPV ⁺ HNC or HPV ^" HNC after treatment with a treatment regimen, wherein a similarity of the subject's salivary miRNA profile after treatment to the reference salivary miRNA profile indicates the itkeithood that the treatment regimen is effective for changing the health status of the subject to the desired health state.

[0023] Still another aspect of the present invention provides methods for correlating a reference salivary miRNA profile with an effective treatment regimen for a condition selected from pHN, HNC, HPV ⁺ HNC or HPV ^" HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)) selected from the group consisting of; miR-9, miR-127, miR- 196a, miR-lMb, miR-2lG, miR-455, miR-191 and miR-222. These methods generally comprise, consist or consist essentially of; (a) determining a sample salivary miRNA profile from a subject with the condition prior to treatment, wherein the sample salivary miRNA profile evaluates for at least one salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and correlating the sample salivary miRNA profile with a treatment regimen that is effective for treating the condition in the subject

[0024] In another aspect, the present invention provides methods for determining whether a treatment regimen is effective for treating a subject with a condition selected from pHN, HNC, HPV ⁺ HNC or HPV- HNC. These methods generally comprise, consist or consist essentially of: (a) correlating a reference salivary miRNA profile prior to treatment with an effective treatment regimen for the condition, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA {e.g., 1, 2, 3,

4, 5, 6, 7, 8 or 9 salivary miRNA(s)) selected from the group consisting of: miR-9, miR- 127, mi R- 196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; and (b) obtaining a sample salivary miRNA profile from the subject after treatment, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA, and wherein the sample salivary miRNA profile after treatment indicates whether the treatment regimen is effective for treating the condition in the subject.

[0025] In a further aspect, the present invention provides methods for correlating a salivary miRNA profile with a positive or negative response to a treatment regimen for a condition selected from pHN, HNC, HPV ⁺ HNC or HPV- HNC. These methods generally comprise, consist or consist essentially of : (a) obtaining a salivary miRNA profile from a subject with the condition following commencement of the treatment regimen, wherein the salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)) selected from the group consisting of: miR- 9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; and (b) correlating the salivary miRNA profile from the subject with a positive or negative response to the treatment regimen.

[0026] Another aspect of the present invention provides methods for determining a positive or negative response to a treatment regimen by a subject with a condition selected from pHN, HNC, HPV ⁺ HNC or HPV- HNC. These methods generally comprise, consist or consist essentially of: (a) correlating a reference salivary miRNA profile with a positive or negative response to the treatment regimen, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA {e.g., 1, 2, 3, 4,

5, 6, 7, 8 or 9 salivary miRNA(s)) selected from the group consisting of: miR-9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR-191 and miR-222; and (b) determining a sample salivary miRNA profile from the subject, wherein the subject's sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA and indicates whether the subject is responding to the treatment regimen,

[0027] In some embodiments, the methods for determining a positive or negative response to a treatment regimen further comprise; determining a first sample salivary miRNA profile from the subject prior to commencing the treatment regimen, wherein the first sample salivary miRNA profile evaluates at least one salivary miRNA (e.g,, 1, 2, 3, 4, 5, 6, 7, 8 Or 9 salivary mlRNA(s)) selected from the group consisting of : miR-9, miR-127, miR-i96a, miR-l96b, miR-210, miR-455, miR-191 and miR-222; and comparing the first sample salivary miRNA profile with a second sample salivary miRNA profile from the subject after commencement of the treatment regimen, wherein the second sample salivary miRNA profile evaluates for an individual salivary miRNA in the first sample salivary miRNA profile a corresponding salivary miRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a graphical representation showing differential expression of five salivary miRNAs between HNSCC patients and healthy controls. (A) Relative expression levels of five miRNAs determined by microarray and RT-qPCR technologies, respectively. Microarray and RT-qPCR data sets were normalized to SNGRD96A, (B) Expression levels of mlR-222; miR-134, mi R ^» 1.27, .miR-9 and miR-191 in saliva collected from HNSCC patients (n-2I) and healthy controls (n-21.) determined by RT-qPCR (Con firmato ry study) .

[0029] Figure 2 is a graphical representation showing differential expression of five salivary miRNAs between HNSCC patients and healthy controls. (A) Expression levels of m!R-222, miR-134, miR-127,. miR-9 and miR-191 in saliva collected from HNSCC patients (,η -35) and healthy controls (n=35) determined by RT-qPCR. Differences were determined using Wilcoxon Statistical test and statistical

Significance was obtained for miR-191, miR-9 and miR-134 (P<0.QQ1). (B) Ii!umina HiSeq miRNAseq data were downloaded from The Cancer Genome Atlas (TCGA) portal (HNSCC) of 334 tumors and 39 norma! tissues. The Y-axis represents reads per million and the X-axis represents five selected miRNAs investigated.

[0030] Figure 3 is a graphical representation showing receiver operating characteristic (ROC) curve analysis of the independent validation study using saliva- derived miR-9 (a), miR-134 (b) and miR-191 (c) for discriminating HNSCC patients from normal subjects (P <0.001).

[0031] Figure 4 is a graphical representation showing miScript™ microarray signature cluster profiles of five selected miRNAs in saliva from healthy controls (n = 56) and HNSCC patients (n=56).

[0032] Figure 5 is a graphical representation showing differential expression of nine salivary miRNAs between Patients with pHN, HPV ⁺ HNSCC patients (HPV ⁺ ), HPV ^" HNSCC patients (HPV ^" ), and healthy controls. Expression levels of miR-210, miR-455, miR~196a _f miR-196b, miR-θ, miR-191, miR-134, miR-222 and miR-127 in saliva collected from pHN patients (n=30), HPV* HNSCC patients (n=50), HPV ^" HNSCC patients (n-SO), and healthy controls (n-60) determined by RT-qPCR.

[0033] Figure 6 is a graphical representation showing box and whisker plots of individual fold changes for salivary miR-210 (A), miR-222 (B) and miR-127 (C) between HPV ⁺ and HPV- HNSCC patients. Circles present outliers on the 5 ^th and 95 ^th percentiles. P values show the significance,

[0034] Figure 7 is a graphical representation showing box and whisker plots of individual fold changes for salivary miR-455 and miR-134 (B) between healthy controls, HNSCC patients and subjects with pHN. Circles present outliers on the 5 ^th and 95 ^th percentiles. P values show the significance.

[0035] Figure 8 is a graphical representation showing box and whisker plots of individual fold changes for salivary miR-222, miR-i9i (B) and miR-9 (C) between healthy controls, subjects with pHN and HNSCC patients. Circles present outliers on the 5 ^th and 95 ^th percentiles, P values show the significance.

[0036] Figure 9 is a graphical representation showing ROC curve analysis of an independent validation study using .salivary rn.iR-2.lG, msR-455, mi R- 196a, mi.R-196b, miR-9, miR-191, miR-134, miR-222 and rnsR-127 for discriminating pHN patients from normal subjects (P <0.00l). Area under the curve (AUCJ = 0.962; sensitivity = 96.7%; specificity = 90,0%,

[0037] Figure 10 is a graphical representation showing ROC curve analysis of an independent validation study using salivary miR-2iQ, miR-455, mi R- 196a, miR-lSSb, miR-9, miR-191, miR-134, miR-222 and miR-127 for discriminating pHN patients from all HNSCC patients (P <0.0Q1). Area under the curve (AUC) = 0.983; sensitivity = 95.0%; specificity = 93.3%.

[0038] Figure 11 is a graphical representation showing ROC curve analysis of an independent validation study using salivary miR-2lQ _f miR-455, miR-i96a, miR- 196b, miR-9, miR-191, miR-134, miR-222 and miR-127 for discriminating pHN patients from HPV ^÷ HNSCC patients (P <0.00i). Area under the curve (AUC) = 1.00; sensitivity = 100.0%; specificity = 100.0%.

[0039] Figure 12 is a graphical representation showing ROC curve analysis of an independent validation study using salivary miR~210, miR-455, miR-1963,, miR-196b, miR-9, miR-191, rolR-134, miR-222 and miR-127 for discriminating pHIM patients from HPV HNSCC patients (P <0.001). Area under the curve (AUC) = 0.933; sensitivity = 80.0%; specificity = 100,0%.

DETAILED DESCRIPTION OF THE INVENTION

1, Definitions

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are deft below.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the a rtiele. By way of example, "an element" means one element or more than one element.

[0042] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0043] The term ^tt about ^ft _/ as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used In conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of iO%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by end points include all numbers and fractions subsumed within that range Ce.gr. , 1 to 5 includes 1,. 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about".

[0044] The term "accuracy", as used herein, means a statistical measure for the correctness of classification or identification of sample types. The accuracy is the proportion of true results (both true positives and true negatives).

[004S] The term "biomarker", as used herein, represents a characteristic that can be objectively measured and evaluated as an indicator of normal and disease processes or pharmacological responses. A biomarker is a parameter that can be used to measure the onset or the progress of disease or the effects of treatment. The parameter can be chemical, physical or biological. The term "surrogate biomarker", as used in the context of the present invention, represents a biomarker, whether alone or in

combination with other markers, intended to substitute for a clinical endpoint. It is a measure of a clinical condition or a measure of effect of a certain treatment that may correlate with the real clinical condition {e.g., healthy, or diseased such as HNC, HPV ⁺ HNC and HPV HNC) but doesn't necessarily have a guaranteed relationship. [0046] Throughout this specification, unless the context requires otherwise, the words comprise-!se\ ^' "comprises " .and "comprising" wilt be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0047] The term "carcinoma" would be understood by persons skilled in the art as tumor comprising cells derived from putative epithelial cells that have become malignant. The term also encompasses a carcinoma in situ, which is often used to describe a carcinoma in its pre-malignant stage; that is, having the cytological appearance of a malignant carcinoma but showing no signs of i nvasion through the epithelial basement membrane. Carcinomas are typically characterized by their histological appearance or their presumptive organ of origin. Persons skilled tn the art would be familiar with the different types of carcinoma. Bcamples include

adenocarcinoma {e.g., renal cell carcinoma, hepatocellular carcinoma), squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), adenosquamous carcinoma and basal cell carcinoma. The term carcinoma also refers to metastases derived from the primary tumor; that is, cells that have metastasized to other areas of the body, including those that may be found the circulation (e.g., circulating within blood vessels or the lymphatics). Thus, reference to a carcinoma is to be understood as a reference to the primary tumor and any metastases _* in some embodiments disclosed herein, the carcinoma is a squamous cell carcinoma. In non-limiting examples of this type, the carcinoma is a head and neck squamous ceil carcinoma.

[0048] The term "(clinical) condition" (biological state or health state), as used herein, means a status of a subject that can be described by physical, mental or social criteria. It includes so-called "healthy" and "diseased" conditions. For the definition of "healthy" and "diseased" conditions it is referred to the international classification of diseases (ICD) of the WHO (http://www.int/classrfJcations/icd/en/inde^ [00493 The term ^" control subject" as used in the context of the present invention, may refer to a subject known to be affected with a disease condition (e.g., HNC, HPV ⁺ HNC or HPV HNC, i.e., diseased) of disease susceptible cohditiori (e.g., pHN, /«.&, susceptible to disease) (positive control), or to a subject known to be not affected or diagnosed with the disease condition or with the disease susceptible condition (negative control), i.e., healthy. It may also refer to a subject known to be effected by another disease/condition (see definition "(clinical) condition"). It should be noted that a control subject that is known to be healthy, i.e., not suffering from the disease condition or with the disease susceptible condition, may possibly suffer from another disease not tested/known. The term "control population", as used herein, refers to a population of control subjects affected with the same disease condition Of disease susceptible condition (positive control population) or known to be not affected with the disease condition or with the disease susceptible condition (negative control population), it is also understood that the control subject, normal control, and healthy control, include data obtained and used as a standard, i.e. it can be used over and over again for multiple different subjects. In other words, for example, when comparing a subject sample to a control sample, the data from the control sample could have been obtained In a different set of experiments, for example, it could be an average obtained from a number of healthy subjects and not actually obtained at the time the data for the subject was obtained.

[0050] The term "correlating" generally refers to determining a relationship between one type of data with another or with a state. In various embodiments, correlating a miRNA profile with the presence or absence of a condition (e.g., a condition selected from a healthy condition, pHN, HNC, HPV ⁺ HNC or HPV- HNC) comprises determining the presence, absence or amount of at least one miRNA in a subject that suffers from that condition; or in persons known to be free of that condition, in specific embodiments, a profile of miRNA levels, absences or presences is correlated to a global probability or a particular outcome, using receiver operating characteristic (ROC) curves.

[0051] By "corresponding miRNA" or "corresponding miRNA biomarker" is meant a miRNA biomarker that is structurally and/or functionaify sirnilar to a reference miRNA biomarker. Representative corresponding miRNA biomarkers include expression products of allelic variants (same locus), homo!ogs (different locus), and orthologs (different organism) of reference miRNA biomarker genes. Nucleic acid variants of reference miRNA biomarker genes and encoded miRNA biomarker expression products can contain nucleotide substitutions, deletions, inversions and/or insertions. Generally, variants of a particular miRNA biomarker gene or expression product will have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as

determined by sequence alignment programs know in the a rthee art using default parameters. In some embodiments, the miRNA biomarker gene or expression product displays at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleotide sequence selected from any one of SEQ ID NO: 1.-9, Corresponding miRNA biomarkers also include nucleic acid sequences that hybridize to reference miRNA biomarkers, or to their complements, under stringent hybridization conditions, as described for example below,

[0052] The term "diagnosis" as used in the context of the present invention refers to the process of determining the presence or absence of a possible condition and therefore is a process attempting to define the (clinical) condition of a subject. The determination of the expression level of a set of miRNAs according to the present invention correlates with the (clinical) condition of a subject (e.g., one of healthy condition, pHN, HNC, HPV ⁺ HNC or HPV HNC),

[0053] The term -differential expression" of miRNAs as used herein, means qualitative and/or quantitative differences in the temporal and/or local miRNA expression patterns, e.g., between a biological sample taken from subjects with a condition as compared to a comparable sample taken from subjects lacking the condition. Thus, a differentially expressed miRNA may qualitatively have its expression altered, including an activation or inaetivation in, for example, saliva from a subject with a disease: condition (e.g., HNC, HP\T HNC or HPV- HNC) or disease susceptible condition (e.g., pHN) versus saliva from a healthy subject. The difference in miRNA expression may also be

quantitative, e.g., in that expression is modulated, i.e., either up-regulated, resulting in an increased amount of miRNA, or down -regulated, resulting in a decreased amount of miRNA. The degree to which miRNA expression differs need only be large enough to be quantified via standard expression characterization techniques, e.g., by quantitative hybridization (e.g., to a microarray, to beads), amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche 454 GS FL), flow cytometry (eg. LUMINEX, Firefly Bioworks) and the like. For example, a miRNA molecule is differentially expressed between the samples if the amount of the miRNA molecule in one sample is significantly different (i.e., p<0.05) from the amount of the miRNA molecule in the other sample. It should be noted that if the miRNA molecule or other marker is detectable in one sample and not detectable in the other, then the miRNA molecule can be considered to be differentially present.

[0054] The term "downregulated," "underexpressed" and the like refer to a downward deviation in the level of expression of a miRNA biomarker as compared to a baseline expression level of a corresponding miRNA biomarker in a control sample. [00553 The term "eariy diagnosis" as used herein with "early detection" refers to specific screening / monitoring processes that allow detection and evaluation of pHN, HNC, HPV ⁺ HNC or HPV HNC at an early point in disease development and/or

progression. For example, early detection of HNC, including HPV ⁺ HNC and HPV- HNC, can be achieved through a plurality of evaluations of patients with pHN to detect a transition to HNC.

[0056] The term ^expression" refers to the transcription and stable

accumulation of sense (mRNA) or functional RNA {e.g., miRNA). Expression may also refer to the production of protein. Thus, as will be dear from the context, expression of a coding sequence results from transcription and translation of the coding sequence.

Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0057] As used herein, the term ^ mi RNA gene expression product" is any product of transcription from an miRNA gene including, the primary transcript, the pri- miRNA, the pre-miRNA, miRNA*, or the mature miRNA.

[0058] As used herein,, "head and neck cancer" refers to a cancer or tumor that occurs in the neck and the head except for the brain and the eyes. In general, it includes oral cancer, paranasal sinus and nasal cancer, labial cancer, pharyngeal cancer, laryngeal cancer, head tumor, cancer of ears, cancer of nasopharynx, oropharynx, or hypopharyrtx, or cancer of salivary gland. Head and neck cancers may begin in the squamous cells that line the moist, mucosal surfaces inside the head and neck (for example, inside the mouth, the nose, and the throat). These squamous cell cancers may be referred to as squamous cell carcinomas of the head and neck. Head and neck cancers may also begin in the salivary glands. Salivary glands contain many different types of cells that can become cancerous, so there are many different types of salivary gland cancer.

[0059] The terms ^" "head and neck squamous ceil carcinoma" and "HNSCC" include, but are not limited to, cancers of the mouth, lip, nasal cavity, paranasal sinuses, pharynx, larynx, nasopharynx, throat and trachea.

[0060] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a PNA-DNA hybrid or a DNA-RNA hybrid.

Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with (3. In RNA, U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands.

Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. [0061] As used herein, the term "likelihood" is used as a measure of whether subjects with a particular miRNA profile actually have a condition (or not) based on a given mathematical model. An increased likelihood for example may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased risk may be expressed as simply determining the subject's level of a given miRNA and placing the test subject in an "increased risk" category, based upon previous population studies. Alternatively, a numerical expression of the test subject's increased risk may be determined based upon miRNA level analysis.

[0062] The terms "microRNA" or "miRNA" refer to single-stranded RNA molecules of about 21-22 nucleotides in length, though lengths of 16 and up to 35 nucleotides have been reported. MicroRNAs regulate gene expression and are encoded by genes from whose DMA they are transcribed but miRNAs are not translated into protein (i.e., miRNAs are non-coding RNAs). The genes encoding miRNAs are longer than the processed mature miRNA molecules, The miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70 nucleotide stem-loop structures known as pre-miRNAs in the ceil nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-mi RNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA- induced silencing complex (RISC). When Dieer cleaves the pre-miRNA stem-loop, two

complementary short RNA molecules are formed, but only one is integrated into the RISC. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5" end. The remaining strand, known as the miRNA", anti-guide (anti-strand)* or passenger strand, is degraded as a RISC substrate. Therefore, the miRNA"s are derived from the same hairpin structure like the "normal" miRNAs. So if the "normal ^* miRNA is then later called the ^,v mature miRNA" or "guide strand", the miRNA" is the "anti-guide strand" or

^passenger strand". Reference to specific miRNAs disclosed herein include miRNA molecules that correspond to those miRNAs. Such miRNA molecules are also referred to herein as ^tt cor responding miRNAs" or ^corresponding miRNA biomarkers".

[0063] The term "miRBase" refers to a well established repository of validated miRNAs. The miRBase (www.mirbase.org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references arid annotation. All sequence and annotation data are also available for download.

[0064] The terms "miRNA profile"' and "miRNA expression profile" are used interchangeably herein to refer to the determination of a miRNA expression level or a measure that correlates with the miRNA expression level in a biological sample (e.g. , saliva). The miRNA expression profile may be generated by any convenient means, e.g., nudeic acid hybridization (e.g., to a microarray, bead -based methods), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-j^roughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ΑΒΪ SOLID, Illumina Genome Analyzer, Roche/454 GS FLX), flow cytometry (e.g. LUMINEK, Firefly Bioworks) and the like, that allow the analysis of differential miRNA expression levels between samples of a subject with a first condition (e.g., pHN, HNC, HPV ⁺ HNC or HPV HNC) and a control subject (e.g., healthy). The sample material measure by the aforementioned means may be total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cONA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the

aforementioned RNA/DNA species. By determining the miRNA expression profile, each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA. As used herein, an miRNA profile represents the expression level/expression data of a single miRNA or a collection of expression levels of at least two miRNAs, suitably of least ¾ 3, 4, 5, 6, 7, 8, 9 or more, or up to all known miRNAs.

[0065] The term -neoplastic ceil" is used to denote a cell that shows aberrant cell growth. In some embodiments, the aberrant ceil growth of a neoplastic cell is increased cell growth. A neoplastic cell may be a hyperpiastic ceil, a cell that shows a lack of contact inhibition of growth in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastasis in vivo and that may recur after attempted removal.

[0066] As used herein, a "non-coding RNA" (ncRNA) is a functional RNA molecule that is not translated into a protein, Less-frequently used synonyms are non- protein-coding RNA (npcRNA), non-messenger RNA (nmRNA), small non-messenger RNA (snmRNA), functional RNA (fRNA). The term small RNA (sRNA) is often used for bacterial ncRNAs. The DNA sequence from which a non-coding RNA is transcribed as the end product is often called an RNA gene or non-coding RNA gene.

[0067] As used herein, the term "nucleotides" refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals: adenine, thymine, guanine, arid cytosine) l lecule of sugar and one of phosphoric acid. The term "nucleosides" refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phpsphorylated by specific kinases in the ceil on the sugar's primary alcohol group (- CH2-OH), producing nucleotides, which are the molecular building blocks of DNA and RNA.

[0068] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including eDNA, a DNA fragment, genomic DNA, synthetic (e.g. , chemically synthesized) DNA, plasmid DNA, mRNA, miRNA and anti-sense RNA, any of which can be single stranded or double stranded. The terms "nucleotide sequence" "nucleic acid", "nucleic acid molecule", "oligonucleotide" and ^polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides, and include RNA or DNA that is linear or branched, single or double stranded, Of a hybrid thereof. The term also encompasses RNA/DNA hybrids.. Nucleic acid sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR 1.821 - 1.825 and the World Intellectual Property Organteation (WIPO) Standard ST.25.

[0069] As used herein, the term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides and/or ribonucleotides,; and suitably more than three, Its exact size will depend upon many factors which in turn, depend upon the ultimate function and use of the oligonucleotide. The oligonucleotides may be from about 3 to about 1,000 nucleotides long. Although oligonucleotides of 5 to 100 nucleotides are useful in the invention, preferred oligonucleotides range from about 5 to about 15 bases in length, from about 5 to about 20 bases in length, from about 5 to about 25 bases in length, from about 5 to about 30 bases in length, from about 5 to about 40 bases in length or from about 5 to about 50 bases in length. More specifically, the detecting oligonucleotides molecule used for detecting the miRNA molecules fo the present invention may comprise any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26" 27, 28, 29, 30, 35, 40, 45, 50 bases in length. It should be further noted that the term "oligonucleotide" refers to 9 single stranded or double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally- occurring bases, sugars and covalent internueleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly. [0070] The term " precancer" refers to a lesion from which a malignant tumor is presumed to develop in a significant number of instances and that may or may not be recognizable clinically or by microscopic changes in the affected tissue. In particular, without being bound by the following, a precancer may be classified by: (1) evidence that the precancer is associated with an increased risk of cancer; (2) when a precancer progresses to cancer, the resulting cancer arises from cells within the precancer; (3) a precancer differs from the normal tissue from which it arises; (4) a precancer differs from the cancer into which it develops, although it has some, but not all, of the molecular and phenotypic properties that characterize the cancer; (5) there is a method by which the precancer can be diagnosed. In the context of the present invention, precancers of the head and neck include but are not restricted to leukoplakia, proliferative verruciform leukoplakia, erythroplakia, smokeless tobacco keratosis, lichen planus, submucous fibrosis and recurring melanotic macule.

[0071] By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3' end of the template sequence. In certain embodiments, primers can be large polynucleotides, such as from about 35 nucleotides: to several kilobases or more. Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. Desirably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-eomplementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non- complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.

[0072] As used herein, the term "probability" refers strictly to the probability of class membership for a sample as determined by a given mathematical model and is construed to be equivalent likelihood in this context.

[0073] "Probe" refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.

[0074] The term "receiver operating characteristic (ROC) curves" means a graphical measure of sensitivity (y-axis) vs. 1 -specificity (x-axis) for a clinical test. An important measure of the accuracy of the clinical test is the area under the ROC curve value (AUC value). If this area is equal to 1.0 then this test is 100% accurate because both the sensitivity and specificity are 1.0 so there are no false positives and no false negatives. On the other hand a test that cannot discriminate that is the diagonal line from 0,0 to 1,1. The ROC area for this line is 0.5. ROC curve areas (AUC-vaiues) are typically between 0,5 and 1.0, but also ROC values below 0,5 can - according to information theory - be as good, if the result is interpreted inversely. Therefore, according to the present invention an AUC -value close to 1 (e.g., 0.95) represents the same good measure for a clinical test as an AUC-value close to 0 (e.g., 0.05).

[0075] The terms "sample", "biological sample", and the like mean a material known or suspected of expressing or containing one or more sa livary miRNA or head and neck cancer biomarkers. A test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The sample is suitably derived from saliva, saliva extracts, saliva fractions, including cell fractions (e.g., comprising tumor cells) or lysates thereof, cell-free or ce!!-depfeted fractions, and the like. The sample can be treated prior to use, such as diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, extraction, concentration, inaettvation of interfering components (e.g., inhibiting nucleases such as RNases and DNases), the addition of reagents, and the like.

[0076] The term ^sensitivity", as used herein, refers to the probability that a diagnostic or predictive method or kit of the present invention gives a positive result when the sample is positive, e.g. , having the predicted diagnosis. Sensitivity is calculated as the number of true positive results divided by the sum of the true positives and false negatives. Sensitivity essentially is a measure of how weii the present invention correctly identifies those who have the predicted diagnosis from those who do not have the predicted diagnosis. The statistical methods and models can be selected such that the sensitivity is at least about 60%, and can be, e.g., at least about 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0077] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by~nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of

comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, Q, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Giy, Val _y Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity, For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2,5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0078] Terms used to describe sequence relationships between two or more polynucleotides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity" and "substantial identity", A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides, in length. Because two polynucleotides may each comprise (1) a sequence (/,e , only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two

polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a

"comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions {i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family .of programs as for example disclosed by Altschul et a/., 1997, Nucl, Adds Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., -'Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994- 1998, Chapter 15.

[0079] The term "specificity", as used herein, refers to the probability that a diagnostic or predictive method or kit of the present invention gives a negative result when the sample is not positive, e.g., not having the predicted diagnosis, Specificity is calculated as the number of true negative results divided by the sum of the true negatives and false positives. Specificity essentially is a measure of how we!i the present invention excludes those who do not have the predicted diagnosis from those who do have the predicted diagnosis. The statistical methods and models can be selected such that the specificity is at least about 60%, and can be, e.g. , at least about 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0080] The term "stringent hybridization conditions", as used herein, means conditions under which a first nucleotide sequence {e.g. , polynucleotide in its function as a probe for detecting a miRNA or miRNA*) will hybridize to a second nucleotide sequence (e.g., target sequence such as nucleotide sequence of a miRNA or miRNA*), such as in a complex mixture of nucleotide sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength, pH. The Trh may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20° C for short probes (e.g., about 10-35 nucleotides) and up to 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, SxSSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1% SDS at 65° C; or 6xSSPE, 10% formamide, 0.01%, Tween 20, O.lxTE buffer, 0.5 mg/mL BSA, 0.1 mg/mL herring sperm DNA, incubating at 42° C with wash in O.SxSSPE and 6xSSPE at 45° C.

[0081] The terms " subject", "individual" and ^patient" are used interchangeably herein to refer to any subject, particularly a mammalian subject. The subject may be diagnosed to be affected by a disease (e.g., HNC, HPV ⁺ HNC or HPV- HNC), or may be diagnosed to be not affected by the disease condition, i.e., healthy. Alternatively, the subject may be diagnosed at risk of developing HNC or a specific form of HNC (e.g., HPV ⁺ HNC or HPV- HNC) as it has been surprisingly found that miRNAs representative for pHN are present in saliva before HNC or a specific form of HNC (e.g., HPV ⁺ HNC or HPV- HNC) occurs. It should be noted that a subject that is diagnosed as being healthy, i.e., not suffering from HNC or a specific form of HNC (e.g., HPV ⁺ HNC or HPV HNC), may possibly suffer from another disease not tested/ known. The subject may be any mammal. The mammal is suitably a human. Alternatively, the mammal is a primate, a livestock animal (e.g., bovine, porcine, ovine, caprine, etc), a companion animal (e.g., canine, feline, etc.) or a laboratory animal (e.g., murine).

[0082] The terms "treat" and "treating" as used herein, unless otherwise indicated, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, either partially or completely, ameliorate or slow down (lessen) the targeted condition or disorder (e.g., pHN, HNC, HPV ⁺ HNC, HPV ^* HNC), or one or more symptom associated therewith. The terms are also used herein to denote delaying the onset of, inhibiting (e.g., reducing or arresting the growth of}, alleviating the effects of, or prolonging the life of a patient suffering from, cancer, in particular, HNC, preferably HNSCC including HPV ⁺ and HPV- forms thereof . Those in need of treatment include those diagnosed with the disorder, those suspected of having the disorder, those predisposed to have the disorder as well as those in whom the disorder is to be prevented. Hence, the subject to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder. In some embodiments, treatment refers to the eradication, removal, modification, or control of primary, regional, or metastatic cancer tissue that results from the administration of one or more therapeutic agents according to the methods of the invention. In other embodiments, such terms refer to the minimizing or delaying the spread of cancer resulting from the administration of one or more therapeutic agents to a subject with such a disease. In other embodiments, such terms refer to elimination of disease causing eel Is. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating. [00833 As used herein, the term ^treatment regimen" refers to prophylactic and/or prophylactic regimen (i.e., before the onset of HNC or specific form thereof, e.g., HPV ⁺ HNC or HPV HNC, e.g. - if the subject is affected with pHN), or to a therapeutic regimen (/.e„ after the onset of HNC or specific form thereof, e.g., HPV' HNC or HPV- HNC). The term "treatment regimen" encompasses natural substances and

pharmaceutical agents (i.e., "drugs") as well as any other treatment regimen including but not limited to chemotherapy, radiotherapy, proton therapy, immunotherapy, hormone therapy, phototherapy, cryotherapy, cryosurgery, toxin therapy or pro- apoptosis therapy, high intensity focused ultrasound, dietary treatments, physical therapy or exercise regimens, surgical interventions, and combinations thereof.

[0084] The term -unregulated," "overexpressed" and the like refer to an upward deviation in the level of expression of a miRNA biomarker as compared to a baseline expression level of a corresponding miRNA biomarker in a control sample,

2. Abbreviations

[0085] The followi ng abbreviations are used throughout the a pplication:

HNC * head and neck cancer

pHN ~ precancer of the head and neck

HPV ⁺ HNC = human papillomavirus positive head and neck cancer

HPV HNC = human papillomavirus negative head and neck cancer

HNSCC = head and neck squamous ceil carcinoma

ROC = receiver operating characteristic

h - hours

min = minutes

s = seconds

3. Salivary miRNA profiles and their uses

[0086] The present invention provides kits and methods that employ select miRNAs as biomarkers in determining whether a patient has a condition selected from a healthy condition, pHN, HNC, HPV ⁺ HNC Or HPV HNC, or at least aiding in that determination. The select miRNA biomarkers are conveniently obtained from patient saliva samples and can be assayed and compared to standards or controls as described herein. The miRNA biomarkers can be obtained and assayed in order to determine sensitivity and specificity for indicating the likelihood that a condition selected from a healthy condition, pHN, HNC, HPV ⁺ HNC or HPV HNC is present or absent in a patient. Once an increased likelihood of the presence of a disease condition {e.g., HNC, HPV ⁺ HNC or HPV HNC, i.e., diseased) or a disease susceptible condition (e.g., pHN, i.e., susceptible to disease) Has been identified through miRNA biomarker analysis, therapeutic intervention can be undertaken to treat the conditiQn.

[0087] The present discloses: nine miRNA biomarkers that are differentially expressed between healthy subjects and subjects affected by pHN, between pHN subjects and HNC affected subjects and between pHN subjects and subjects affected with different forms of HNC, including HPV ⁺ HNC and HPV- HNC. The miRNA biomarkers are suitably selected from miR-9, miR-127, miR-196a, miR-196b, miR-210, miR-455, miR- 191 and miR-222.

[0088] A miRNA biomarker may be determined to be differentially ^' expressed in a variety of ways, for example, between subjects or group of subjects with different conditions if the presence or absence or mean or median level or concentration of the biomarker in the different subjects or group of subjects is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wailis, Wilcoxon, Mann-Whitney and odds ratio.

[0089] In certain embodiments, the presence, absence, elevation, or reduction in the level of expression of a particular miRNA or set of miRNAs in a sample salivary miRNA profile is correlated with a condition, as compared to the expression level of that miRNA or set of miRNAs in a reference salivary miRNA profile. This correlation allows for diagnostic methods to be carried out when the expression level of an miRNA is measured in a sample miRNA profile being assessed and then compared to the expression level in a reference miRNA profile. It is specifically contemplated that miRNA profiles for patients, particularly those suspected of having a particular disease or condition such as a pHN, HNC, or particular forms of HNC including HPV ⁺ HNC and HPV" HNC, can be generated by evaluating any miRNA or sets of the miRNAs disclosed herein. The miRNA profile that is generated from the patient will be one that provides information regarding the particular disease or condition.

[0090] In some embodiments, the methods and kits of the present invention involve: (1) correlating a reference salivary miRNA profile with the presence of a condition selected from a healthy condition, pHN, HNC, HPV * HNC or HPV- HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA biomarker(s)) selected from the group consisting of: miR-9, miR-127, miR-134, miR-191, miR-196a, mi R- 196b, miR-210, miR-222 and miR- 455; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; and (3) determining a likelihood of the subject having or not having the condition based on the sample salivary miRNA profile and the reference salivary miRNA profile. [0091] In accordance with the present invention, miRNA profiles chafaeteristie of the conditions listed above and elsewhere herein are obtained from saliva samples, which offer an inexpensive, non-invasive and accessible bodily fluid to act as an ideal medium for investigative analysis- Representative saliva samples include whole saliva, a cell fraction of saliva,, and a ceil free or cell depleted fraction of saliva, or nucleic acid (suitably RNA) extracts thereof.

[0092] The level or abundance of mi RIM A can be assayed to detect differences in miRNA expression or levels between two samples, or a sample and a reference {e.g., a saliva sample reference or a digital reference representative of a condition). Specifically contemplated applications include identifying and/or quantifying differences between miRNA from a saliva sample that is normal {e.g. , healthy condition) and from a saliva sample that is not normal, between a cancerous condition (e.g., HNC, HPV ⁺ HNC or HPV- HNC) and a non-cancerous condition (e.g., healthy condition or pHN), between two differently treated samples (e.g., a pretreatment versus a post-treatment sample) or between cancerous samples with differing prognosis. Also, miRNA may be compared between a sample believed to be susceptible to a particular therapy, disease, or condition and one believed to be not susceptible or resistant to that therapy, disease, or condition. A sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition (e,g,, pHN, HNC, HPV ⁺ HNC or HPV- HNC) or one believed to be not normal with respect to that disease or condition. It may be compared to a saliva sample that is normal with respect to that disease or condition. Phenotypic traits include symptoms of a disease or condition of which a component is or may or may not be genetic or caused by a neoplastic cell or cells.

[0093] It is specifically contemplated that the invention can be; used to evaluate differences between pre-cancer (e.g., pHN) and cancer (e.g., HNC), or between different forms of cancer (e.g., HPV ⁺ HNC and HPV HNC).

[0094] Phenotypic traits also include characteristics such as longevity, morbidity, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity.

[0095] In certain embodiments, miRNA profiles are generated to evaluate and correlate those pnofiies with pharmacokinetics. For example, miRNA profiles may be created and evaluated for patient saliva samples prior to the patient being treated or during treatment to determine if there are miRNAs whose expression correlates with the outcome of treatment Identification of differential miRNAs can lead to a diagnostic assay involving them that can be used to evaluate saliva samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical tri l If iRNA profile is determined to be correlated with drug efficacy or drug toxicity that may be relevant to whether that patient is an appropriate patient for receiving the drug or for a particular dosage of the drug.

3.1 Evaluation of miRNA levels

[0096] The level or abundance of salivary miRNA biomarkers can be evaluated using any suitable assay. The assays may include internal or external standards to permit quantitative or semi-quantitative determination of those biomarkers, to thereby enable a valid comparison of the level of the salivary miRNA biomarkers in a sample with the corresponding salivary miRNA biomarkers in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols. In specific examples, absolute values for the level or functional activity of individual expression products are determined.

[0097] In semi-quantitative methods, a threshold or cut-off value is suitably determined, and is optionally a predetermined value. In particular embodiments, the threshold value is predetermined in the sense that it is fixed, for example, based on previous experience with the assay and/or a population of affected and/or unaffected subjects. Alternatively, the predetermined value can also indicate that the method of arriving at the threshold is predetermined or fixed even if the particular value varies among assays or may even be determined for every assay run.

[0098] In some embodiments, the level of a salivary miRNA biomarker is normalized against a housekeeping biomarker. The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., one or more RNA biomarkers), which are typically found at a constant level in the cell type(s) being analyzed and across the conditions being assessed.

[0099] Many methods exist for evaluating miRNA levels by amplifying all or part of miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and pri mary mi RN As, Suitable nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. In certain embodiments, more than one amplification method may be used, such as reverse transcription followed by real time PCR.

[0100] A typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species.A typical PCR reaction includes three steps: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PGR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence. Typical PGR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. Since mature miRNAs are single stranded, a reverse transcription reaction (which produces a complementary eDNA sequence) is performed prior to PGR reactions. Reverse transcription reactions include the use of, e,g>, a RNA-based DNA polymerase (reverse transcriptase) and a primer.

[0101] In PGR a nd q-PCR methods, for example, a set of primers is used for each target sequence. In certain embodiments, the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified, A primer is suitably about 15 to about 35 nucleotides in length. In illustrative examples, a forward primer can comprise at least one sequence that anneals to a target miRNA and alternatively can comprise an additional 5' non-complementary region. Suitably, a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA sequence, and multiple miMAs may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA.

[0102] In some embodiments, two or more miRNAs Or nucleic acids are amplified in a single reaction volume or multiple reaction volumes. Suitably, one or more miRNA or nucleic may be used as a normalization control or a reference nucleic acid for normalization. Normalization may be performed in separate or the same reaction volumes as other amplification reactions. One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least one miRNA of interest and at least one reference nucleic acid in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that uniquely binds each nucleic acid, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs. Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.

[0103] A single combined reaction for q-PCR, may be used to: (I) decrease risk of experimenter error, (2) reduce assay-to-assay variability, (3) decrease risk of target or product contamination, and (4) increase assay speed. The q RT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase. When two polymerases are used, a "hot start" approach may be used to maximize assay performance (U.S. Pat, HQS. 5,411 ,876 and 5,935,619). For example, the components for a reverse

transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Pat. Nos. 5,550,044, 5,413,924, and 6,403,341).

[0104] To assess the expression of microRNAs, real-time RT-PCR detection can be used to screen nucleic acids or RNA isolated from samples of interest and a related reference such as normal adjacent tissue (NAT) samples.

[0105] A panel of amplification targets is chosen for real-time RT-PCR quantification. The selection of the pane! or targets can be based on the results of microarray expression analyses, such as mirVana™ miRNA Bioarray VI , Ambion and miScript™, Qiagen, Suitably, the pane! of targets includes one or more miRNA described herein. One example of a normalization target is 5S rRNA and the small nucleolar RNA, SNORD96A), and others can be included. Reverse transcription (RT) reaction components are typically assembled on ice prior to the addition of RNA template. Total RNA template is added and mixed . RT reactions are incubated in an appropriate PCR System at an appropriate temperature ( 15-70° C, including all values and ranges there between) for an appropriate time, 15 to 30 minutes or longer, then at a temperature of 35 to 42 to 50° C for 10 to 30 to 60 minutes, and then at SO to 85 to 95° C for 5 minutes, then placed on wet ice. Reverse Transcription reaction components typically include nuclease- free water, reverse transcription buffer, dNTP mix, RT Primer, RNase Inhibitor, Reverse Transcriptase, and RNA.

[0106] PCR reaction components are typically assembled on ice prior to the addition of the cDNA from the RT reactions. Following assembly of the PCR reaction components a portion of the RT reaction is transferred to the PCR mix. PGR reaction are then typicaliy incubated in an PCR system at an elevated temperature (e.g. , 95° C) for 1 minute or so, then for a number of cycles of denaturing, annealing, and extension (e.g. , 40 cycles of 95° C for 5 seconds and 60° C for 30 seconds). Results can be analyzed, for example, with SDS V2.3 (Applied Biosystems). Real -time PCR components typically include Nuclease-free water, MgClj, PCR Buffer, dNTP mix, One or more primers, DNA Polymerase, cDNA from RT reaction and one or more detectable label.

[0107] Software tools such as NormFinder (Andersen ef a!, , 2004. Cancer Res. ,

64( 15) : 5245-5250) are used to determine targets for normalization with the targets of interest and sample set. For normalization of the real -time RT-PCR results, the cycle threshold (CO value (a log value) for the microRNA of interest is subtracted from the geometric mean C _t value of normalization targets. Fold change can be determined by subtracting the AQ normal reference (N) from the corresponding AQ sample being evaluated (T), producing a ΔΔC _t (T-N) value for each sample. The average MC _t (T-N) value across all samples is converted to fold change by 2ΔΔCt. The representative p-values are determined by a two-tailed paired Student's t-test from the ΔCt values of sample and normal reference.

[0108] In other embodiments, miRNA levels are evaluated using nucleic acid arrays. Accordingly, the present invention also contemplates the preparation and use of mi RIM A arrays or miRNA probe arrays, which are ordered macroarrays or miGroarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and are positioned on a support or support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.

Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.

[0109] Representative methods and apparatus for preparing a microarray have been described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231; 5,242,974;

5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807;

5,432,049; 5,436,327; 5,445,934; 5,468,613; 5,470,710; 5,472,672; 5,492,806;

5,503,980; 5,510,270; 5,525,464; 5,527,681; 5,529,756; 5,532,128; 5,545,531;

5,547,839; 5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732;

5,593,839; 5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134; 5,639,603;

5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695 _/ 940; 5,700,637;

5>744,3Q5; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928; 5,847,219;

5,876,932; 5,919,626; 6,004,755; 6,087,102; 6, 368,799; 6,383,745; 6,617,112;

6,638,717; 6,720,138, as well as VVO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO 96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO 09936760; WO0i38580; WO 0168255; WO 03020898; WO

03040410; WO 03053586; WO 03087297; WO 03091426; W0O31OOQ12; WO 04020085; WO 04027093; EP 373 203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of which are all herein incorporated by reference. Moreover, a person of ordinary skill in the art could readily analyze data generated using art array. Such protocols are disclosed above, and include information found in WO 9743450; WO 03023058; WO 03022421; WO 03029485; WO 03067217; WO 03066906; WO 03076928; WO 03093810 ; WO Q3100448A1, all of which are specifically incorporated by reference. [0110] After an array or a set of miRNA probes is prepared arid the miRNA in the sample is labeled, the population of target nucleic acids is contacted with the array or probes under hybridization conditions _/ where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et ah (In: Molecular doning: a laboratory manual, 3 ^rd Ed,, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001) and WO 95/21944. Of particular interest in embodiments of the present invention is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.

3.2 Determination of miRNA profiles

[0111] The expression levels of salivary miRNA biomarker in reference control groups (e.g., healthy control, pHN control, HNC control, HPV* HNC control and/or HPV ^* HNC control groups) are used to generate profiles of salivary miRNA biomarker levels reflecting differences between the level of individual miRNA biomarkers in one control group as compared to the level of corresponding miRNA biomarkers in another control group. Thus, a particular miRNA biomarker or corresponding miRNA biomarker may be more abundant or less abundant in one control group as compared to another control group. The profile may be represented as an overall signature score or the profile may be represented as a barcode or other graphical representation to facilitate analysis or diagnosis or determination of likelihood. The salivary miRNA biomarker levels from a test subject may be represented in the same way and the similarity with the signature score or level of *fff to a signature barcode or other graphical representation may be determined. In other embodiments, the levels of a particular salivary miRNA biomarker are analyzed and a downward or an upward trend in salivary miRNA biomarker level determined*

[0112] A salivary miRNA profile provides a compositional analysis (e.g., concentration or mole percentage (%) of respective salivary miRNAs) in which two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or a greater number of salivary miRNA biomarkers are evaluated.

[0113] The salivary miRNA profile can be quantitative, semi-quantitative and/or qualitative. For example, the salivary miRNA profile can evaluate the presence or absence of salivary miRNA b«omarkef(s), can evaluate the presence of salivary miRNA biomarker(s) above or below a particular threshold, and/or can evaluate the relative or absolute amount of salivary miRNA(s). In particular embodiments, a ratio among two, three, four or more salivary miRNA biomarkers is determined. Changes or perturbations in salivary miRNA biomarker ratios can be advantageous in indicating where there are blocks (or releases of such blocks) or other alterations in cellular pathways associated with a condition of the invention, response to treatment, development of side effects, and the like.

[0114] Representative differences in patterns of expression of individual salivary miRNA biomarkers of the invention between controi groups may include any one or more of the following :

[0115] Expression level of salivary miR-9 is: higher in HNC than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls; higher in HPV ⁺ HNC controls than in HPV- HNC controls.

[0116] Expression level of salivary mlR-127 is; higher in HNC controls than in healthy controls; higher in pHN controls than in healthy eontrols; higher in HPV- HNC controls than in pHN controls; lower in HPV ⁺ HNC controls than in HPV- HNC controls; lower in HPV ⁺ HNC controls than in pHN controls; higher In HPV ⁺ HNC controls than in healthy controls.

[0117] Expression level of salivary miR-134 is: lower in HNC controls than in healthy controls; lower in pHN controls than in healthy controls; lower in HPV- HNC controls than in pHN controls; lower in HPV ⁺ HNC controls than in pHN controls.

[0118] Expression level of salivary miR-196a is: higher in HNC controls than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls ; higher in HPV ⁺ HNC controls than in HPV- HNC controls.

[0119] Expression level of salivary miR~196b is: higher in HNC controls than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls; higher in HPV ⁺ HNC controls than in ΗΡΨ HNC controls.

[0120] Expression level of salivary miR-210 is: higher in HNC controls than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls; lower in HPV ⁺ HNC controls than in HPV- HNC controls; lower in HPV ⁺ HNC controls than in pHN controls; higher in HPV ⁺ HNC controls than in healthy controls.

[0121] Expression level of salivary m!R-222 is: higher in HNC controls than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls ; lower in HPV" HNC controls than in HPV- HNC controls ; higher in HPV ⁺ HNC controls than in pHN controls.

[0122] Expression level of salivary miR-455 is: higher in HNC controls than in healthy controls; higher in pHN controls than in healthy controls; higher in HPV- HNC controls than in pHN controls; higher in HPV" HNC controls than in pHN controls. [0123] The present invention thus provides several reference salivary miRNA profiles for determining the likelihood of the presence of a condition selected from a healthy condition, pHN, HNC, HPV' HNC, and HPV- HNC, which are also referred to herein as reference healthy salivary miRNA profile, reference pHN salivary miRNA profile, reference HNC salivary miRNA profile, reference HPV ⁺ HNC salivary miRNA profile, and reference HPV- HNC salivary miRNA profile, respectively.

[0124] The salivary miRNA patterns of expression or profiles thus enable methods of determining the likelihood of the presence of a condition, as broadly described above, which comprise: comparing the level of at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)) in a subject's sample salivary miRNA profile to the level of a corresponding salivary miRNA in a reference salivary miRNA profile selected from a reference healthy salivary miRNA profile, a reference pHN salivary miRNA profile, a reference HNC salivary miRNA profile, a reference HPV ⁺ HNC salivary miRNA profile, or a reference HNC salivary miRNA profile, a reference HPV- HNC salivary miRNA profile, wherein a similarity between the level of the at least one salivary miRNA in the sample salivary miRNA profile and the level of the corresponding salivary miRNA in the reference healthy salivary miRNA profile identifies that the subject has a salivary miRNA profile that correlates with the presence of a healthy condition, or alternatively the absence of pHN, HNC, HPV ⁺ HNC or HPV- HNC, wherein a similarity between the level of the at least one salivary miRNA in the sample salivary miRNA profile and the level of the corresponding salivary miRNA in the pHN salivary miRNA profile identifies that the subject has a salivary miRNA profile that correlates with the presence of pHN, or alternatively the absence of a healthy condition, HNC, HPV ⁺ HNC or HPV- HNC, wherein a similarity between the level of the at least one salivary miRNA in the sample salivary miRNA profile and the level of the corresponding salivary miRNA in the HNC salivary miRNA profile identifies that the subject has a salivary miRNA profile that correlates with the presence of HNC, or alternatively the absence of a healthy condition or pHN, wherein a similarity between the level of the at least one salivary miRNA in the sample salivary miRNA profile and the level of the: corresponding salivary miRNA in the HPV ⁺ HNC salivary miRNA profile identifies that the subject has a salivary miRNA profile that correlates with the presence of ^' HPV ⁺ HNC* or alternatively the absence of a healthy condition, pHN or HPV HNC, and wherein a similarity between the level of the at least one salivary miRNA in the sample salivary miRNA profile and the level of the

corresponding salivary miRNA in the HPV- HNC salivary miRNA profile identifies that the subject has a salivary miRNA profile that correlates with the presence of HPV- HNC, or alternatively the absence of a healthy condition, pHN or HPV ⁺ HNC.

[0125] In some embodiments, the instantly disclosed salivary miRNA

biomarkers are useful for assisting in disti between healthy subjects and Unhealthy subjects that have HNC (i.e., sick subjects with HNC or specific forms of HNC including HPV" HNC and HPV- HNC). Thus, in some embodiments, the methods and kits of the present invention involve determining the likelihood that HNC or a healthy condition a normal condition or a condition in which HNC is absent) is present or absent in a subject. These methods and kits generally comprise or involve: 1) providing a correlation of a reference salivary miRNA profile with the presence or absence of HNC or the healthy condition, wherein the reference salivary miRNA profile evaluates at least one (e.g., I, 2, 3, 4, 5, 6, 7, 8, 9 efc.) salivary miRNA biomarker selected from miR-9, rniR- 127, miR-134, miR-191, miR-196a, miR-196b, miR-210, miR-222 and miR-455; (2) obtaining a sample salivary miRNA profile from the subject, which evaluates for an individual salivary miRNA biomarker in the reference salivary miRNA profile a

corresponding salivary miRNA biomarker, and (3) determining a likelihood of the subject having or not having the healthy condition or HNC based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0126] In illustrative examples of this type, a reference healthy salivary miRNA profile comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 etc.) salivary miRNA biomarker that is downregulated or undefexpressed relative to a reference HNC salivary miRNA profile, illustrative examples pf which include: miR-9, miR-127, miR-191, miR- 196a, miR-196b, miR-210, miR-222 and miR-455.

[0127] In other illustrative examples, a reference healthy salivary miRNA profile comprises at least one salivary miRNA biomarker (i.e., miR-134) that is upregulated or overexpressed relative to a reference HNC salivary miRNA profile.

[0128] In still other illustrative examples, a reference healthy salivary miRNA profile comprises: (1) at least one salivary miRNA biomarker that is downregulated or underexpressed relative to a reference HNC salivary miRNA profile, as broadly described above and (2) at least one salivary miRNA biomarker that is upregulated or

overexpressed relative to a reference HNC salivary miRNA profile, as broadly described above.

[0129] In representative examples, a reference HNC salivary miRNA profile comprises at least one {e,g 1, 2, 3, 4, 5, 6, 7, 8, 9 etc.) salivary miRNA biomarker that is upregulated or overexpressed relative to a reference healthy salivary miRNA profile, illustrative examples of which include: miR-9, miR-127, miR-191, miR-196a, miR-196b, miR-210, miR-222 and miR-455.

[0130] In other representative examples, a reference HNC salivary miRNA profile comprises at least one salivary miRNA biomarker (i.e., miR-134) that is downregulated or underexpressed relative to a reference healthy salivary miRNA profile. [0131] In still other representative examples, a reference HNC salivary miRNA profile comprises: (1) at least one salivary miRNA biomarker that is upregulated or overexpressed relative to a reference healthy salivary miRNA profile, as broadly described above and (2) at least one salivary roiRNA biQmar¾er that is downregulated or underexpressed relative to a referefiee heaithy salivary miRNA profile, as broadly described above.

[0132] The instantly disclosed salivary miRNA bidmarkers have also been identified as being useful for assisting in distinguishing between healthy subjects, subjects with pHN as well as HPV' HNC and HPV- HNC affected subjects. Aeeorilingly, iri some embodiments, the methods and kits of the present invention are useful for determining the likelihood that pHN, HPV ⁺ HNC, HPV- HNC or a healthy condition (e.g., a normal condition or a condition in which pHN, HPV ⁺ HNC, HPV HNC are absent) is present or absent in a subject. These methods and kits generally comprise or involve: i) providing a correlation of a reference salivary miRNA profile with the likelihood of having or not having pHN, HPV ^" HNC, HPV- HNC or a heaithy condition, wherein the reference biomarker profile evaluates at least one (e,gu, 1, 2, 3, 4, 5, 6, 7, 8, 9 etc.) salivary miRNA biomarker selected from miR-9, miR-127, miR-134, miR-191, miR-196a, miR- 196b, miR-210, miR-222 and miR-455; (2) obtaining a sample salivary miRNA profile from the subject, which evaluates for an individual salivary miRNA biomarker in the reference salivary miRNA profile a corresponding salivary miRNA biomarker; and (3) determining a likelihood of the subject having or not having pHN, HPV ⁺ HNC, HPV- HNC or a healthy condition based on the sample salivary miRNA profile and the reference salivary miRNA profile.

[0133] In illustrative examples of this type, a reference healthy salivary miRNA profile comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 etc.) salivary miRNA biomarker that is downregulated or underexpressed relative to a reference pHN salivary miRNA profile, illustrative examples of which include: miR-9, miR-127, miR-191, miR-196a, miR- 196b, miR-210, miR-222 and miR-455.

[0134] In other illustrative examples, a reference healthy salivary miRNA profile comprises at least one salivary miRNA biomarker (/.e. , miR-134) that is upregulated or dvsrexpresseid relative to a reference pHN salivary miRNA profile.

[0135] In still other illustrative examples, a reference healthy salivary miRNA profile comprises: (1) at least one salivary miRNA biomarker that is downregulated or underexpressed relative to a reference pHN salivary miRNA profile, as broadly described above and (2) at least one salivary miRNA biomarker that is upregulated or

overexpressed relative to a reference pHN salivary miRNA profile, as broadly described above. [0136] In representative examples, a reference pHN salivary miRNA profile comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 etc.) salivary miRNA biomarker that is upregulated or overexpressed relative to a reference healthy salivary miRNA profile, illustrative examples of which include: miR-9, miR-127, miR-191, miR-196a, miR-196b, mfR-210, miR-222 and miR-455.

[0137] In other representative examples, a reference pHN salivary miRNA profile comprises at least one salivary miRNA biomarker (i,e, _f miR-134) that is

downregulated or underexpressed relative to a reference healthy salivary miRNA profile,

[0138] In still other representative examples, a reference pHN salivary miRNA profile comprises: (i) at least one salivary miRNA biomarker that is upregulated or overexpressed relative to a reference healthy salivary miRNA profile, as broadly described above and (2) at least one salivary miRNA biomarker that is downregulated or underexpressed relative to a reference healthy salivary miRNA profile, as broadly described above,

[0139] In illustrative examples, a reference pHN salivary miRNA profile comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 etc.) salivary miRNA biomarker that is downregulated or underexpressed relative to a reference HPV ^" HNC salivary miRNA profile, iilustrative examples of which include: miR-9, miR-127, miR-191, miR-196a, miR- 196b _f miR-210, miR-222 and miR-455.

[0140] In other illustrative examples, a reference pHN salivary miRNA profile comprises at least one (e.g., 1, 2 etc.) salivary miRNA biomarker that is upregulated or overexpressed relative to a reference HPV ^" HNC salivary miRNA profile, illustrative examples of which include: miR-127 and miR-210.

[0141] In Still other illustrative examples, a reference pHN salivary miRNA profile comprises: (1) at least one salivary miRNA biomarker that is downregulated or underexpressed relative to a reference HPV ^* HNC salivary miRNA profile, as broadly described above and (2) at least one salivary miRNA biomarker that is upregulated or overexpressed relative to a reference HPV ^" HNC salivary miRNA profile, as broadly described above.

[0142] In representative examples, a reference HPV ^" HNC salivary miRNA profile comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 etc.) salivary miRNA biomarker that is upregulated or overexpressed relative to a reference pHN salivary miRNA profile, iilustrative examples of which include: miR-9, miR-i27, miR-191, miR~196a, miR- 196b, miR-210, miR-222 and miR-455.

[0143] In non-limiting examples, a reference HPV HNC salivary miRNA profile comprises at least one {e.g., i, 2, 3, 4, 5 etc.) salivary miRNA biomarker that is upregulated or overexpressed relative to a reference HPV ^" HNC salivary miRNA profile, illustrative examples of which include: miR-9, miR-127, miR-191 , miR-196a, miR-196b, miR-210, miR-222 and miR-455,

[0144] In other non-limiting examples, a reference HPV ^÷ HNC salivary miRNA profile comprises at least one (e.g., 1, 2, 3 etc) salivary miRNA blomarker (that is downregulated or underexpressed relative to a reference HPV ^" HNC salivary miRNA profile, illustrative examples of which include: miR-127, miR-210 and miR-222.

[0145] As noted above, the level of an individual salivary miRNA in a salivary miRNA profile may be increased or decreased relative to the level of a corresponding salivary miRNA in another salivary miRNA profile, which is also referred to herein as "differential expression". The increase or decrease is suitably statistica lly significant.

[0146] Suitably, the level of an individual salivary miRNA is at least 110%> 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 2000%, 3000%, or 4000% (representing upregulation or overexpression) or no more than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0,5%, 0, 1%, 0.01%, 0,001% or 0.0001% (representing down regulation or

underexpression) of the level of a corresponding salivary miRNA.

[0147] Alternatively, the level of an individual salivary miRNA in a salivary miRNA profile may be similar to the level of a corresponding salivary miRNA in another salivary miRNA profile. In these embodiments, the level of an individual salivary miRNA in the salivary miRNA profile varies from the level of the corresponding salivary miRNA in the other salivary miRNA profile by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%.

[0148] In other embodiments, the methods of determining the likelihood of the presence or absence Of a condition, as broadly described above, comprise comparing the level of a first salivary miRNA in the sample salivary miRNA profile with the level of a second salivary miRNA in the sample salivary miRNA profile to provide a ratio and determining a likelihood of the presence or absence of the condition based on that ratio. In illustrative examples of this type, the determination is carried out in the absence of comparing the level of the first or second salivary miRJMA in the sample salivary miRNA profile to the level of a corresponding salivary miRNA in the reference salivary miRNA profile. Representative pairs of HNC btomarkers that are useful for these embodiments include: (1) a salivary miRNA that is downregulated or underexpressed in a reference HNC salivary miRNA profile, and a salivary miRNA that is upregulated or overexpressed in a reference HNC salivary miRNA profile; and (2) a first salivary miRNA that is

upregulated or overexpressed in a reference HNC salivary miRNA profile and a second salivary miRNA that is upreguiated of overexpressed in a reference HNC salivary miRNA profile, wherein first salivary miRNA Is expressed at a higher level than the second salivary miRNA in the reference HNC salivary miRNA profile. In illustrative examples of this type, the pairs of salivary miRNAs are suitably selected from miR-9 and mi R- 134, miR-122 and miR-134, miR-127 and mi R- 134, miR-191 and miRl-34, miR-l96a and miR-134, miR-196b and miR-134, miR-210 and miR-134, miR-222 and miR-134, miR- 455 and miR-134, miR-9 and miR-191, miR-122 and miR-191, miR-127 and miR-191, miR-196a and miR-191, miR-196b and miR-191, miR-210 and miR-191, miR-222 and miR-191, miR-455 and miR-191, miR-122 and miR-9, miR-127 and miR-9, miR-196a and miR-9, miR-196b and miR-9, miR-210 and miR-9, miR-222 and miR-9, and miR-455 and miR-9.

3.3 Assessing likelihood

[0149] The likelihood that a particular condition is present or absent in a subject is assessed by comparing the level or abundance of individual salivary miRNA biomarkers to one or more preselected or th reshold levels. Thresholds may be selected that provide an acceptable ability to predict diagnosis, prognostic risk, treatment success, etc. In illustrative examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population has a first condition or risk and a second population has a second condition or risk (called arbitrarily, for example, "healthy condition" and "pHN", "healthy condition" and "HNC", "healthy condition" and "HPV ⁺ HNC", "healthy condition" and "ΗΡV HNC, "pHN" and "HPV ⁺ HNC", "pHN" and "HPV HNC"," HPV ⁺ HNC" and "HPV HNC, or "low risk" and "high risk").

[0150] For any particular salivary miRNA biomarker, a distribution of salivary miRNA biomarker levels for subjects with and without a condition will likely overlap. Under such conditions, a test does not absolutely distinguish a first condition and a second condition with 100% accuracy, and the area of overlap indicates where the test cannot distinguish the first condition and the second condition. A threshold is selected, above which (or below which, depending on how a salivary miRNA biomarker changes with a specified condition or prognosis) the test is considered to be "positive" and below which the test is considered to be "negative,'' The area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, e.g., Hanley et al., Radiology 143: 29-36 (1982).

[0151] Alternatively, or in addition, thresholds may be established by obtaining an earlier biomarkef result from the same patient, to which later results may be compared. In these embodiments, the individual in effect acts as their own "control group." In biomarkers that increase with condition severity or prognostic risk, ail increase over time in the same patient can indicate a worsening of the condition or a failure of a treatment regimen, while a decrease over time can indicate remission of the condition or success of a treatment regimen.

[0152] In some embodiments, a positive likelihood ratio, negative iikeiihood ratio, odds ratio, and/or AUC or receiver operating characteristic (ROC) values are used as a measure of a method's ability to predict risk or to diagnose a condition. As used herein, the term "likelihood ratio" is the probability that a given test result would be observed in a subject with a condition of interest divided by the probability that that same result would be observed in a patient without the condition of interest. Thus, a positive likelihood ratio is the probability of a positive result observed in subjects with the specified condition divided by the probability of a positive results in subjects without the specified condition. A negative iikeiihood ratio is the probability of a negative result in subjects without the specified condition divided by the probability of a negative result in subjects with specified condition. The term "odds ratio," as used herein, refers to the ratio of the odds of an event occurring in one group {e.g., a healthy condition group) to the odds of it occurring in another group (e,g,> a pHN group, a HNC group, a HPV ⁺ HNC group, or a HPV ^* HNC group), or to a data-based estimate of that ratio. The term "area under the curve" or "AUC" refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures are useful for comparing the accuracy of a classifier across the complete data range.

Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., a healthy condition salivary miRNA profile and a pHN, HNC, HPV" HNC, or HPV HNC salivary miRNA profile). ROC curves are useful for plotting the performance of a particular feature (e.g. , any of the salivary miRNA biomarkers described herein and/or any item of additional biomedical information) in distinguishing or discriminating between two populations (e.g., cases having a condition and controls without the condition). Typically, the feature data across the entire population (e.g., the cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data are calculated. The sensitivity is determined by counting the number of cases above the value for that feature and then dividing by the total number Of cases. The specificity is determined by counting the number of controls below the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated In cases compared to controls, this definition also applies to scenarios in which a feature is lower in cases compared to the controls {in such a scenario, samples below the value for that feature would be counted). ROC curves can be generated for single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined {e.g., added, subtracted, multiplied, etc.) to produce a single value, and this single value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test. The ROC curve is the plot of the sensitivity of a test against the specificity of the test, where sensitivity is traditionally presented on the vertical axis and specificity is traditionally presented on the horizontal axis. Thus, "AUG ROC values" are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. An AUC ROC value may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wiicoxon test of ranks.

[0153] In some embodiments, a salivary rniRNA biomarker or a panel {e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or more) of salivary miRNA biomarkers is selected to discriminate between subjects with a first condition and subjects with a second condition with at least about 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% accuracy or having a C-statistic of at least about 0.50, 0.55, 0.60, 0-65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95.

[0154] In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "condition" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the condition group; and a value less than 1 indicates that a positive result is more likely in the control group. In this context, "condition" is meant to refer to a group having one characteristic (e.g. , the presence of a healthy condition, pHN, HNC, HPV ⁺ HNC, or HPV ^" HNC) and "control" group lacking the same characteristic. In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "condition" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the "condition" group; and a value less than 1 indicates that a negative result is more likely in the "control" group. In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the condition" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the "condition" group; and a value less than 1 indicates that a positive result is more likely in the "control" group. In the case of an AUC ROC value, this is computed by numerical integration of the ROC curve. The range of this value can be 0.5 to 1.0. A value of 0.5 indicates that a classifier (e,g,, a salivary miRNA profile) is no better than a 50% chance to classify unknowns correctly between two groups of interest, while 1.0 indicates the relatively best diagnostic accuracy. In certain embodiments, salivary miRNA biomarkers and/or salivary miRNA biomarker panels are selected to exhibit a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, at least about 2 or more or about 0.5 or less, at least about 5 or more or about 0.2 or less, at least about 10 or more or about O. i or less, or at least about 20 or more or about 0.05 or less.

[0155] In certain embodiments, salivary miRNA biomarkers and/or salivary miRNA biomarker panels are selected to exhibit an odds ratio of at least about 2 or more or about 0.5 or less, at least about 3 or more or about 0.33 or less, at least about 4 or more or about 0.25 or less, at feast about 5 or more or about 0.2 or less, or at least about 10 or more or about 0.1 or less.

[0156] In certain embodiments, salivary miRNA biomarkers and/or salivary miRNA biomarker panels are selected to exhibit an AUG ROC value of greater than 0,5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.

[0157] In some cases, multiple thresholds may be determined in so-called "tertile," "quartile," or "quintile" analyses. In these methods, the "diseased" and "'control groups" (or ^¾ high risk" and "low risk") groups are considered together as a single population, and are divided into 3, 4, or 5 (or more) "bins" having equal numbers of individuals. The boundary between two of these "bins" may be considered "thresholds," A risk (of a particular diagnosis or prognosis for example) can be assigned based on which "bin" a test subject falls into _*

[0158] In other embodiments, particular thresholds for the salivary miRNA biomarker(s) measured are not relied upon to determine if the biomarker !evel(s) obtained from a subject are correlated to a particular diagnosis or prognosis. For example, a temporal change in the biomarker(s) can be used to rule in or out one or more particular diagnoses and/or prognoses. Alternatively, salivary miRNA biomarker(s) are cofreiated to a condition, disease, prognosis, ere, by the presence or absence of one or more salivary miRNA biomarkers in a particular assay format. In the case of salivary miRNA biomarker panels, the present invention may utilize an evaluation of the entire profile of salivary miRNA biomarkers to provide a single result value {e.g. , a "panel response" value expressed either as a numeric score or as a percentage risk). In such embodiments, an increase, decrease, or other change (e.g. , slope over time) in a certain subset of salivary miRNA biomarkers may be sufficient to indicate a particular condition or future outcome in one patient, while an increase, decrease, or other change in a different subset of salivary miRNA biomarkers may be sufficient to indicate the same or a different condition or outcome in another patient.

[0159] In certain embodiments, a panel of salivary miRNA biomarkers is selected to assist in distinguishing a pair of groups (i.e., assist in assessing whether a subject has an increased likelihood of being in one group or the other group of the pair) selected from "healthy condition" and "pHN", "healthy condition" and "HNC", "healthy condition" and "HPV HNC", "healthy condition" and "HPV HNC ", "pHN" and "HPV HNC", "pHN" and "HPV HNC", "HPV ⁺ HNC" and "HPV HNC", or "low risk" and "high risk" with at least about 70%, 80%, 85%, 90% or 95% sensitivity, suitably in combination with at least about 70% 80%, 85%, 90% or 95% specificity, In some embodiments, both the sensitivity and specificity are at least about 75%, 80%, 85%, 90% or 95%.

[0160] The phrases "assessing the likelihood" and "determining the likelihood," as used herein, refer to methods by which the skilled artisan can predict the presence or absence of a condition (e.g., a condition selected from healthy condition, pHN, HNC,

HPV ⁺ HNC, or HPV- HNC) in a patient The skilled artisan will understand that this phrase includes within its scope an increased probability that a condition is present or absence in a patient; that is, that a condition is more likely to be present or absent in a subject. For example, the probability that an individual identified as having a specified condition actually has the condition may be expressed as a " positive predictive value" or "PPV." Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. PPV is determined by the characteristics of the predictive methods of the present invention as well as the prevalence ..of the condition in the population analysed. The statistical algorithms can be selected such that the positive predictive value in a population having a condition prevalence is in the range of 70% to 99% and can be, for example, at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0161] In other examples, the probability that an individual identified as not having a specified condition actually does not have that condition may be expressed as a "negative predictive value" or "NPV." Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predjctiye value is determined by the characteristics of the diagnostic or prognostic method, system, or code as well as the preva lence of the disease in the population analysed. The statistical methods and models can be selected such that the negative predictive value in a population having a condition prevalence is in the range of about 70% to about 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,

[0162] In some embodiments, a subject is determined as having a significant likelihood of having or not having a specified condition, By "significant likelihood" is meant that the subject has a reasonable probability (0.6, 0.7, 0.8, 0.9 or more) of having, or not having, a specified condition.

[0163] The salivary miRNA biomarker analysis of the present invention permits the generation. of high-density sets that can be evaluated using informatics approaches. High data density informatics analytical methods are known and software is available to those in the art, e.g,, cluster analysis (Pirouette, Informetrix), class prediction (SIMCA-P, Umetrics), principal components analysis of a computationally modeled dataset (SIMCA-P, Umetrics), 2D cluster analysis (GeneLinker Platinum, Improved Outcomes Software}, and metabolic pathway analysis

(biotech.icmb.utexas.edu). The choice of software packages offers specific tools for questions of interest (Kennedy et al., Solving Data Mining Problems Through Pattern Recognition. Indianapolis: Prentice Hall PTR, 1997; Golub et aU, (2999) Science 286; 531- 7) Eriksson et al., Multi and Megwari ate Analysis Principles and Applications: Umetrics, Umea, 2001). In general, any suitable mathematie analyses can be used to evaluate at least one {e.g., 1, 2, 3, 4, 5, 6 _» 7, 8, 9, etc.) salivary miRNA biomarker in a salivary miRNA profile with respect to a condition selected from healthy condition, pHN, HNC, HPV ^÷ HNC, or HPV- HNC. For example, methods such as multivariate analysis of variance, multivariate regression, and/or multiple regression can be used to determine

relationships between dependent variables (e.g., clinical measures) and independent variables (e.g., levels of salivary miRNA biomarkers). Clustering, including both hierarchical and non-hierarchical methods, as well as non-metric Dimensional Scaling can be used to determine associations or relationships among variables and among changes in those variables.

[0164] In addition, principal component analysis is a common way of reducing the dimension of studies, and can be used to interpret the variance-covariance structure of a data set Principal components may be used in such applications as multiple regression and cluster analysis. Factor analysis is used to describe the covartartce by constructing "hidden" variables from the observed variables. Factor analysis may be considered an extension of principal component analysis _* where principal component analysis is used as parameter estimation along with the maximum likelihood method. Furthermore* simple hypothesis such as equality of two vectors of means can be tested using Hotelling¾ T squareid statistic.

[0165] In some embodiments, the data sets corresponding to salivary miRNA profiles are used to create a diagnostic or predictive rule or model based on the application of a statistical and machine learning algorithm. Such an algorithm uses relationships between a salivary miRNA profile and a condition selected from healthy condition, pHN, HNC, HPV ⁺ HNC, or HPV- HNC observed In control subjects or typically cohorts of control subjects (sometimes referred to as training data), which provides combined control or reference salivary miRNA profiles for comparison with salivary miRNA profiles of a subject. The data are used to infer relationships that are then used to predict the status of a subject, including the presence or absence of one of the conditions referred to above.

[0166] Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the present invention. The data presented in the Tables, Figures and Examples herein has been used to generate illustrative minimal combinations of salivary miRNA biomarkers (models) that differentiate between two conditions selected from healthy condition, pHN, HNC, HPV" HNC, or HPV- HNC using feature selection based on AUC maximization in combination with support vector machine classification. Figure 10 illustrates the ability of each salivary miRNA bidmarker to i distinguish between at least two of the conditions. Illustrative models comprising at least about 2 salivary miRNA biomarkers were able to discriminate between two control groups as def i ned above with significantly improved positive predictive values compared to conventional

methodologies.

[0167] Thus, the salivary miRNA profiles disclosed herein enable the

assessment of likelihood that a particular condition is present or absent in a subject. For example, the likelihood that a healthy condition is present in a subject can be determined by detecting in the sample salivary miRNA profile a decrease in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference HNC salivary miRNA profile, wherein the salivary rniRNAs are selected from miR-9, miR-127, miR-191, miR-lsea, miR~196b, miR-210, miR-222 and miR-455. Alternatively or in addition, the likelihood that a healthy condition is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of mi R- 134, as compared to the level of a corresponding salivary miRNA in the reference HNC salivary miRNA profile.

[0168] In some embodiments, the likeiihQod that a healthy condition is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary mlRNA(s), as compared to the level of a corresponding salivary miRNA in ¾he reference pHN salivary miRNA profile, wherein the salivary rniRNAs are selected from miR-9, miR-127, miR-191, miR-196a, miR-196b, miR- 210, miR-222 and miR-455, Alternatively or in addition, the likelihood that a healthy condition is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of miR-134, as compared to the level of a corresponding salivary miRNA in the reference pHN salivary miRNA profile. [0169] In some embodiments, the likelihood that a healthy condition is present in a subject is determined by detecting in the sample salivary miRNA profile a similar level of at least I, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference healthy salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, mi R- 134, miR-191, miR-196a, miR-196b, miR-210, miR-222 and miR-455.

[0170] In some embodiments, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference healthy salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-191, miR-196a, miR~196b, miR-210, miR-222 and miR-455. Alternatively or in addition, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of miR-134, as compared to the level of a corresponding salivary miRNA in the reference healthy miRNA biomarker profile,

[0171] In some embodiments, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profiie a decrease in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary miRNA(s), as compared to the level of a

corresponding salivary miRNA in the reference HPV ^" HNC salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-191, miR-196a, miR-196b, miR-210, miR-222 and miR-455, Alternatively or in addition, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of miR-134, as compared to the level of a corresponding salivary miRNA in the reference HPV ^" HNC salivary miRNA biomarker profile,

[0172] In some embodiments, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of miR-210, as compared to the level of a corresponding salivary miRNA in the reference HPV ^÷ HNC salivary miRNA biomarker profile.

[0173] In certain embodiments, the likelihood that pHN is present in a subject is determined by detecting in the sample salivary miRNA profiie a similar level of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference pHN salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-134, miR-191, miR-195a, miR-196b, miR-210, miR-222 and miR-455.

[0174] In some embodiments, the likelihood that HNC is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference healthy salivary miRNA profile:, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-191, miR-i96a, miR-196b, miR-210, miR-222 arid miR-455, Alternatively or in addition, the likelihood that HNC is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of rniR-134, as compared to the level of a corresponding salivary miRNA in the reference pHN miRNA biomarker profile,

[0175] In certain embodiments, the presence or risk of development of HNC is determined by detecting in the sample salivary miRNA profile a similar level of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNACs), as compared to the level of a rorresponding salivary miRNA in in the reference HNC salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-134, miR-191, mi R- 196a, miR-196b, miR-210, miR-222 and miR-455.

[0176] In some embodiments, the likelihood that HPV HNC is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of at least 1, 2, 3, 4, 5, 6, 7 or 8 salivary rniRNACs), as compared to the level of a corresponding salivary miRNA in the reference pHN salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-191, miR-196a, miR~196b, miR- 210, miR-222 and miR-455, Alternatively or in addition, the likelihood that HPV HNC is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of miR-134, as compared to the level of a corresponding salivary miRNA in the reference pHN miRNA biomarker profile.

[0177] In certain embodiments _/ the likelihood that HPV- HNC is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of at least 1, 2, 3 or 4 salivary m{RNA(s), as compared to the level of a

corresponding salivary miRNA in the reference HPV ⁺ HNC salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-191, miR-196a and m|R-196b, Alternatively or in addition, the likelihood that HPV- HNC is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of miR-210 or miR22, as compared to the level of a corresponding salivary miRNA in the reference HPV ⁺ HNC miRNA biomarker profile.

[0178] In some embod i ments, the presence or risk of development of H PV ^* H NC is determined by detecting in the sample salivary miRNA profile a similar level of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in in the reference HPV- HNC salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-134, miR-191, mi R- 196a, miR-196b, miR-210, miR-222 and miR-455. [0179] In some embodiments, the likelihood that HPV ⁺ HNC is present in a subject is determined by detecting in the sample salivary miRNA profile an increase in the level of at least 1, 2, 3 or 4 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference HPV- HNC salivary mtRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-191, mi R- 196a arid miR-i96b.

Alternatively or in addition, the likelihood that HPV ⁺ HNC is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of at least 1, 2, 3 or 4 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in the reference pHN miRNA biomarker profile.

[0180] In certain embodiments, the likelihood that HPV ⁺ HNC is present in a subject is determined by detecting in the sample salivary miRNA profile a decrease in the level of at least 1, 2 or 3 salivary miRNACs}, as compared to the level of a corresponding salivary miRNA in the reference HPV- HNC salivary miRNA profile, wherein; the salivary miRNAs are selected from miR-127, miR-210 and miR-222.

[0181] In specific embodiments, the presence or risk of development of HPV ⁺

HNC is determined by detecting in the sample salivary miRNA profile a similar level of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s), as compared to the level of a corresponding salivary miRNA in in the reference HPV ⁺ HNC salivary miRNA profile, wherein the salivary miRNAs are selected from miR-9, miR-127, miR-134, miR-191, miR- 196a, miR-196b, miR-210, miR-222 and miR-455.

4. Kits

[0182] Any of the compositions or components described herein may be comprised in a kit. In non-limiting examples, reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array, nucleic acid amplification, and/or hybridization can be included in a kit, as well reagents for preparation of samples from saliva samples. The kit may further include reagents for creating or synthesizing miRNA probes. The kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. In certain aspects, the kit can include amplification reagents. In other embodiments, the kit may include various supports, such as glass, nylon, polymeric beads, magnetic beads, and the like, and/or reagents for coupling any probes and/or target nucleic acids. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support. [01833 Kits for implementing methods of the invention described herein are specifically contemplated. In some embodiments, there are kits for preparing: miRNA for multi-labeling and kites for preparing miRNA probes and/or miRNA arrays. In these embodiments, kits comprise, in suitable container means, i, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more of the following : (1) pofy(A) pofymerase; (2) unmodified nucleotides (G, A, T, C, and/or U); (3) a modified nucleotide (labeled or unlabeled); (4) poly(A) polymerase buffer; and, (5) at least one microfi!ter; (6) label that can be attached to a nucleotide; (7) at least one miRNA probe; (8) reaction buffer; (9) a miRNA array or components for making such an array; (10) acetic acid; (11) alcohol; (12) solutions for preparing, isolating, enriching, and purifying miRNAs or miRNA probes or arrays; (13) one Of more salivary miRNA biomarkers (which may be used as a positive control); (14) one or more primers that specifically hybridizes to salivary miRNA biomarker(s); (15) an enzyme suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq _t Sequenase™. DNA ligase etc. depending on the nucleic acid amplification technique employed); (16) deoxynucleotides. Other reagents may include those generally used for manipulating RNA, such as formamide, loading dye, ribonuclease inhibitors, and DNase,

[0184] In specific embodiments, kits of the invention include an array containing miRNA probes. An array may have probes corresponding to all known miRNAs of an mammal or a particular tissue, fluid or organ in particular conditions, or to a subset of such probes. The subset of probes on arrays may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application. For example, the array may contain one or more probes that is indicative or suggestive of (1) a disease or condition (pHN, HNC, HPV" HNC, HPV- HNC), (2) susceptibility or resistance; to a particular drug or treatment;; (3) susceptibility to toxicity from a drug or substance; (4) the stage of development or seventy of a disease or condition (one aspect of prognosis); (5) the likelihood of cancer recurrence (one aspect of prognosis) and (6) genetic predisposition to a disease or condition.

[0185] In other embodiments, kits of the invention include a set of miRNA primers. A set of miRNA primers may have primers for amplifying all known miRNAs of an mammal or of a particular tissue, fluid or organ in particular conditions, or to a subset of such probes. The subset of primers may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application. For example, the primer set may contain primers that specifically amplify salivary miRNA biomarkers that are

Indicative or Suggestive of (1) a disease Or condition (pHN, HNC, HPV ⁺ HNC, HPV- HNC), (2) susceptibility or resistance to a particular drug or treatment; (3) susceptibility to toxicity from a drug or substance; (4) the stage of development or severity of a disease or condition (one aspect of prognosis); (5) the likelihood of cancer recurrence (one aspect of prognosis) and (6) genetic predisposition to a disease or condition.

[0186] For arty kit embodiment, including an array or prime? ^' set, there can be nucleic acid molecules that contain or can be used to amplify a sequence that is a variant of, identical to or complementary to all of part of any of SEQ ID NOs described herein. Any nucleic acid discussed above may be implemented as part of a kit.

[0187] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0188] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.

[0189] However, the components of the kit may be provided as dried

powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent It is envisioned that the solvent may also be provided in another container means. In some embodiments^ labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500 _> 600, 700, 800, 900, 1000 ug or at least or at most those amounts of dried dye are provided in kits of the invention. The dye may then be resuspended in any suitable solvent, such as DMSO.

[0190] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid

formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. [0191] The kits of the present invention will also typically include a means for containing the vials in close confinement for commerci sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained,

[0192] Such kits may also include components that facilitate isolation of the labeled miRNA. It may also include components that preserve or maintain the miRNA or that protect against its degradation. Such components may be RNase-free or protect against RNases. Such kits generally will comprise, In suitable means, distinct containers for each individual reagent or solution.

[0193] A kit will generally also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented,

[0194] Kits of the invention may also include one or more of the following: Control RNA; nuclease-free water; RNase-free containers, such as 1.5 ml tubes; RNase- free elution tubes; PEG or d extra n; ethanol; acetic add; sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acid size marker; RNase-free tube tips; and RNase or DNase inhibitors.

[0195] It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.

5. Methods of treatment

[0196] The diagnostic methods of the present invention are also suitable for identifying patients that may require treatment; that is, patient stratification. Thus, the invention further provides methods for treating, preventing or inhibiting the development or progression of a condition in a subject wherein the condition is selected from p/HN, HNC, HPV ⁺ HNC Or HPV HNC (also referred to herein as "treatment methods"). These methods generally comprise^ consist or consist essentially of : exposing the subject to a treatment regimen for treating the conditon based on results obtained from any one or more of the methods of determining the likelihood of the presence or absence of the condition, as broadly described above and elsewhere herein (also referred to herein as "determination methods"), using a sample salivary miRNA profile from the subject for the determination, wherein the results indicate that the condition is present in the subject. In some embodiments, the treatments methods further comprise conducting a

determination method as broadly described above and elsewhere herein on the subject prior to exposing the subject to the treatment regimen.

[0197] Saliva samples or nucleic acid extracts thereof can be analyzed at the point of care or they can be sent to laboratories to conduct the analysis. Thus, in some embodiments, the determination method is performed by a person who exposes the subject to the treatment regimen. In other embodiments, a saliva sample from the subject or nucleic acid extract thereof is provided to another person {e.g., a person in a laboratory) who performs the determination; method and provides the results of the determination method to the person who exposes the subject to the treatment regimen.

[0198] In some embod intents the treatment methods comprise ; (1) providing a correlation of a reference salivary mi RN A profile with the presence of a condition selected from a healthy condition, pHN, HNC, HPV ⁺ HNC or HPV ^* HNC, wherein the reference salivary miRNA profile evaluates at least one salivary miRNA (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 salivary miRNA(s)}; (2) obtaining a sample salivary miRNA profile from a subject, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA in the reference salivary miRNA profile a corresponding salivary miRNA; (3) determining a likelihood of the subject having or not having the condition based on the sample salivary miRNA profile and the reference salivary miRNA profile, and (4) administering to the subject, on the basis that the subject has an increased likelihood of having pHN an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of pHN, or administering to the subject, on the basis that the subject has an increased likelihood of having HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of HNC> or administering to the subject, on the basis that the subject has an increased likelihood of having HPV ⁺ HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of HPV+ HNC, or administering to the subject, on the basis that the subject has an increased likelihood of having HPV- HNC an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the: development of HPV- HNC.

[0199] Following diagnosis, treatment is often decided according to the type of condition present in the subject. For example, if the subject is diagnosed with the likelihood that a precancer (i.e., pHN) is present in the subject, the subject is suitably exposed to a precancer treatment regimen (e.g., surgical excision of precancerous lesions, chemoprevention, etc.) or left untreated and monitored for any transition or progression of the precancer to cancer. In these embodiments the determination methods and kits of the present invention are typically used at a frequency that is effective to monitor the early development of HNC, HPV ⁺ HNC or HPV- HNC, to thereby enable early therapeutic intervention and treatment of those conditions. In illustrative examples, the determination methods or kits are used at least at 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hour intervals or at least 1, 2, 3, 4, 5 or 6 day intervals, or at least weekly> fortnightly or monthly. Accordingly, the present invention encompasses the use of the methods and kits of the present invention for early diagnosis of HNC, HPV ⁺ HNC and HPV HNC.

[0200] in other embodiments in which the subject is diagnosed with the likelihood that a cancer selected from HNC, HPV ⁺ HNC or HPV- HNC is present in the subject, the subject is suitably exposed to an appropriate treatment regimen.

Accordingly, the present invention also contemplates exposing the subject to a treatment regimen if the subject tests positive for the likelihood of the presence of a cancer selected from HNC, HPV ⁺ HNC or HPV ^* HNC. Non-limiting examples of such cancer treatment regimens include radiotherapy, surgery,, chemotherapy, hormone ablation therapy, pro-apoptosis therapy and immunotherapy.

[0201] Radiotherapies include radiation and waves that induce DNA damage for example, γ- irradiation. X-rays, UV irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DMA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.

[0202] Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks) _t to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0203] Non-limiting examples of radiotherapies include conformal external beam radiotherapy (50-100 Grey given as fractions over 4-8 weeks), either single shot or fractionated, high dose rate brachytherapy, permanent interstitial brachytherapy, systemic radio-isotopes (e.g., Strontium 89). in some embodiments the radiotherapy may be administered in combination with a radiosensitizing agent. Illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etanidazole, fluosol, misomdazqle, nimorazole, temopprfin and tirapazamine.

[0204] Chemotherapeutic agents may be selected from any one or more of the following categories:

[020S] (i) anti prol iferative/antineopi astic drugs and co mbi nations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carbopiatin, cyclophosphamide, nitrogen mustard, me!phalan, chlorambucil, busulphan and

nitrosoureas); antimetabolites (for example antifolates such as fluoropyridines like 5- fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and

hydroxyurea; anti -tumor antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin^ daunomycin, epirubicin, idarubicin, mitomycin -C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinoreibine and taxoids like paclitaxel and docetaxel; and topoisomerase inhibitors (for example epipodophyilotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin);

[0206] (ii) cytostatic agents such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), oestrogen receptor down regulators (for example fu!vestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorozo!e and exemestane) and inhibitors of 5a~reductase such as finasteride;

[0207] (iii) agents which inhibit cancer cell invasion (for example

metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

[020S] (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti- erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab

[C225]), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quin azolin-4-amine

(gefitinib, AZD1839), N-(3~ethyny!phenyl)-6,7-bts{2-methoxyethoxy)quina2olin-4-ami ne (erlotinib, OSI-774) and 6-acrylamido-N-(3-ch!oro-4-fluorophenyl)-7-(3- morpholinopropGxy)quinazori- n-4-amine (CI 1033)), for example inhibitors of the plateiet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family;

[0209] (v) anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endotheliai cei! growth factor antibody bevaeizumab [Avastin™], compounds such as those disclosed in

International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin ανβ3 function and angiostatin);

[0210] (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/021&6, WOOO/40529, WO 00/41669, WOOl/92224, WO02/04434 and WO02/08213; [0211] (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; and

[0212] (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacteria) nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy,

[0213] Immunotherapy approaches, include for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interSeukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine- transfected tumor cell lines and approaches using anti-idiotypic antibodies. These approaches generally rely on the use of immune effector cells and molecules to target and destroy cancer cells, The immune effector may be, for example, an antibody specific for some marker on the surface of a maiignant cell. The antibody alone may serve as an effector of therapy or it may recruit other ce!!s to actually facilitate cell killing. The antibody also may be conjugated to a drug or toxin (chemotrterapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a malignant cell target. Various effector cells include cytotoxic T cells and MK cells,

[0214] Examples of other cancer therapies include phototherapy, cryotherapy, toxin therapy or pro-apoptosis therapy. One of skill in the art would know that this list is not exhaustive of the types of treatment modalities available for cancer and other hyperplastic lesions.

[021S] Typically, therapeutic agents as described for example above will be administered in pharmaceutical compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of pHN, HNC or particular forms of HNC, including HPV ⁴ HNC and HPV ^" HIMC. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment or prevention of pHN, HNC or particular forms of HNC, including HPV ⁺ HNC and HPV- HNC, the medical practitioner may evaluate severity of any symptom associaftsd with the presence of pHN, HNC or particular forms of HNC, including HPV ⁺ HNC and HPV- HNC including the presence of lumps, sores that do not heal, sore throats that do not go away, difficulty in swallowing, a change or hoarseness in voice, inflammation, etc. in any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.

6. Methods of monitoring treatment

[0216] The present invention can be practiced in the field of predictive medicine for the purposes of diagnosis or monitoring the presence or development of a condition selected from pHN, HNC or particular forms of HNC, including HPV ⁴ HNC and HPV- HNC in a subject, and/or monitoring response to therapy efficacy.

[0217] The salivary miRNA profiles of the present invention further enable determination of endpoints in pharmacotranslational studies. For example, clinical trials can take many months or even years to establish the pharmacological parameters for a medicament to be used in treating or preventing pHN, HNC or pa rtieular forms of HNC, including HPV ⁺ HNC and HPV- HNC. However, these parameters may be associated with an salivary miRNA profile associated with a health state {e.g., a healthy condition).

Hence, the clinical trial can be expedited by selecting a treatment regimen (e.g., medicament and pharmaceutical parameters), which results in a salivary miRNA profile associated with the desired health state {e.g., healthy condition). This may be

determined for example by (1) providing a correlation of a reference salivary miRNA profile with the likelihood of having the healthy condition; (2) obtaining a corresponding salivary miRNA profile of a subject having pHN, HNC, or a particular form of HNC selected from HPV ^÷ HNC and HPV- HNC, after treatment with a treatment regimen, wherein a similarity of the subject's salivary miRNA profile after treatment to the reference salivary miRNA profile indicates the likelihood that the treatment regimen is effective for changing the health status of the subject to the desired health state {e.g. _f healthy condition). This aspect of the present invention advantageously provides methods of monitoring the efficacy of a particular treatment regimen in a subject (for example, in the context of a clinical trial) already diagnosed with a condition selected from pHN, HNC or particular forms of HNC, including HPV ⁺ HNC and HPV HNC. These methods take advantage of salivary miRNA biomarkers that correlate with treatment efficacy, for example, to determine whether the salivary miRNA profile of a subject undergoing treatment partially or completely normalizes during the course of or following therapy or otherwise shows changes associated with responsiveness to the therapy. [0218] The salivary rni RNA profiles further enable stratification of patients prior to enrolment in pharrr^cptransiationat studies. For example, a clinical trial cart be expedited by selecting a priori patients with a particular salivary miRNA profile that would most benefit from a particular treatment regimen (e.g., medicament and pharmaceutical parameters). For instance, patient enrolment Into a clinical trial testing the efficacy of a new HPV cancer therapeutic would best include patients with a salivary miRNA profile that indicated that they had HPV ⁺ HNC rather than HPV HNC, and as such the selected patients would most likely benefit from the new therapy.

[0219] Thus, the invention provides methods of correlating a reference salivary miRNA profile with an effective treatment regimen for a condition selected from pHN, HNC, or a particular form of HNC selected from HPV ⁺ HNC and HPV- HNC, wherein the reference salivary miRNA profile evaluates at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) salivary miRNA biomarker. These methods generally comprise: (a) determining a sample salivary miRNA profile from a subject with the condition prior to treatment (i.e., baseline), wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA biomarker in the reference salivary miRNA profile a corresponding salivary miRNA biomarker; and correlating the sample salivary miRNA profile with a treatment regimen that is effective for treating that condition.

[0220] The invention further provides methods of determining whether a treatment regimen is effective for treating a subject with a condition selected from pHN, HNC, or a particular form of HNC selected from HPV ⁺ HNC and HPV- HNC. These methods generally comprise: (a) correlating a reference salivary miRNA profile prior to treatment (i.e., baseline) with an effective treatment regimen for the condition, wherein the reference salivary miRNA profile evaluates at least one (e.g., X, 2, 3, 4, 5, 6, 7, 8, 9, etc.) salivary miRNA biomarker; and (b) obtaining a sample salivary miRNA profile from the; subject after treatment, wherein the sample salivary miRNA profile evaluates for an individual salivary miRNA biomarker in the reference salivary miRNA profile a

corresponding salivary miRNA biomarker, and wherein the sample salivary miRNA profile after treatment indicates whether the treatment regimen is effective for treating the condition jn the subject.

[0221] The invention can also be practiced to evaluate whether a subject is responding (i.e., a positive response) or not responding (i.e., a negative response) to a treatment regimen. This aspect of the invention provides methods of correlating a salivary miRNA profile with a positive and/or negative response to a treatment regimen. These methods generally comprise: (a) obtaining an salivary miRNA profile from a subject with a condition selected from pHN, HNC, or a particular form of HNC selected from HPV ⁺ HNC and HPV- HNC foiiowing commencement of the treatment regimen. wherein the salivary miRNA profile evaluates at least one {e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc,) salivary miRNA biomarker; and (b) correlating the salivary miRNA profile; from the subject with a positive and/or negative response to the treatment regimen.

[0222] The invention aiso provides methods of determining a positive and/or negative response to a treatment regimen by a subject with a condition selected from pHN, HNC, or a particular form of HNC selected from HPV ⁺ HNC and HPV HNC. These methods generally comprise: (a) correlating a reference salivary miRNA profile with a positive and/or negative response to the treatment regimen, wherein the reference salivary miRNA profile evaluates at least one (e.g., i, 2, %, 4, 5, 6, 7, 8, 9, etc.) salivary miRNA biomarker; and (b) determining a sample salivary miRNA profile from the subject, wherein the subject's sample salivary miRNA profile evaluates for an individual salivary miRNA biomarker in the reference salivary miRNA profile a corresponding salivary miRNA biomarker and indicates whether the subject is responding to the treatment regimen.

[0223] In some embodiments, the methods further comprise determining a first sample salivary miRNA profile from the subject prior to commencing the treatment regimen (/,e., a baseline profile), wherein the first sample salivary miRNA profile evaluates at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) salivary miRNA biomarker; and comparing the first sample salivary miRNA profile .with a second sample salivary miRNA profile from the subject after commencement of the treatment regimen, wherein the second sample salivary miRNA profile evaluates for an individual salivary miRNA biomarker in the first sample salivary miRNA profile a corresponding salivary miRNA biomarker. This aspect of the invention can be practiced to identify responders or non- responders relatively early in the treatment process, i.e., before clinical manifestations of efficacy. In this way, the treatment regimen can optionally be discontinued, a different treatment protocol can be implemented and/or supplemental therapy can be

administered. Thus, in some embodiments, a sample salivary miRNA profile is obtained within about 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, S weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, six months or longer of commencing therapy.

[0224] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES

EXAMPLE 1

SALIVARY MlCRORNA BlOMARKERS FOR DETECTION OF HEAD AND NECK CANCERS

Materials and Methods

Participants and samples

[0225] Unstimulated saliva samples were collected from HNSCC patients (n = 61, including the 5 samples used in the miRNA PCR arrays) at various stages (Π-IV) of the disease, who presented to the Head and Neck Clinic at the Princess Alexandra Hospital, Woolloongabba, Australia, Similarly,, saliva samples were col lected from healthy individuals (n -61, including the 5 samples used in the miRNA PCR arrays) with no previous history of any malignancy, and who were non- smokers with a good oral hygiene. All samples were immediately stored in dry ice and transferred to the laboratory for processing. The participants' demographics are listed in Table 1. All participants provided written informed consent before sample collection. This study was approved by the University of Queensland Medical Ethical Institutional Board and by the Princess Alexandra Hospital Ethics Review Board.

Table 1 : Participant demographics

Sample characteristics

[0226] Whole mouth resting saliva was collected in sterile 50 ml Falcon tubes

(Sarstedt, Australia) as reported before (IMagadia, R.„ et al., 2013. Ceil. Oncol. 36: 1-7; Mohamed, R., et ak, 2012. Gift. TransL Med, 1: 19). Cell-free salivary supernatants allow the detection of miRNAs that are secreted into the saliva, whereas whole mouth salivas allow the detection of both secretory and non-secretory miRNAs. Park et al. (Park, N.JL, et al, 2009, Clin, Cancer Res, 15:5473-5477) concluded from their work that the miRNA levels in salivary cell-free supernatants are lower than those in whole mouth salivas and, in addition, that more heterogeneous miRNA populations can be found in whole saliva. Moreover, Patel etal. (Patel, R.S, _f et al., 2011, Arch. Oral Biol. 56: 1506-1513) reported that they were able to obtain high resolution microRNA signatures from whole saliva samples. miRNA extraction and cDNA synthesis

[0227] QIAzoi lysis reagent (Qiagen, Valencia, CA, USA) was used as an extraction solvent to isolate total RNA and a NucleoSpin miRNA. kit (Macherey-Nagel, Duren, Germany) was used to enrich for rniRNAs, When using a NucleoSpin miRNA kit, there are 3 options: (I) to isolate total RNA (rniRNAs and large RNAs) in the same fraction, (it) to isolate miRNAs and large RNAs in two separate: fractions or (iii) to only isolate rniRNAs. Option (iii) was used. In brief, 800 μί. Q!Azo! lysis reagent was added to 200 pL whole saliva. This mixture was briefly vortexed and incubated for 5 min at room temperature (RT). Next, 200 pL chloroform was added, and the mixture was vortexed vigorously and incubated at RT for another 5 min. The samples were then centrifuged at 10,000 x g for 10 min at 4° C. The upper aqueous layer (800 pL) was transferred into a new microcentrifuge tube, another 200 pL chloroform was added and the mixture was vortexed again. After this, the samples were again centrifuged at 10,000 x g at 4° C. The upper aqueous layer (800 pL), containing total RNA, was carefully removed and transferred to a new microcentrifuge tube and 200 pL 100 % ethanol was added. This mixture was used for miRNA enrichment using a NucleoSpin miRNA kit. To this end, the solution containing total RNA was passed through a NucleoSpin column (blue ring) by centrifugation at 11,000 x g for 30 s at RT. The flow through was collected and 800 pL MX buffer was added. Next, the sample was loaded onto a NucleoSpin column (green ring) and centrifuged at 11,000 x g for 30 s at RT. The coiumns were then washed with 600 pL MWl buffer and centnfuged at 11,000 x g for 30 s. The wash was repeated with 700 pL MWl buffer followed by 250 pL MW2 buffer and centrifugation at 11,000 x g for 30 s. The coiumns were centrifuged at 11,000 x g for another 2 min to remove aft the ethanol. Then, the miRNA was eluted in 30 pL RNase free water. The quantity of the isolated miRNA was determined using a Nanodrop ND-1,000 spectrophotometer (Thermo scientific Wilmington DE), In supplementary Table 2 the concentrations of the rniRNAs are listed. QD 260:280 ratios≥ 1.8 were accepted as pure.

[0228] Using the isolated rniRNAs as template (input 250 ng), cDNA synthesis was carried out via a miScript II RT Kit with Hlspec buffer (Qiagen, Valencia, CA, USA) according to the manufacturers' instructions. In brief, rniRNAs were polyadenylated with polymerase A and transcribed into cDNA using oligo-dT primers (in parallel in the same tube). The oligo-dT had a S'degenerate anchor and a universal tag sequence at the 5'end allowing amplification of mature miRNA through a real-time PCR step. The

polyadenylation and universal tag primer ensure that genomic DNA is not amplified. Therefore, DNase treatment was omitted, miRNA PGR array assay

[0229] miScript™ niiRNA PCR arrays (Qiagen, catalogue number HXHS^lOZZ) were used; accords ng to the manufacturer's instructions. Briefly, 100 pL saliva: samples from five HMSCC patients and from five healthy controls: were pooled separateiy to reduce biological variation. From these two saliva sample pools, miRNA was extract&d and converted to eDNA as described above, Next, the cONA was diluted IMO in

RNAse/pN Ase free water and used as a template for miRNA FCR array amplification;, Before that, the PCR array plate was sealed and centrifuged at 1,000 x g for 1 min at RT to remove a i r b ub bles that may interfere with the FCR amplification process. Then, PCR feactiorts were performed using a thermo cycler £Bso"Rad, Hercules, CA, USA), The reaction mixtures were Incubated at 95° G for 10 min to activate the HptStart; DMA Taq polymerase" followed: by 40 cycles of 95° G for 15 s and 60 ⁶ C for 60 s, monitored by melt curve analysis- Ct values >35 were excluded from the analyses.

[0230] miScript™ miRNA PGR Arrays allow the profiling of 84 miRNAs that were selected based their presumed correlation with the diagnosis, staging, progression and/or prognosis of various cancers. A set of controls present on this array enables data analysis using the MCt method, the assessment of reverse transcription performance and the assessment of PCR performance. The endogenous ndrmaiizer SNORD96A (Choi _/ Y<, ef at, 2013. PL&S One &:e76fQl; 5.C. Sahu, microRNAs in: Toxicology and Medicine, 1st edri. (Wiley, 2013), pg 486) showed a relatively stable expression across control and HNSCC patient samples. The miScript miRNA PCR Array Data Analysis Web Portal

(http://pcrdataanalysis. sa biosciences.com/mi ma/arrayanalysis.php) was used to analyze the microarray data (Qiagen, Germany). [0231] To confirm the microarray- based expression data, saliva collected from

21 HNSCC patients and 21 healthy controls were first analyzed using RT-qPCR. Next, the cohorts of HNSCC patients and controls were expanded with 35 each fea validate the clinical utility of the identified mjRNAs. To this end, the cPN As were diluted 1 :10 in RNase/ONAse free water and used as a template for PGR amplification. The PCRs were run in. duplicate using a miScript Syber green PCR master mix (Qiagen, Valencia, CA, USA) arid carried out in a thermo cyder (Bio- Rad , Hercules, CA, USA) The reactioh mixtures were incubated at 95° C for 15 min to: activate the hot start Tag DIM

polymerase* followed by 40 cycles of 94 °C for 15 s, 55° C for 30s and 70° C for 30s, monitored by melt curve analysis. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (Bustiti, S. A., et ai, 2009. Clm, Chem, 55:611-622) , the threshold value (Q) was determined for each well and the values were averaged for each gene. Samples with C _t values > 35 were excluded from the analysis.

Independent validation usi ng TCGA data

[0232] Additionally,. The Cancer Genome Atlas (TCGA) mlRNAseq. data were used to validate the findings. In tn-s study, saliva samples were used for miRNA profiling. There are two ways by which tumour specific molecules can enter the saliva (i) via direct release or (ii) via. the secretion of exosomes/ microvesicles ( Carvalho, A.L, ei al., 2008. Clin. Cancer Res. 14:97-107; Ei-Mag.gar, A.K., eta/., 1995. Cancer Res, 55:2656-2659; Huang, M,F„ et a!,, 1999, Oral QncoL 35:296-301; Ei-Naggar, A.K„ et aL, 2001. J, Mot. Diagn, 3: 164-170). From the TCGA data portal (HNSCC cancer) ISIumina HiSeq mlRNAseq data was downloaded from 334 tumours and 39 norma! tissues, and performed PCA expression analysis (see below) for both tumour and normal tissues, Eight samples (4 from tumours and 4 from normal tissues) that were found to be outliers were excluded.

Statistical analysis

[0233] All statistical analyses were performed using GraphPad Prism 5 software version 5.03 (GraphPad Software Inc., USA). Data analyses were performed using the miScript mi R:N A PGR Array Data Analysis Excel web portal

{http://pcrdataanalysis.sabioseiences.com./mirna/arrayana lysis.php) _i including

quantification by the ΔΔΟτ method (Livak, K.J., et aL, 2001. Methods 25:402-408). Data normalization was performed using an endogenous control (SNORD96A) to correct for sample variation. miRNA expression levels were compared using the Mann Whitney Litest for non-parametric analysis. Receiver-operating characteristics: (ROC) curves were generated for five selected miRNAs to determine their clinical utility as diagnostic biomarkers. For TCGA data analysis, differential expression was evaluated on the reads per million counts (on a log 2 scale) using the Wilcoxon rank sum test. The BonferronJ method was used for multiple hypothesis correction.

Results

High miRNA yields from small saliva samples

[0234] A robust and reproducible method has been established herein to isolate high yield miRNAs from both fresh and archival saliva samples based on QIAzoi (Qiagen) extraction followed by solid phase enrichment of miRNAs on silica columns. This method allowed isolation of miRNAs from as iittie as 200 pL of whole mouth saliva. This method also turned out to be robust for isolating high yield miRNAs from archival saliva samples stored at: -80° C for up to 2 years. The amounts of miRNA isolated from 200 pL of saliva ranged from 11.50 ng to 644 ng. miRNA expression in saliva from HNSCC patients and healthy controls

[0235] After mi Script™ miRNA PCR Array based expression a nalysis using pooled samples- from five HNSCC patients and five healthy controls, a subset of five differentially expressed miRNAs (based on > 2-fold change) was selected for confirmation and validation in Independent cohorts using RT- qPCR. These miRNAs were: miR-9, mi.R- 127, mi R* 134, miR~191 and miR-222, For the confirmatory study saliva from 21 HNSCC patients and 21 healthy controls was used and for the validation study saliva from an independent cohort of 35 HNSCC patients and 35 healthy controls was used. When comparing the microarray and RT-qPCR results side- by-sice after normalizing both data sets with the same control (SNORD96A) it was found found that, with the exception of miR-191, in the confirmatory cohort (n∞ 21) all other miRNA microarray data were in agreement with the RT~qPCR data (Figure 1A). The average SNORD96A Ct values for the saliva collected from the healthy controls (C _t - 22.9) and the HNSCC patients (C _t = 22.5) were similar (Hann-Whitney U- Test), thus confirming Its applicability as a normalizer. In Figure IB the RT-qPCR based fold-changes for the five selected miRNAs are listed. miR- 127 (P = 0,07), miR-222 (P = 0, 11), mlR~19i (P < 0.01) and mlR-9 (P =0>68) were found to be υρ-regulated in the saliva from HNSCC patients as compared to the healthy controls (fold changes 2.3, 1.92, 8.2 and 3.7,, respectively). In contrast, miR-134 was ^• found to be down-regulated in the microarray data set but not in the RT- qPCR data set. In the second independent validation study (n = 35), except for miR-134, the other four miRNAs were consistently up -regulated in saliva from the HNSCC patients as compared to the saliva from the healthy controls (Figure 2A},

Independent validation using TCGA data

[0236] TCGA HNSCC miRNAseq data revealed that four of the five miRNAs; (miR-9, miR-127, miR -R-191 and mlR~222) were differentially expressed (P < 0.01) after multiple hypothesis correction. miR-9, .miR-191 and miRNA-222 showed a significant over- expression in- tumours derived from HNSCC patients compared to normal tissues collected from the same patients. In contrast to salivary miRNA expression data for miRNA 127 and miRNA 134, both of these miRNAs were higher in normal tissues as compared to tumour samples from the same patients (Figure 2B).

Discriminatory power of miR-9, miR-134 and miR-191

[0237] Receiver operating characteristic (ROC) curves were generated to evaluate the discriminatory power of miR-9, miR-134 and miR-191 for their ability to differentiate between control and disease groups. All three miRNAs were found to provide a good discriminative ability with AUC values of 0.85 (P <0.000l),. 0.74 (P < 0.001 ) and 0.98 (P <0,GGDi},, respectively. When combining ail three miRNAs, the AUC was 0.74 (P < 0,0001), In contrast,. miR-127 and miR-222 yielded AUCs values of 0,73 (P < 0,01} and 0.58 (P =0,24), respectively. Figure 3 shows ROC analyses for these three miRNAs using salivary samples from ail 56 HNSCC patients and healthy controls, Figure 4 depicts the signature cluster profiles of five selected salivary miRNAs,

[0238] The study also evaluated whether the miRNA expression profiles can discriminate HNSCC patients based on anatomical sites (Table 2). To this end, again ROCs were generated, It was found that the fold changes of miR-222 (P<0.05>, miR-127 (P<0.05) and miR-191 (P<0.G5) could differentiate HNSCC patients belonging to Group 1 and Group 4 (Group 1= oral cavity and Group 4=pharynx). The expression of miR~9 could <P<0.0S) differentiate HNSCC patients in Group 2 and Group 3 (Group

2~ oropharynx and Group 3 ^™ larynx) with an AUG of 0.77, Similarly, it was found that the expression of miR-191 could (P<0.05) differentiate HNSCC patients from Group 2 and Group 4 with an AUG of 0.94,· and that the .expression of miR-12? cou!d (P<0,0i) differentiate HNSCC patients from Group 3 and Group 4 with an AUG of 1,00.

Table 2: The fold-changes for the five miRNAs to discriminate the expression differences based on the anatomical sites,

DDscussion

[0239] Currently, the early diagnosis of head and neck squamous ceil carcinoma (HNSCC) is severely hampered by the lack of suitable piomarkers. A protocol has been; developed to isolate high yields of miRNAs from as little as 200 pL of fresh saliva, as well as from archival samples stored at --SOº C By using these samples, a novel salivary biomarfcer panel has been identified and validated, comprising miR-9, miR- 191 and miR-134, to reliably detect HNSCC, Through an independent validation using the TCGA data base, the clinical utility of this panel was further confirmed.

[0240] From the present study, it can be concluded that miR~9 _f miR-191 and miR-134 may serve as novel non-invasive biomarkers in HNSCC, Notably, the expression of salivary miR-9, miR-191 and miR-134 was independently validated using a HNSCC cohort consisting of 334 tumour samples and 39 normal adjacent tissues, curated in the TCGA database.

EXAMPLE 2

SALIVARY MICRORNA BIOMARKERS FOR DISTINGUISHING BETWEEN HEALTHY CONDITION PRECANCER OF THE HEAD AND NECK AND HPV ⁺ OR HPV ^" HEAD AND NECK CANCER

[0241] Using miScript™ miRNA PGR Arrays from Qiagen, a subset of five differentially expressed miRNAs (based on the fold change >2~foid) was selected to further validate them in independent cohorts, RNA input was normalized to endogenous SNORD96A and 5 miRNAs identified (hsa-miR-222-3p, hsa-miR-134, hsa-miR-i27-5p _f hsa-miR-191-5p and hsa-miR-9-5p) using the miScript™ miRNA PGR Arrays (cat number: MIHS-3102Z) and these were tested in a separate cohort of saliva samples (n~6G controls, precancer n=30 and HNSCC n~ 10G (HPV ⁺ and HPV ^" 50 each). Other miRNAs (hsa~miR-196a, hsa-miR-196b, hsa-miR-210 and hsa-miR-455) were based on the Cancer Genome Atlas Data base (TCGA).

[0242] All statistical analyses were performed using GraphPad Prism 5 software version 5.03 (GraphPad Software Inc., USA). Data anaiysis was performed using miScript miRNA PGR Array Data Analysis Excel web portal (http://

wvvw.sabiosciences.com/pcrarraydataanalysis.php). This automatically performs the quantification using the ΔΔC _Τ method (Livak, KJ., et aL, 2001. supra) to relatively quantify miRNAs. Data normalization was performed using endogenous control

(SNORD96A) to correct for sample variations. The expression ievels of miRNAs between the HNSCC patients and the controls were compared using the Mann Whitney U- test for non-parametric analysis. Receiver-operating characteristics (ROC) curves were generated for the five miRNAs to determine their clinical utility as diagnostic biomarkers.

[0243] The data presented in Table 3 were generated using the Qiagen miScript miRNA PGR Array Data Analysis Excel web porta! (http://

www. sabiQSCiences.com/pcrarraydataanaSysis.php). This software compares the average relative expression for controls and average relative expression for patients and calculates the ΔΔC _τ method. Table 3. Fold changes of fosa÷mi&~2l 0, nsa-miRr4S5, tisa-miR÷l 96a, hsa^mlR^i 96b, hsa-miR-9,†®a~rreR~i91, hsa~mtR-i34, --^^m^lllmKi- hss-rt^^-^yr^sUY^ to a healthy control group between pHN, HPV- HNSCC ahd HPV+ HNSCC

95% CI= 95% confidence intervals. Numbers In bold typeface represent upreguiated miRNAs compared with a healthy control group (control group expression is 1); Numbers in underlined typeface represent downregulated miRNAs compared with a healthy control group (control group expression is 1).

[0244] The results presented in Table 3 clearly show that hsa-miR-210, hsa- miR-455, hsa-miR-196a, hS3-m!R"i96b, hsa-miR-9, hsa-miR-191, hsa-miR-134, hsa- miR ^T -222 and hsa-miR-127 are differentially expressed between each of the conditions (i.e., healthy condition, pHN (i.e., precancer) HPV- HNSCC and HPV ⁺ HNSCC) and can thus be used to differentiate between them. These results are shown graphicaliy in Figure 5. The differentia] expression of the biomarkers is also presented in Figures 6 to 8 in the form of "Box and Whisker" plots where the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartiie (the median) of miRNA expression.

[0245] ROC curves were generated to evaluate the discriminatory power of hsa- miR-210, hsa-miR-455, hsa-miR-196a, hsa-miR-196b, hsa-rniR-9, hsa-miR-191, hsa- mtR-134, hsa-miR-222 and hsa-miR-127 for their ability to differentiate between healthy control and pHN (Figure 9), between pHN and HNSCC generally {i.e., regardless of HPV infection status) (Figure 10)., between pHN and HPV HNSCC (Figure 11) and between pHN and HPV HNSCC (Figure 12). All nine mtRNAs were found to provide excellent discriminative ability with AUG values of 0.962, 0,983, 1.000 and 0,933 ,. respectively.

[0246] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0247] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0248] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.