WO2025111485A9 - Point-of-care device for detecting oral cancer - Google Patents

Abstract

A method for detecting oral cancer in a subject can comprise: (a) providing a POC, lateral flow assay device comprising: a healthy channel and a suspect channel, each channel including first and second labeled binding ligands that specifically bind to BD-2 and BD-3, respectively, and a control indicator; first and second inlets in fluid communication with the suspect and healthy channels, respectively; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect and healthy samples to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (e) characterizing the subject as having oral cancer based on the BDI; wherein at least step (c) is performed at about room temperature.

Description

PATENT APPLICATION POINT-OF-CARE DEVICE FOR DETECTING ORAL CANCER RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Serial No.63/601,519, filed November 21, 2023, as well as U.S. Provisional Patent Application Serial No.63/559,996, filed March 1, 2024, the entirety of each of which is hereby incorporated by reference for all purposes. GOVERNMENT FUNDING [0002] This invention was made with government support under CA253108 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The present disclosure relates generally to diagnostic point-of-care (POC) devices, systems, and methods for detecting oral cancer and, in particular, to POC devices, systems, and methods based on a lateral flow assay for detecting the presence of oral cancer in a subject. BACKGROUND [0004] Head and neck cancer (HNC) is the 6th most prevalent malignancy in the world, and the third most common cancer in developing countries. HNC makes up approximately 5% of all cancers worldwide and 3% of all malignancies in the U.S. There are approximately 640,000 cases of HNC per year, resulting in about 350,000 deaths worldwide. In the U.S., ~65,000 new cases were estimated to have been diagnosed in 2020. Oral squamous cell carcinoma (OSCC) is the most frequent (approximately 90%) malignant tumor of HNC. It arises from the mucosal surfaces of the oral cavity, is often associated with excessive tobacco, alcohol and/or betel- nut usage, resulting in mutations of key tumor suppressor genes, primarily p53. The accurate diagnosis of early malignant or premalignant OSCC lesions remains challenging, as we currently rely on histology with inherent variability in inter-rater reliability. Moreover, biopsies are costly, invasive, and may lead to increased complications. Biopsies are also not feasible if repeated screenings of the same lesion are required. SUMMARY [0005] The present disclosure relates generally to diagnostic point-of-care (POC) devices, systems, and methods for detecting oral cancer and, in particular, to POC devices, systems, and methods based on a lateral flow assay for detecting the presence of oral cancer in a subject. [0006] One aspect of the present disclosure can include a POC, lateral flow assay device for detecting oral cancer in a subject. The device can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells. [0007] Another aspect of the present disclosure can include a system for detection of oral cancer in a subject. The system can comprise a POC, lateral flow assay device and a mobile device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI). The device can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells. [0008] Another aspect of the present disclosure can include a method for detecting oral cancer in a subject. The method can comprising the steps of: (a) providing a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (e) characterizing the subject as having oral cancer based on the BDI; wherein at least step (c) is performed at about room temperature. [0009] Another aspect of the present disclosure can include a method for detecting and treating oral cancer in a subject. The method can comprise the steps of: (a) providing a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; (e) characterizing the subject as having oral cancer if the BDI is greater than 1; and (f) treating the oral squamous cell carcinoma with a therapy selected from the group consisting of surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy; wherein at least step (c) is performed at about room temperature. [0010] Another aspect of the present disclosure can include a kit for detection of oral cancer in a subject. The kit can comprise: a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; and instructions for using the device for detection of oral cancer in the subject; optionally, means for obtaining healthy and suspect samples from the subject; optionally, separate cartridges for receiving healthy and suspect samples, each of which contains a cell lysis solution; optionally, means for delivering lysed suspect and healthy samples to the device; optionally, a mobile electronic device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI). BRIEF DESCRIPTION OF THE DRAWINGS [0011] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which: [0012] Fig.1A is a schematic illustration showing a point-of-care, lateral flow assay device for detecting oral cancer in a subject, constructed in accordance with one aspect of the present disclosure; [0013] Fig.1B is a schematic illustration showing an exemplary implementation the point-of-care, lateral flow assay device in Fig.1A; [0014] Figs.2A-E show immunofluorescence microscopy and flow-cytometry of DEFB103/hBD-3 and DEFB4/hBD-2. Expression of DEFB103 (Fig.2A) and DEFB4 (Fig.2B) across all TCGA cancer types (tumor [red] and normal [blue]) (Figs.2A-B). Immunofluorescence microscopic data for the expression of hBD-2 and -3 in 54 subjects (24, non-cancerous, 27 CIS and 3 OSCC). Data presented as mean immunofluorescence intensity (IMF) +SD. (p-value was calculated by non- parametric Mann-Whitney test) (Fig.2C). Representative immunofluorescence images of normal, CIS and OSCC samples; hBD-3: Green, hBD-2: Red and DAPI: Blue (Fig.2D). Representative flow-cytometric data showing hBD-3 and hBD-2 expressing cells in respective OSCC tumors excised from 3 additional patients (Fig. 2E); [0015] Figs.3A-F show cytological and flow-cytometric analysis of non-invasively collected cytobrushed cells. Cytological images of epithelial cells obtained by cytobrushing of cancerous (Fig.3A) and contralateral (Fig.3B) sites of the same patient. The cancer cells in Fig.3A (outlined in red) are present as three- dimensional clusters with marked variation in nuclear size and shape (blue arrows), and single cells and small clusters with abnormal cytoplasmic shapes (green arrows), and nuclear enlargement and multi-nucleation in occasional cells (black arrows). In contrast, the epithelial cells obtained from the contralateral site (Fig.3B) have features of normal superficial squamous cells, including single cells and cell clusters in flat sheets with small, uniform nuclei. Scale bar = 20 µm. Representative flow-cytometric data showing hBD-3 and hBD-2 expressing cells among E-cadherin+ epithelial cell population in non-invasively collected cytobrushed cells from cheek and tongue of a healthy subject (Fig.3C). Expression levels, as MFI (Mean Florescence Intensity) of hBD-3 and -2 in cytobrush samples collected from lesional and contralateral normal sites of OSCC patients (N=4). (p value was calculated by non-parametric Mann-Whitney test) (Fig.3D). Representative FACS data of one of the OSCC patients from Fig.3B (Pink: Cells collected from lesional site; Green: Cells collected from contralateral site; Maroon: Cells isolated from excised tumor of the same patient) (Fig.3E). The ratio of hBD-3 and hBD-2 in contralateral and lesional cytobrush samples (N=4). (p-value was calculated by non-parametric Mann-Whitney test) (Fig.3F); [0016] Figs.4A-C show Beta Defensin Index (BDI) study design. Cartoon presentation and formula for the BDI (Fig.4A). Proof of concept for the BDI. Cytobrush samples were collected from 4 OSCC and 4 non-cancerous lesions. Diagnosis was determined by biopsy followed by pathology review. BDI values, as determined in Fig.4A, were calculated following ELISA (p value was calculated by non-parametric Mann-Whitney test) (Fig.4B). Flow diagram of ELISA based BDI biomarker for proof of concept, discovery and validation studies. Pie chart shows the percentage of non-cancerous/benign (green) and cancerous lesions (orange) for each cohort. UC= University of Cincinnati, CWRU: Case Western Reserve University; WVU=West Virginia University (Fig.4C); [0017] Figs.5A-D show the BDI discovery and validation study. BDI values for 15 non-cancerous and 25 OSCC lesions from the discovery cohort. Black horizontal line for each cohort is the median BDI values. Dashed gray line is the cut-off value (1.25) for cancer diagnosis (p value was calculated by non-parametric Mann-Whitney test) (Fig.5A). Receiver Operating Curve (ROC) (Fig.5B). Sensitivity/specificity vs BDI curves for the discovery cohort (Fig.5C). BDI values for 23 non-cancerous and 29 OSCC lesions from the three validation cohorts (Blue: Internal Validation Cohort [N=21]; Green: External Validation Cohort 1 (N=19) and Orange: External Validation Cohort 2 (N=12)). Dashed gray line is the BDI cut-off value (1.25) for cancer diagnosis (Fig.5D). Diagnoses of all the lesions are found in Table 4; [0018] Figs.6A-D show one example of a microfluidic intact cell analysis (MICA) device. Schematic illustration of the MICA device with embedded micropillar arrays forming narrow openings from 160 μm down to 20 μm along the flow direction for cell aggregate filtering and individual cell retention (Fig.6A). Photograph of an assembled MICA device (Fig.6B). Cytobrush samples were collected from the lesion and the contralateral normal sites of patients, and pumped through micropillar arrays within respective troughs of the MICA device. Representative microscopic image showing typical distribution of the retained cells in the MICA microchannel (Fig.6C). The area within the dashed red lines (seen in Fig.6C) indicates the area of interest (Fig.6D); [0019] Figs.7A-C show MICA-BDI proof of concepts. Representative results of phase-contrast, hBD-2 and -3 fluorescence and DAPI staining of nuclei of the cells collected from a healthy participant’s right and left cheek. A violin plot showing no significant difference in hBD-3 to hBD-2 ratio in cells collected from left vs right side of the healthy participant (Fig.7A). Representative results of phase-contrast, hBD-2 and -3 fluorescence and DAPI staining of nuclei of the cells collected from a cancerous patient’s lesional and contralateral side. A violin plot showing a significantly higher hBD-3 to hBD-2 ratio in cells collected from the lesional side compared to the contralateral side (Fig.7B). Patients diagnosed with oral squamous cell carcinoma (OSCC) had significantly greater BDI values compared to healthy participants (p=0.033, Mann-Whitney, N=5 in each group) (Fig.7C); [0020] Figs.8A-D are a series of graphs showing TCGA data analysis. Expression of DEFB103 (Fig.8A) and DEFB4 (Fig.8B) across all TCGA cancer types (tumor (red) and normal (blue)) (as TPM, Transcript per million) in HNSC compared to normal samples in TCGA database). Differential expression of DEFB103 across different subtype of HNSC (Fig.8C). Differential expression of DEFB103 transcript across 4 different grades of HNSC and compared to normal samples. [* p<0.05] (Fig.8D); [0021] Figs.9A-H are a series of plots showing DEFB103 expression and correlation analysis. Expression of DEFB103 based on cancer stage (Fig.9A), patient’s gender (Fig.9B), age (Fig.9C) and race (Fig.9D). [*p<0.05]. Correlation of DEFB103 transcript with other genes in HNSC (Figs.9E-F). Differential expression of LCE3D and LCE3E between HNSC (T) and normal (N). [* p<0.05] (Fig.9G). Gene ontology (GO) analysis (Fig.9H), using rho value >0.7, to identify 38 positively correlated genes with DEFB103 (Fig.9F); [0022] Figs.10A-B are a series of plots showing BDI of tonsillar/oropharyngeal cytobrush samples and salivary levels of beta-defensins. BDI values of 5 benign and 7 cancerous lesions obtained from tonsillar/oropharyngeal region (Pathological diagnosis of each subjects are also shown on left) (p=0.03; Mann-Whitney test) (Fig. 10A). Salivary levels of hBD-2 , hBD-3 and ratio of hBD-3 to hBD-2 in cancerous (OSCC, N=17) and non-cancerous (N=13) subjects. (ns= Not significant (non- parametric Mann-Whitney test) (Fig.10B); [0023] Figs.11A-E are a series of plots showing BDI with respect to smoking status, gender, anatomical locations and OSCC Stages. Distribution of BDI values based on smoking status (Fig.11A) and gender (Fig.11B). NS= Not Significant (Mann-Whitney test). Pie charts showing the percentage of OSCC (left panel) and non-cancerous (right panel) lesions based on anatomical sites within the oral cavity where they appeared (Fig.11C). The BDI based on anatomical locations (Cheek, tongue, and others). (ns=not significant, Kruskal–Wallis test) (Fig.11D). BDI values with respect to cancer stages. (ns= not significant; Kruskal–Wallis test) (Fig.11E); [0024] Figs.12A-E are a series of charts showing BDI benchmarking. Fig.12A shows BDI benchmarking against published OSCC biomarkers and in use (and/or published) OSCC biomarker platforms: IL-8: Interleukin 8; IL-1B: Interleukin 1 Beta, DUSP: Dual-specificity phosphatase; S100P: Calcium-Binding Protein S100P, solCD44: Soluble CD44, CPLANE1: Ciliogenesis and planar polarity effector complex subunit 1; NUS1+RCN1: Nuclear Undecaprenyl Pyrophosphate Synthase 1 + Reticulocalbin 1; GAS6: Growth Arrest Specific 6, LBC: Liquid based cytology, CDOT: Cancer Detect for Oral & Throat cancer POCOCT: Point Of Care Oral Cytology Test. Detection methods for each of the biomarkers/platforms are described in Table 7. Figs.12B-E show a comparison of sensitivity and specificity values (with 95% confidence intervals, when available) of BDI with published OSCC biomarkers and in use (and/or published) OSCC biomarker platforms. Data for IL-8, IL-1B, S100P, DUSP1, vizilite, velscope and CDx are from multiple single studies, POCOCT and BDI are single multi-center studies, while the rest are from single center studies. Note: In Figs.12B and 12C, black circles represent salivary biomarkers and black squares represents serum biomarkers. In Figs.12D and 12E, green circles represent commercially available/in use platforms; [0025] Fig.13 shows detection of epidermal growth factor receptor (EGFR) by MICA. Representative phase-contrast and fluorescent images of the cells collected from either the lesion site or the contralateral normal site of patients. Scale bars represent a length of 100 μm; [0026] Fig.14 is a flow chart of image analysis and BDI generation; and [0027] Fig.15 is a schematic illustration showing another exemplary implementation of a single lane or channel of the point-of-care, lateral flow assay device in Figs.1A-B. DETAILED DESCRIPTION [0028] Definitions [0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the present disclosure pertains. [0030] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step. [0031] In the context of the present disclosure, the term “about”, when expressed as from “about” one particular value and/or “about” another particular value, also specifically contemplated and disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub- ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed. [0032] Optionally, in some aspects, when values or characteristics are approximated by use of the antecedents “about,” “substantially,” or “generally,” it is contemplated that values within up to 15%, up to 10%, up to 5%, or up to 1% (above or below) of the particularly stated value or characteristic can be included within the scope of those aspects. [0033] As used herein, phrases such as “between X and Y” and “between about X and Y” can be interpreted to include X and Y. [0034] As used herein, phrases such as “between about X and Y” can mean “between about X and about Y”. [0035] As used herein, phrases such as “from about X to Y” can mean “from about X to about Y”. [0036] It will be understood that when an element is referred to as being “on”, “attached” to, “connected” to, “coupled” with, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature. [0037] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms can encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. [0038] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts/steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise. [0039] As used herein, the terms “optionally” and “optional” can mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present. [0040] As used herein, the term “diagnosis” can encompass determining the nature of disease in a subject, as well as determining the severity and probable outcome of disease or episode of disease and/or prospect of recovery (prognosis). “Diagnosis” can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy (e.g., adjustment of dose and/or dosage regimen), and the like. [0041] As used herein, the term “epithelial cell” can refer to a cell comprising epithelial tissue in the body of a subject, and which may be found, for example, on the outside surface of skin, the linings of all digestive organs, the lining of the heart, the inner and outer membranes of the respiratory system, vessel walls, and the internal body cavities. The types of epithelial tissue, and thus epithelial cells, include simple squamous, stratified squamous, simple cuboidal, stratified cuboidal, simple columnar, and stratified columnar. Unique types of epithelial tissue include pseudostratified epithelium and transitional epithelium. Epithelial cells tightly connected to each other via various types of junctions. They have an apical surface that is exposed to a lumen or outside environment. They have a basal surface that is connected to basement membrane, which adheres them to the underlying structure. Non-limiting functions of epithelial cells include absorbing nutrients, providing mechanical protection, secreting various substances, and providing a matrix to house sensory receptor cells. [0042] As used herein, the term “intact epithelial cell” can refer to an epithelial cell that is mechanically intact, not disrupted, nor collapsed or burst. [0043] As used herein, “oral cancer” means any of the following: oral carcinogenesis, oral squamous cell carcinoma (OSCC), oral cancer, pre-cancer, pre- malignant lesion, oral adenocarcinoma, squamous cell carcinoma of the head and neck (SCCHN), any cytological or genetic abnormality of an oral cell, and any disease or disorder of oral cells. [0044] Also, as used herein, ”epithelial cancer” can refer to any cancer derived from epithelial cells. This group includes many of the most common types of cancer, include nearly all those developing in the skin, head and neck, breast, prostate, lung, pancreas, and colon. A common type of epithelial cancer is squamous cell carcinoma. In some embodiments, the methods of the present disclosure are directed to detecting epithelial cancer at a site of origin, while in other embodiments of the methods are directed to detecting epithelial cancer that has migrated as a result of metastasis. In some embodiments, the methods of the present disclosure are directed to detecting epithelial cancer selected from the group consisting of head and neck cancer, oral cancer, esophageal cancer, and skin cancer. Head and neck cancer is cancer that starts in the lip, oral cavity (mouth), nasal cavity (inside the nose), paranasal sinuses, pharynx, larynx or parotid glands. Most head and neck cancers are biologically similar. 90% of head and neck cancers are squamous cell carcinomas, so they are called head and neck squamous cell carcinomas (HNSCC). These cancers commonly originate from the mucosal lining (epithelium) of these regions. Oral cancer or mouth cancer is a type of head and neck cancer, and includes any cancerous tissue growth located in the oral cavity. Typically, oral cancer is oral epithelial cancer. Finally, methods of the present disclosure can also be used to detect epithelial skin cancer, which is epithelial cancer that arises from the skin, including basal-cell carcinoma and squamous-cell carcinoma. [0045] As used herein, the terms “subject” and “patient” can be used interchangeably and refer to a vertebrate, such as a mammal (e.g., a human). Mammals can include, but are not limited to, humans, dogs, cats, horses, cows, and pigs. [0046] As used herein, “beta defensin” or “BD” can refer to a class of cationic antimicrobial peptides that play an important role in innate and adaptive immunity, as well as other non-immunological processes (Machado L R, Ottolini B, Front Immunol., 6:115 (2015)). Defensins are an ancient and diverse family of proteins, and are present in most multicellular organisms. The amino acid sequence for human BD-2 and BD-3 are known (see Schroder J M, Harder J., Int J Biochem Cell Biol., 31(6):645-51 (1999) and Harder et al., J Biol Chem.276(8):5707-13 (2001), respectively), and their 3D structures have been determined (Schibli et al., J. Biol. Chem.277, 8279-89 (2002) and Bauer et al., Protein Sci.10, 2470-9 (2001)). In addition, beta defensins have been characterized in a wide variety of other species, such as birds, fish, and pigs. One skilled in the art can therefore conduct cancer diagnosis using corresponding beta defensins and beta defensin orthologs in a variety of species. As such, a beta defensin (e.g., BD-2 and BD-3) detectable by the devices and methods of the present disclosure can be associated with any one of a variety of species including, but not limited to, humans, birds, fish, pigs, dogs, cats, and other livestock. [0047] Point-of-care (POC), lateral flow assay devices [0048] As shown in Fig.1A, one aspect of the present disclosure can include a POC, lateral flow assay device 10 for detecting oral cancer in a subject. [0049] In one embodiment, a lateral flow assay may be employed in a POC device 10 to detect the presence or absence of beta definsin-2 (hBD-2) (e.g., human BD-2 or hBD-2) and beta definsin-3 (BD-3) (e.g., human BD-3 or hBD-3) within a test or biological sample. As discussed in more detail below, the readout may be done visually, i.e., presence or absence of one or more colored test lines (also referred to as test stripes), and the confirmation/validation of the test may be done by the presence and/or absence of one or more colored indicator lines/stripes. The test may be qualitative (presence or absence) as well as quantitative, and the detection/quantification may be aided by reading equipment, or can be purely visual detection by the eye of the user of the lateral flow assay. [0050] If desired, a reading device, such as an optical reader may be used in some embodiments to measure the intensity of the binding ligands. The actual configuration and structure of the optical reader may generally vary depending on the binding ligands, which are to be measured. For example, optical detection techniques that may be utilized include, but are not limited to, luminescence (e.g., fluorescence, phosphorescence, etc.), absorbance (e.g., fluorescent or non- fluorescent), diffraction, and so on. Qualitative, quantitative, or semi-quantitative determination of the presence or concentration of hBD-2 and hBD-3 may be achieved in accordance with the present disclosure. For instance, the amount of hBD-2 and hBD-3 may be quantitatively or semi-quantitatively determined by using the intensities of the signals produced by binding ligands bound to hBD-2 and hBD-3 present in a test or biological sample. [0051] Referring to Fig.1A, the device 10 can comprise a housing 12 that includes: a healthy channel 14 and a suspect channel 16, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator 18; a first inlet 20 in fluid communication with the suspect channel; and a second inlet 22 in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells. [0052] The housing 12 may be based on, or made of, a capillary bed (such as porous paper or sintered polymer) or, in some instances, on a series of capillary beds in fluid communication with each other. The capillary beds have the capacity to transport fluid by action of capillary forces. [0053] The housing 12 is configured to obtain and receive healthy and suspect samples, which means that the housing should be capable of absorbing the healthy and suspect samples, and any one or combination of materials capable of doing so may be suitable. Such materials used for the housing may include, but are not limited to, natural, synthetic or naturally occurring materials that are synthetically modified, such as polysaccharides (e.g., cellulose materials such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose), polyether sulfone, polyethylene, nylon polyvinylidene fluoride (PVDF), polyester, polypropylene, cotton, or cloth. [0054] The housing 12 can have a planar, sheet-like configuration. The housing 12 can have a thickness equal to or less than 4 mm (such as less than 4, 3, 2, 1 mm), and a width and a length both greater than the thickness. In some embodiments, the width and length of the housing 12 are both greater (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 50 times greater or up to 4, 5, 6, 7, 8, 9, 10, 50 times greater) than the thickness. In some embodiments, the housing 12 is square-shaped or rectangular, and in other embodiments the housing is circular. If the housing 12 is an irregular shape, i.e., different from a square or rectangle, then the width, length and thickness refers to the maximum values for such an irregular shape. For example, the width of a circle will be the diameter. Examples of widths and lengths may be 4, 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 mm, such as, e.g., a range of widths and lengths from 5-30 mm. [0055] The housing 12 may optionally comprise a backing layer (not shown) adhered thereto, which is liquid-impermeable so that fluid flowing through device 10 does not leak through the housing. Examples of suitable materials for the backing layer include, but are not limited to, glass; polymeric materials, such as polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxides, methacrylates, and polymelamine. [0056] Referring again to Fig.1A, the housing 12 includes a healthy channel 14 and a suspect channel 16. The healthy channel 14 includes a second inlet 22 in fluid communication therewith and is adapted to receive a biological sample comprising a healthy sample. The suspect channel 16 includes a first inlet 20 in fluid communication therewith and is adapted to receive a biological sample comprising a suspect sample. [0057] Each of the healthy channel 14 and the suspect channel 16 includes a first labeled binding ligand that specifically binds to BD-2 (e.g., hBD-2), a second labeled binding ligand that specifically binds to BD-3 (e.g., hBD-3), and a control indicator 18. A variety of binding ligands are known to those skilled in the art, such as antibodies, antibody fragments, and aptamers. [0058] In another aspect, the first and second labeled binding ligands are antibodies. Antibodies include polyclonal and monoclonal antibodies, as well as antibody fragments that contain the relevant antigen binding domain of the antibodies. The term “antibody” as used herein refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain. The term “antibody” as used herein is intended to include whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, multi-specific antibodies, single chain antibodies, epitope- binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, and totally synthetic and recombinant antibodies. The antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. [0059] Monoclonal antibodies may be produced in animals such as mice and rats by immunization. B cells can be isolated from the immunized animal, for example from the spleen. The isolated B cells can be fused, for example with a myeloma cell line, to produce hybridomas that can be maintained indefinitely in in vitro cultures. These hybridomas can be isolated by dilution (single cell cloning) and grown into colonies. Individual colonies can be screened for the production of antibodies of uniform affinity and specificity. Hybridoma cells may be grown in tissue culture and antibodies may be isolated from the culture medium. Hybridoma cells may also be injected into an animal, such as a mouse, to form tumors in vivo (such as peritoneal tumors) that produce antibodies that can be harvested as intraperitoneal fluid (ascites). The lytic complement activity of serum may be optionally inactivated, for example by heating. [0060] Biological analytes (e.g., BD-2 or BD-3) may be used to generate antibodies. One skilled in the art will recognize that the amount of polypeptides used for immunization will vary based on a number of factors, including the animal which is immunized, the antigenicity of the polypeptide selected, and the site of injection. The polypeptides used as an immunogen may be modified as appropriate or administered in an adjuvant in order to increase the peptide antigenicity. In some embodiments, polypeptides, peptides, haptens, and small compounds may be conjugated to a carrier protein to elicit an immune response or may be administered with and adjuvant, e.g., incomplete Freund’s adjuvant. [0061] Protocols for generating antibodies, including preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp.55-120, and A. M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984). [0062] The term “antibody fragment” as used herein is intended to include any appropriate antibody fragment that comprises an antigen-binding domain that displays antigen binding function. Antibodies can be fragmented using conventional techniques. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab¹ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, Fv, dsFv, Fd, dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. [0063] Antibodies are designed for specific binding, as a result of the affinity of complementary determining region of the antibody for the epitope of the biological analyte. An antibody “specifically binds” when the antibody preferentially binds a target structure, or subunit thereof, but binds to a substantially lesser degree or does not bind to a biological molecule that is not a target structure. In some embodiments, the antibody specifically binds to the target analyte (e.g., BD-2 or BD- 3) with a specific affinity of between 10⁻⁸ M and 10⁻¹¹ M. In some embodiments, an antibody or antibody fragment binds to BD-2 or BD-3 with a specific affinity of greater than 10⁻⁷ M, 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹ M, between 10⁻⁸ M-10⁻¹¹M, 10⁻⁹ M- 10⁻¹⁰ M, and 10⁻¹⁰ M-10⁻¹¹M. In one aspect, specific activity is measured using a competitive binding assay as set forth in Ausubel FM, (1994), Current Protocols in Molecular Biology (Chichester: John Wiley and Sons). [0064] In one example, antibodies present in the healthy and suspect channels 14 and 16 can be fluorescently-labeled antibodies. [0065] In some embodiments, the binding ligand is an aptamer. Aptamers can be used as an alternative to antibodies for immunoassays (see Chen A1, Yang S2, Biosens Bioelectron.71, 230-42 (2015). An aptamer is a nucleic acid that binds with high specificity and affinity to a particular target molecule or cell structure, through interactions other than Watson-Crick base pairing. Aptamer functioning is unrelated to the nucleotide sequence itself, but rather is based on the secondary/tertiary structure formed, and are therefore best considered as non-coding sequences. Aptamers of the present disclosure may be single-stranded RNA, DNA, a modified nucleic acid, or a mixture thereof. The aptamers can also be in a linear or circular form. Accordingly, in some embodiments, the aptamers are single-stranded DNA, while in other embodiments they are single-stranded RNA. [0066] The length of the aptamer of the present disclosure is not particularly limited, and can usually be about 10 to about 200 nucleotides, and can be, for example, about 100 nucleotides or less, about 50 nucleotides or less, about 40 nucleotides or less, or about 35 nucleotides or less. When the total number of nucleotides present in the aptamer is smaller, chemical synthesis and mass- production will be easier and less costly. In addition, in almost all known cases, the various structural motifs that are involved in the non-Watson-Crick type of interactions involved in aptamer binding, such as hairpin loops, symmetric and asymmetric bulges, and pseudoknots, can be formed in nucleic acid sequences of 30 nucleotides or less. [0067] The aptamers are capable of specifically binding to biological analytes, such as BD-2 and BD-3. Specific binding refers to binding which discriminates between the selected target and other potential targets, and binds with substantial affinity to the selected target. Substantial affinity represents an aptamer having a binding dissociation constant of at least about 10⁻⁸ M, but in other embodiments, the aptamers can have a binding dissociation constant of at least about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, or at least about 10⁻¹² M. [0068] Aptamers can include structural analogs of the original aptamer. Examples of structural analogs include aptamers modified at the 2′-position hydroxyl group of pyrimidine or purine nucleotides with a hydrogen atom, halogen, or an —O- alkyl group. Wild-type RNA and DNA aptamers are not as stable as would be preferred because of their susceptibility to degradation by nucleases. Resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2′-position. Examples of other modifications of aptamer nucleotides include 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5- bromo or 5-iodo-uracil; backbone modifications, and phosphorothioate or alkyl phosphate modifications. [0069] Each of the healthy and suspect channels 14 and 16 includes a control indicator 18, which can function as a control to verify that the lateral flow assay has been conducted properly and/or to provide a standard value (e.g., a standard fluorescent value) for comparison to detected BD-2 and BD-3 levels. [0070] In one embodiment, each of the healthy and suspect channels 14 and 16 do not include binding ligands that specifically bind to and immobilize intact epithelial cells. The fact that such binding ligands are not present in the healthy and suspect channels 14 and 16 is advantageous because the device 10 and associated lateral flow detection assay of the present disclosure uses lysed cells, thereby obviating the conventional need for binding ligands to bind whole cells. [0071] In another embodiment, each of the healthy and suspect channels 14 and 16 is free of a filter. The fact that each of the healthy and suspect channels 14 and 16 is free of a filter is advantageous because all cells subject to the device 10 and associated assay of the present disclosure undergo lysis, obviating the need for filtration. [0072] Each of the healthy and suspect channels 14 and 16 further include a detector zone 24. Each detector zone 24 includes a first line or stripe 26 where one or more binding ligands that specifically bind BD-3 has been immobilized, and a second line or stripe 28 where one or more binding ligands that specifically bind BD- 2 has been immobilized. As discussed in more detail below, each of the first and second stripes 26 and 28 changes color when, and if, BD-3 and BD-2 (respectively) are present in the test or biological samples and flowed through the channels 14 and 16. [0073] The control indicator 18 can be located upstream or downstream of the first and second stripes 26 and 28. The stripes 26 and 28 in the detector zone 24 may be disposed in a direction that is substantially perpendicular to the lateral flow (L) of the healthy and suspect samples. In some embodiments, the stripes 26 and 28 may be positioned in a direction that is substantially parallel to the flow (L) of the healthy and suspect samples. The stripes 26 and 28 in the detector zone 24 do not need to be lines or stripes, and can also be other shapes, such as dots or patterns. [0074] Methods [0075] Another aspect of the present disclosure can include a method for detecting oral cancer in a subject. [0076] A first step of the method can comprise providing a POC, lateral flow assay device 10, such as the one illustrated in Fig.1A and described above. [0077] Either before, contemporaneous with, or after the device 10 is provided, the method can include the step of obtaining a healthy sample and a suspect sample from a subject (e.g., a human subject). [0078] The present method involves determining the levels of BD-2 (e.g., hBD-2) and BD-3 (e.g., hBD-3) in biological samples. A “biological sample”, as used herein, can refer to any tissue or fluid obtained from a subject that includes at least one epithelial cell. Biological samples can include, but are not necessarily limited to, bodily fluids such as saliva, urine, and blood-related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), cerebral spinal fluid, bronchoalveolar lavage, and the like. Biological samples can also include a skin sample or a mucosal sample (e.g., derived or obtained from an oral cavity, a cervix, or an anus). Because the present disclosure is directed to diagnosing oral cancer, in some embodiments, the suspect sample and the healthy sample both include epithelial cells. Biological samples can be obtained by any known means including needle stick, needle biopsy, swab, and the like. In an exemplary method, the biological sample is a mucosal sample (e.g., from an oral cavity, such as the inside of a cheek), which may be obtained for example by using a cytobrush. In some embodiments, the head of the cytobrush is sized to correspond to the size of the suspect tissue or lesion. [0079] A biological sample may be fresh or stored (e.g., blood or blood fraction stored in a blood bank). Samples can be stored for varying amounts of time, such as being stored for an hour, a day, a week, a month, or more than a month. The biological sample may be a skin sample or a mucosal sample expressly obtained for the assays of this disclosure or a sample obtained for another purpose, which can be sub-sampled for the assays of this disclosure. [0080] The biological samples of the present disclosure can be distinguished based on the role they play in the method. In the present disclosure, the biological sample can be a suspect sample or a healthy sample. When carrying out the method, both a suspect sample and a healthy sample are used. A “suspect sample,” as used herein, refers to a biological sample obtained from a tissue site on the subject where oral cancer may reside, or is suspected of residing, such as a skin or mucosal lesion. The site may be suspect as a result of visible irregularities, pain, or as the result of other diagnostic tests, or it may simply be the site where testing is being carried out. For example, in some embodiments, the suspect sample is obtained from an oral lesion, while in other embodiments the suspect sample is obtained from a skin lesion. If the method indicates that the subject has, or is at increased risk of having, oral cancer, the cancer would be present at the suspect site. [0081] The method of the present disclosure also involves obtaining a healthy sample, which is a biological sample obtained from a tissue site where there is no reason to suspect that oral cancer exists, or preferably where the tissue is clearly healthy and non-cancerous based on other available information. In some embodiments, the suspect sample and the healthy sample are obtained from the same subject, which can provide an advantage in terms of serving as an internal standard for more reliable diagnosis. But, in some embodiments, the suspect sample and the healthy sample may be obtained from different subjects. The suspect sample and the healthy sample can be obtained from different tissue sites. Accordingly, in one embodiment, for example, where oral cancer is being diagnosed, the suspect sample is obtained from an oral lesion, while the healthy sample is skin, whole blood, serum, plasma, or mucosal tissue from an oral or cervical region that appears healthy. [0082] In some embodiments, an agent is added to the biological sample that reduces electrostatic interaction between β-defensin and negatively-charged moieties in the sample without affecting binding of detection antibodies and/or fragments thereof to the β-defensins (see U.S. Patent Publication No. 2010/0022025). Negatively-charged moieties in the biological sample can include anionic glycoproteins, such as mucins, a family of large, heavily glycosylated proteins, and calprotectin, a calcium-binding protein secreted predominantly by neutrophils. [0083] The agent can include a positively-charged moiety and/or ions capable of reducing the electrostatic interaction between β-defensin and negatively-charged moieties in the biological sample. The positively-charged moiety and/or ions can be provided in the sample by administering a salt to the sample that upon addition can readily dissociate and form cations or positively charge electrolytes that are capable of associating with the negatively-charged moieties. In one example, the salt upon dissociation can form divalent cations capable of associating with the negatively- charged moiety. Examples of salts capable of forming divalent cations are MgCl2 and CaCl2. MgCl2 and CaCl2 upon addition to a biological sample can dissociate and form Mg²⁺ and Ca²⁺ cations. [0084] The agent can be added to the biological sample at an amount effective to reduce electrostatic interaction between β-defensin and negatively-charged moieties in the sample without affecting binding of detection antibodies and/or fragments thereof to the β-defensins. By way of example, the electrostatic interaction between β-defensin and negatively-charged moieties in the bodily sample can be reduced by adding CaCl2 to a biological sample at a concentration of about 50 mmol/L to about 250 mmol/L. Positively-charged moieties can be added to the biological sample before antibodies are contacted with the sample and/or simultaneously with antibody contact. [0085] In one embodiment, the suspect sample is obtained from a lesional site and the healthy sample is obtained from a corresponding contralateral normal site. For example, the suspect sample is obtained from a lesional site comprising skin or mucosa and the healthy sample is obtained from a corresponding contralateral normal skin or mucosal site. [0086] After obtaining the suspect and healthy samples, each of the samples is contacted with a cell lysis solution for a time and in an amount to ensure that any epithelial cells present in the samples are lysed. In other words, following contact with the cell lysis solution, the suspect and healthy samples do not include intact epithelial cells. Examples of such cell lysis solutions are known in the art and routinely contain, for example, mild detergents (e.g., SDS), protease inhibitors and chelating agents. In one example, a cell lysis solution employed by the assay of the present disclosure is cOmplete Lysis-M (Roche, Cat. No.04719956001). [0087] Following preparation of the healthy and suspect samples (i.e., such that the samples do not include any intact epithelial cells), the suspect and healthy samples are delivered to the first and second inlets 20 and 22 of the device 10, respectively. The amount of healthy and suspect samples delivered to the device 10 can be readily determined by one skilled in the art and can be done, for example, using a pipette, syringe, etc. Once the healthy and suspect samples are delivered, the samples can migrate through the healthy and suspect channels 14 and 16, respectively, to the detector zones 24 where HB-2 (e.g., hBD-2) and HB-3 (e.g., hBD-3) in the healthy sample can bind the immobilized binding ligands (e.g., antibodies), and HB-2 and HB-3, if present in the suspect sample, can bind the immobilized binding ligands. Binding of HB-2 and HB-3 to the respective binding ligands (e.g., fluorescent antibodies) results in the production of a fluorescence emission, which can be detected (as discussed below) where the antibodies used to detect BD-2 and BD-3 emit at different fluorescent wavelengths so that their fluorescent signals can be easily distinguished. [0088] In one embodiment, delivery of the suspect and healthy samples to the device 10, and the subsequent period of time needed for the samples to migrate through the device, is done at room temperature, which advantageously obviates the need for external sources to heat and/or cool samples. [0089] Next, a beta defensin index (BDI) can be determined based on the detected levels (e.g., fluorescence intensity or FI) of BD-2 and BD-3 in the healthy channel (also referred to as the “C-lane”) and BD-2 and BD-3 in the suspect channel (also referred to as the “L-lane”) by the device 10. In one embodiment, this can be done, for example, by: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI. In another embodiment, determining the BDI can be done using the following formula: [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane). [0090] The comparison of beta defensin levels or between ratios can be conducted by any suitable method known to those skilled in the art. For example, the comparison can be carried out mathematically or qualitatively by an individual operating an analytic device or by another individual who has access to the data provided by the analytic device. Alternately, the steps of determining and comparing the levels of BD-2 and BD-3 can be carried out electronically (e.g., by an electronic data processor). [0091] The method can also include the step of providing a report indicating that the subject is in need of oral cancer therapy if the BDI is greater than one, for example. [0092] The present method may also be useful for determining if and when therapy for treating epithelial cancer should be administered to a subject. In some embodiments, the method includes providing an oral cancer therapy to the subject if the BDI is greater than one. A variety of methods for treating oral cancer are known to those skilled in the art. Examples of therapy for oral cancer include surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy. [0093] One example of the method is illustrated in Fig.1B and described below with reference to Steps 1-8. [0094] Step 1: wash the mouth of a subject using, e.g., water. [0095] Step 2: place two cartridges (e.g., red and green) on a flat surface, each of which includes a cell lysis solution. [0096] Step 3: peel open a first bag containing a first cytobrush and remove. Rub and rotate the brush on the suspected lesion site (e.g., 20 times with pressure). Pick up red cartridge and hold steady in on hand. Slowly rotate the brush into the liquid of the cartridge (e.g., about 15 times, with occasional flicking between rotations, while holding the top of the cartridge). The purpose is to dislodge collected cells from the brush head into the liquid in the cartridge. Remove and discard the first cytobrush. [0097] Step 4: peel open a bag containing second cytobrush and remove. Rub and rotate the brush on the contralateral site (e.g., about 15 times, opposite the suspected lesional site). Pick up the green cartridge and hold steady in one hand. Slowly rotate the brush into the liquid of the cartridge (e.g., about 15 times, with occasional flicking between rotations, while holding the top of the cartridge). Remove and discard the second cytobrush. [0098] Step 5: keep both the red and green cartridges at room temperature for about 5 minutes. [0099] Step 6: after about 5 minutes, flick both the cartridges, one by one, with finger until it is evident that liquid has accumulated at the bottom of each cartridge. [00100] Step 7: place about 3 drops of liquid from red cartridge onto the inlet marked “L” on the device 10. Place about 3 drops of liquid from green cartridge onto the inlet marked “C” on the device 10. Wait for about 10 minutes. [00101] Step 8: if all six lines or stripes are present as shown in Case #1-5, determine fluorescence intensities (FI) of the hBD-2 and hBD-3 bands using a portable fluorescence reader. Calculate BDI value using the following formula: [HBD-3FI/HBD-2FI]/(L-lane)/HBD-3FI/HBD-2FI](C-lane). [00102] Systems [00103] Another aspect of the present disclosure can include a system for detection of oral cancer in a subject. [00104] In one embodiment, the system can comprise a POC, lateral flow assay device 10 (e.g., as shown in Fig.1A and described above), as well as a mobile device (not shown) configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine the beta defensin index (BDI). [00105] In one embodiment, the processor is operatively coupled to the camera and programmed to: acquire fluorescence intensity (FI) values for the suspect sample (L-lane) and the healthy sample (C-lane); generate a BDI value using the formula, [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane); and output the generated BDI value to a display device (e.g., a computer, handheld electronic device (e.g., cell phone), etc.). [00106] Kits [00107] In another aspect of the present disclosure, a diagnostic kit is provided for detection of oral cancer in a subject. The diagnostic kit can include any one or combination of the following components: a POC, lateral flow device 10 (such as the one shown in Fig.1A and described above); means (e.g., cytobrushes) for obtaining healthy and suspect samples from a subject; cartridges for receiving healthy and suspect samples and further containing a cell lysis solution; means (e.g., syringes, pipettes) for delivering lysed suspect and healthy samples to the device; a mobile electronic device (as described above, e.g., being configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine the beta defensin index (BDI); and instructions for using the device (e.g., according to the above- described method) for detection of oral cancer in a subject. [00108] Exemplary Aspects [00109] In view of the described compositions, devices, and methods and variations thereof, herein below are certain more particularly described aspects of the present disclosure. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language literally used therein. [00110] Aspect 1: A POC, lateral flow assay device for detecting oral cancer in a subject can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein neither the healthy channel nor the suspect channel include binding ligands that specifically bind to and immobilize intact epithelial cells. [00111] Aspect 2: The device of Aspect 1, wherein each of the healthy and suspect channels is free of a filter. [00112] Aspect 3: The device of any one of Aspects 1-2, wherein the first and second labeled binding ligands are antibodies. [00113] Aspect 4: The device of Aspect 3, wherein the antibodies are fluorescently-labeled antibodies. [00114] Aspect 5: A system for detection of oral cancer in a subject, comprising the device of Aspect 1 and a mobile device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI). [00115] Aspect 6: The system of Aspect 5, wherein the processor is operatively coupled to the camera and programmed to: acquire fluorescence intensity (FI) values for the suspect sample (L-lane) and the healthy sample (C-lane); generate a BDI value using the formula, [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane); and output the generated BDI value to a display device. [00116] Aspect 7: A method for detecting oral cancer in a subject, the method comprising: (a) providing the POC, lateral flow assay device of Aspect 1; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (e) characterizing the subject as having oral cancer based on the BDI; wherein at least step (c) is performed at about room temperature. [00117] Aspect 8: The method of Aspect 7, wherein step (d) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI. [00118] Aspect 9: The method of any one of Aspects 7-8, wherein the suspect sample is obtained from a lesional site and the healthy sample is obtained from a corresponding contralateral normal site. [00119] Aspect 10: The method of Aspect 9, wherein the suspect sample is obtained from a lesional site comprising skin or mucosa. [00120] Aspect 11: The method of Aspect 9, wherein the suspect and healthy samples are not saliva. [00121] Aspect 12: The method of any one of Aspects 7-11, wherein the suspect and healthy samples delivered to the first and second inlets at step (c) do not include intact epithelial cells. [00122] Aspect 13: The method of any one of Aspects 7-12, wherein: at step (b), the healthy and suspect samples are obtained non-invasively by cytobrushing and comprise healthy and suspect cells, respectively; and following step (b), the healthy and suspect cells are lysed prior to step (c). [00123] Aspect 14: A method for detecting and treating oral cancer in a subject, the method comprising the steps of: (a) providing the device of Aspect 1; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; (e) characterizing the subject as having oral cancer if the BDI is greater than 1; and (f) treating the oral squamous cell carcinoma with a therapy selected from the group consisting of surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy; wherein at least step (c) is performed at about room temperature. [00124] Aspect 15: A kit for detection of oral cancer in a subject, the kit comprising: a point-of-care, lateral flow device as recited in Aspect 1; and instructions for using the device for detection of oral cancer in the subject; optionally, means for obtaining healthy and suspect samples from the subject; optionally, separate cartridges for receiving healthy and suspect samples, each of which contains a cell lysis solution; optionally, means for delivering lysed suspect and healthy samples to the device; optionally, a mobile electronic device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI). [00125] Aspect 16: The kit of Aspect 15, wherein the instructions include the following steps: (a) obtain a healthy sample and a suspect sample from the subject; (b) deliver the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (c) determine the BDI based on detected levels of BD-2 and BD-3 by the device; and (d) characterize the subject as having oral cancer based on the BDI; wherein at least step (b) is performed at about room temperature. [00126] Aspect 17: The kit of any one of Aspects 15-16, wherein step (c) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI. [00127] Aspect 18: The kit of any one of Aspects 15-17, wherein the instructions further recite: at step (a), obtain the healthy and suspect samples non-invasively by cytobrushing so that the healthy and suspect samples comprise healthy and suspect cells, respectively; and following step (a), lyse the healthy and suspect cells prior to step (b). [00128] The following Examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto. EXAMPLE 1 [00129] An experiment was performed in which the inventors: (i) analyzed the TCGA database to understand and annotate the expression of hBD-3 transcript (DEFB103) in the context of head and neck squamous cell carcinoma (HNSCC); (ii) validated the expression of hBD-3 and hBD-2 in retrospectively collected carcinoma- in-situ (CIS) and OSCC tissue using immunofluorescence microscopy; (iii) demonstrated differential hBD-3 and -2 expression in cell populations from excised OSCC tumors by fluorescent-activated cell sorting (FACS); (iv) showed by FACS that cytobrush collected cells from OSCC lesions have similar defensin expression profiles as cells excised by biopsy from OSCC tumor tissue; (v) designed an ELISA- based BDI assay platform to discriminate OSCC from benign lesions; (vi) validated the BDI platform through multi-center clinical studies of three non-overlapping cohorts of subjects; and (vii) shown the BDI in a microfluidic intact cell assay (MICA) system. [00130] Methods [00131] Study design [00132] We followed guidelines from Standards for Reporting Diagnostic Accuracy (STARD) 2015 for this study. Detailed exclusion and inclusion criteria are described in Table 1. Table 1: Inclusion and Exclusion Criteria [00133] For the tissue immunofluorescence microscopy study, tissue samples (N=54) were retrospectively collected from the Department of Oral Pathology, Case Western Reserve University School of Dental Medicine, and a waiver of informed consent was approved by the Case Western Reserve University Comprehensive Cancer Center (CCCC) Institutional Review Board (IRB). For Flow Cytometry analysis, four paired cytobrush samples and respective biopsy tissues were obtained from the Dept. of Otolaryngology, University Hospitals Cleveland Medical Center UHCMC (Cleveland). For BDI proof-of-concept, discovery phase, internal validation studies and POC proof of concepts studies, paired cytobrush samples were collected from 8, 40, 21 and 5 subjects, respectively, from the Dept. of Otolaryngology, UHCMC (Fig.4C). The study was approved by the IRB of UHCMC (07-15- 03C/CASE4315). For external validation study 1, the study was approved by the University of Cincinnati Medical Center’s (UCMC) IRB [# 2019-0595], and 19 subjects were recruited from the greater Cincinnati area. For external validation study 2, the study was approved by the West Virginia University (Morgantown) IRB [#1904526911], and 12 subjects were recruited from multiple primary care dental clinics in West Virginia. Informed consent was obtained for all participants in all the studies. Incisional biopsies of all lesions were conducted as the standard of care, after cytobrushing the lesions, Board-certified pathologists determined respective diagnoses. [00134] Immunofluorescence microscopy [00135] Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens were obtained from the Department of Oral Pathology, Case Western Reserve University School of Dental Medicine. We previously described methods for immunofluorescence microscopy (Kawsar, H.I. et al., Oral Oncol.45, 696-702 (2021); DasGupta, T. et al., Oncotarget 7, 27430-27444 (2016); Jin, G. et al., PLoS One 5, e10993 (2010). Briefly, each section (5 µm) was de-paraffinized in xylene and hydrated with serially diluted ethanol, followed by antigen retrieval at 98°C in a high pH Target Retrieval Solution (Dako Co) and blocking with 10% donkey serum containing 0.25% Triton X-100, overnight at 4°C. After washing with PBS, each section was incubated with the respective (anti-hBD-3; NOVUS Biologicals, anti- hBD-2; Santa Cruz Biotechnology) primary antibody (1 h, room temperature), washed in PBS (3×10 min), and then stained with the compatible fluorescent dye- conjugated secondary antibody. For double immunofluorescence, consecutive staining by different primary and secondary antibodies was performed. Isotype controls were conducted using isotype-matched IgGs, corresponding to each primary antibody. Sections were mounted on slides using VECTASHIELD Fluorescent Mounting Media (Vector Lab Inc., Burlingame, CA) containing DAPI to visualize nuclei. Immunofluorescent images were generated using a Leica DMI 6000B fluorescence microscope (Leica Microsystems, Bannockburn, IL) or an Olympus BX51 fluorescence microscope mounted with the Olympus DP71 camera (Olympus America Inc., Center Valley, PA). [00136] Immunofluorescence images were processed using the NIH ImageJ program (Collins, T.J. ImageJ for microscopy. Biotechniques 43, 25-30 (2007)). To quantify expression levels of each defensin, normal, CIS and OSCC immunofluorescent images of hBD-2 and hBD-3 were acquired in 16-bit gray scale, respectively. Fluorescent densities on each of the antibody treated sections were measured with ImageJ as described previously (Collins, T.J. ImageJ for microscopy. Biotechniques 43, 25-30 (2007); Kawsar, H.I. et al., Oral Oncol.45, 696-702 (2021)). The expression of hBD-2 and hBD-3 was represented as the ratio of relative fluorescence intensity of hBD-2 and hBD-3 over that of nuclei, respectively. [00137] Cytobrush sample collection procedure [00138] All cytobrush samples were obtained using ROVERS ORCELLEX BRUSH (Rovers Medical Devices, Oss, Netherlands) for cytobrushing oral mucosa based on the Kajun et. al., J Oral Sci 60, 45-50 (2018) report showing good quality mucosal cell collection with this cytobrush. Each participant was first asked to rinse his/her mouth with water. Before performing a biopsy, the clinician applied the cytobrush onto the mucosal surface of the suspicious lesion and, with pressure, turned the brush head 10 turns to collect as much of the entire thickness of the lesion as possible. The brush was then inserted into a sample collection tube (containing 2 ml of PBS) and placed on ice. The same procedure was then conducted on the contralateral normal side of the oral cavity. The paired samples were stored at - 80°C, until used (for ELISA). For MICA, the samples were collected in PBS and added to ethylenediaminetetraacetic acid (EDTA) containing vacutainer tubes. EDTA was used to prevent sample coagulation since bleeding was observed during sample collection in some cases. For FACS and MICA samples, where intact cells were required, samples were processed within 2-3 hrs. post collection. For all subjects, biopsy samples were collected per standard-of-care clinical procedure. For MICA, the samples were collected in PBS and added to (EDTA) vacutainer tubes. [00139] xE whole exome sequencing [00140] xE whole exome sequencing of paired cytobrush samples (OSCC lesional and contralateral) was performed by Tempus Labs, Inc., (Chicago, IL, USA ) (Lau, D. et al., Nat Commun 13, 4053 (2022); Beaubier, N. et al., Oncotarget 9, 25826-25832, (2018); Beaubier, N. et al., Nat Biotechnol 37, 1351-1360 (2019); Leibowitz, B.D. et al., BMC Cancer 22, 587 (2022)). Tempus xE is a whole exome next-generation sequencing proprietary assay which analyzes the entire coding region (exome) of the patient’s genome, combined with whole transcriptome RNA sequencing. This was done as a tumor:normal matched assay for each tumor and contralateral normal cytobrush specimen, sequenced to an average depth of 250x and 150x, respectively. Whole transcriptome RNA-seq was 50 million reads encompassing 19,396 genes covering ~39 Mb of genomic space. The cytobrush samples from OSSC lesions were matched to corresponding contralateral normal samples to ensure fidelity of somatic variant calling. From DNA sequencing, somatic and single nucleotide variants, insertions and deletions and copy number variant data were generated. [00141] Cytobrush sample processing for flow cytometry of defensins [00142] For flow cytometry staining, single-cell suspensions were obtained from cytobrush samples by resuspending the samples in a PBS-based, enzyme-free cell dissociation buffer (Gibco) at room temperature for 5 minutes, followed by passing suspensions through 100 uM cell-strainers. Excised tumors were processed by collagenase digestion (0.5mg/ml) for 8 minutes at room temperature. The cells were then centrifuged in PBS/BSA before sequential flow-cytometry staining for E- cadherin, hBD-2 and hBD3 proteins. For intracellular staining, the cells were washed after surface E-cadherin staining, fixed, permeabilized and stained with antibodies for intracellular hBD2 and hBD3 proteins. Live-Dead viability staining (Life Technologies/Thermofisher) was used to remove dead cells in the flow cytometry analysis. Data was acquired using a BD Fortessa cytometer and analyzed using FlowJo 9.8 or 10.5.3 software. PE-conjugated anti-human α-E-cadherin polyclonal antibody was purchased from R&D systems. Alexa-flour 647 conjugated hBD2, and FITC conjugated hBD3 polyclonal antibodies were purchased from BIOSS (Woburn, MassachusettsU.S.A.). Standard non-stained and single stain controls were used for flow cytometry to determine gates. At least 1000-3000 E- Cad⁺ viable cells were gated for the flow cytometry analysis of hBD-2 and -3. These were pre-gated on cells stained with a Live-Dead marker to exclude non- viable cells. Fluorescence compensation and data analysis were standard methods performed using appropriate unstained and single-stained controls using BD FACSDiva™ and FlowJo 10 software. [00143] Cytobrush sample processing for ELISA [00144] Briefly, paired samples from each subject were thawed on ice, and cells were dislodged from each cytobrush by flicking and subtle vortexing of the collection tube. After discarding the brush, the fluid (~2ml) was centrifuged at 300 x g for 5 min, and the supernatant was discarded. The remaining cell pellet was lysed in RIPA buffer, followed by centrifugation at 10,000 x g at 4°C for 10 min. The resultant supernatant (cell lysates) was collected and used for the ELISA assay. [00145] Beta Defensin ELISA and determination of BDI [00146] For the proof-of-concept, discovery phase, and internal validation studies, we used a sandwich ELISA with optimized hBD-2 and hBD-3 antibody pairs from Peprotech (NJ) (Ghosh, S.K. et al., Clin Chem 53, 757-765 (2007); Meisch, J.P. et al., Inflamm Bowel Dis 19, 942-953 (2013)). Briefly, 96-well immunoplates (R&D Systems, City, MN) were coated with 50 μl anti-hBD-2 or hBD-3 antibodies diluted to 1 μg/ml, 4°C, overnight, followed by blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The paired cell lysates (20 μl) along with 80μl of RIPA buffer were added to each experimental well (in duplicate) and 100 μl of RIPA buffer was added in two additional wells (blank wells). The plate was then incubated at room temperature (RT) for 1 h. The wells were washed 3 times with PBS containing 0.1% Tween 20 and incubated with 50 μl of the biotinylated secondary antibody (Peprotech, NJ) diluted to 0.1 μg/ml, at RT, 30 min. Each well was washed 3 times, 50 μl/well of streptavidin-peroxidase (R&D Systems, 1:200 in wash buffer) was added to each well and incubated for 20 minutes at RT. Each well was washed again (3 times with wash buffer) and incubated with 100 ul of Reagent A and B (1:1) for 20 minutes, after which the reaction was stopped with a μl/well of stop solution (2N HCL) (R&D Systems). Absorbance was measured at 450 nm with a microplate reader. For the external validation studies, hBD-2 and hBD-3 ELISA kits from Phoenix Pharmaceuticals (Burlingame, CA) were used and ELISAs were performed per vendor’s instruction using 20 μl of cell lysates. [00147] We determined the BDI from ELISA data using the following steps: i) Determined the average OD values for each sample (from OD of duplicate wells) and blank wells, ii) Subtracted OD values of the blank well (hBD-2 ELISA) from OD values of the sample well [(hBD-2 ELISA)= ODhBD-2], iii) Subtracted OD values of the blank well (hBD-3 ELISA) from OD values of the sample well [(hBD-3 ELISA)=ODhBD- 3], iv) Determined the BDI for each subject using the following equation: A= [ODhBD-3]/[OD hBD-2] for sample (#XL) collected from lesional site of subject #X; B= [ODhBD-3]/[OD hBD-2] for sample (#XN) collected from contralateral control (normal) site of the same subject (#X); BDI=A/B. [00148] Microfluidic Intact Cell Analysis (MICA): micropillar array construction and processing of cytobrush samples [00149] The MICA device was fabricated using established soft lithography protocols as previously described (Man, Y. et al., Microcirculation 28, e12662 (2021); Man, Y. et al., Lab Chip 21, 1036-1048 (2021)). Briefly, a 3-in silicon wafer (University Wafers, Boston, MA) was spin-coated with a negative photoresist (SU-8 2035, ThermoFisher Scientific, Waltham, MA) at 1000 rpm/min and soft-baked at 95°C for 20 min. The wafer was then UV-patterned under a photomask, post- exposure baked (95°C, 10 min), developed in a photoresist solvent (propylene glycol monomethyl ether acetate, Sigma Aldrich, St. Louis, MO), and hard-baked at 110°C overnight. Surface passivation of the master wafer was performed under vacuum using trichloro (1H,1H,2H,2H-perfluorooctyl) saline (PFOCTS, Sigma Aldrich). Next, a polydimethylsiloxane (PDMS, ThermoFisher Scientific) pre-polymer was mixed with the curing agent at a volume ratio of 10:1 and degassed to remove any air bubbles. The mixture was then poured over the master wafer and cured at 80°C overnight. Thereafter, PDMS replicas were peeled off, cut into individual pieces, and inlet and outlet holes were punched. Excess saline was removed by sonicating in isopropanol. Finally, the PDMS replicas were covalently bonded on standard microscope glass slides using oxygen plasma to form the micro-channels. [00150] Following tubing assembly, the MICA microchannel was rinsed with 100% ethanol and PBS, and then blocked with 2% bovine serum albumin (BSA, ProSpec- Tany TechnoGene Ltd., East Brunswick, NJ) at 4°C overnight to prevent any non- specific cell adhesion to the microchannel walls. Cytobrush samples collected from lesional and contralateral sites of each patient were placed on ice before testing. The cytobrush sample was injected into the appropriate microchannel (lesion in one; contralateral in the other) under 40 mbar inlet pressure using a Flow-EZ pressure control unit (Fluigent, Lowell, MA) until a reasonable cell density was achieved in the microchannel. Following formaldehyde (Sigma Aldrich) fixation (4% in PBS) for 15 min at room temperature (RT), the microchannel was rinsed with PBS and blocked with 1% BSA for 1 h at RT. Next, the fixed cells were permeabilized with Triton X- 100 (Sigma Aldrich) (0.1% in PBS with 1% BSA) for 15 min at RT, and incubated in a mixture of 4’,6-diamidino-2-phenylindole (DAPI, 10 μg/mL final concentration) and hBD-3 and hBD-2 antibodies (Bioss Antibodies, Woburn, MA; Catalogue: bs-7378R- A488 and bs-4307R-A647) (1% in PBS with 1% BSA) in the dark at RT for 1 h. Finally, the microchannel was washed with PBS and imaged under 10× objective with an inverted microscope (Olympus IX83) and a microscope camera (EXi Blue EXI-BLU-RF-M-14-C). [00151] To quantify the ratio of hBD-3 to hBD-2 of each side for each patient and healthy individual, the retained cells were fluorescently labeled with an hBD-3 antibody (Alexa Fluor 488 conjugated) and an hBD-2 antibody (Alexa Fluor 647 conjugated). Cell nuclei were also labeled with DAPI, and used to distinguish intact cells from debris. Cells in the area of interest (AOI: micropillar arrays with 40, 30 and 20-μm openings) were imaged using 10× objective, for which representative microscopic phase-contrast and fluorescent images are shown in Figs.7A-B. [00152] To determine BDI, image segmentation and fluorescence intensity analyses were carried out (Fig.14), where the average ratio of hBD-3 to hBD-2 of each side was computed based on 9 pairs of fluorescent images. Finally, the patient BDI was computed using the equation: BDI= [hBD-3 pixel/hBD-2 pixel] L/[hBD-3 pixel/hBD-2 pixel] N (L= lesional and N= Contralateral Normal). [00153] Salivary levels of hBD-2 and hBD-3 [00154] Salivary levels of hBD-2 and -3 were determined following the protocol as described by Ghosh, S.K. et al., Clin Chem 53, 757-765 (2007). Briefly, 96-well immunoplates (R&D Systems, MN) were coated with 50 μl anti-hBD-2 or hBD-3 antibodies (Peprotech, NJ) diluted to 1 μg/ml, 4°C, overnight, followed by blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The wells were washed 3 times with PBS containing 0.1% Tween 20. Fifty μl of each saliva _{sample and 50 μl of} 500 mM CaCl₂ were added to each well and incubated at room ^{temperature (RT) for 1 h. The wells were} _{washed 3 times with PBS containing 0.1%} Tween 20 and incubated with 50 μl of the biotinylated secondary antibody (Peprotech, NJ) diluted to 0.1 μg/ml, at RT, 30 min. Each well was washed 3 times, followed by 50 μl/well of streptavidin-peroxidase (R&D Systems, 1:200 in wash buffer). Each well was washed again (3 times with wash buffer) and incubated with 100 μl of Reagent A and B (1:1) for 20 minutes, after which the reaction was stopped with a stop solution (2N HCL). Absorbance was measured at 450 nm with a microplate reader. [00155] Quantification and statistical analysis [00156] ROC curve was generated and cut-off value was calculated using easyROC online software (Goksuluk et al., The R Journal.2016;8(2):213-30). TCGA data were analyzed using multiple online software (GEDS (Xia et al., Cells. 2019;8(7)), GEPIA2 (Tang et al., Nucleic Acids Res.2019;47(W1):W556-w60), UALCAN (Chandrashekar et al., Neopasia.2017;19(8):649-58) and LinkedOmics (Vasaikar et al., Nucleic Acids Res.2018;46(D1):D956-d63)) as indicated in the Results section. Most of the other plots were generated using GraphPad Prism version 6 and statistical calculations were carried out with either non-parametric Mann-Whitney test or Kruskal-Wallis test, as indicated in the figure legends (p value <0.05 were considered significant). [00157] Reagent details [00158] Table 2 below provides pertinent information for selected reagents, including their source and identifier. Table 2 – Reagent Details

[00159] Results [00160] Expression of DEFB103 and DEFB4 transcripts in head and cancer based on the cancer genome atlas (TCGA) [00161] Because hBD-3 and hBD-2 peptides and transcripts (DEFB103 AND DEFB4, respectively) are differentially expressed in multiple cancer types, we mined the TCGA database for DEFB103 and DEFB4 transcript expression levels in HNSCC. Using the online Gene Expression Display Server (GEDS), we found that both transcripts were highest in HNSCC, when compared to other cancer types (Figs.8A-B). Using the UALCAN portal (Chandrashekar, D.S. et al., Neoplasia 19, 649-658 (2017), we discovered that DEFB103, but not DEFB4, was significantly upregulated in HNSCC tumor tissue when compared to normal tissue (p=0.048 vs. p=0.56) (Figs.8A-B). We further analyzed the HNSCC-TCGA data using GEPIA2 (Tang, Z. et al., Nucleic Acid Res 47, W556-560 (2019) for differential expression of DEFB103 with respect to different molecular subtypes of HNSCC (as defined by Walter, V. et al., PLoS One 8, e56823 (2013)) and found that expression of DEFB103 was significantly higher in the “basal” subtype when compared to the other subtypes (i.e., mesenchymal, atypical and classical) (Fig.8C). The basal subtype of HNSCC is characterized by genes associated with; (a) epidermal development, (b) ErbB signaling (ErbB; a family of four receptor tyrosine kinases, of which epidermal growth factor receptor (EGFR) is the first discovered member), and (c) growth/transcription factor signaling (Walter, V. et al., PLoS One 8, e56823 (2013)). With respect to HNSCC tumor grade, the data revealed that DEFB103 expression was highest in grade 1; i.e., the “well-differentiated” HNSCC subtype, compared to the other HNSCC subtypes (Fig.8D), and its expression was also significantly higher in well-differentiated grade 1 HNSCC compared to normal/healthy sites (p<0.00003) (Fig.8D). We further analyzed DEFB103 expression based on other clinicopathological characteristics, such as cancer stage, gender, age and race (Figs. 9A-D). As shown in Fig.9A, DEFB103 expression is significantly higher in Stage 1 and 3 cancers compared to normal. Regarding gender, both male and female DEFB103 are significantly higher in HNSC compared to normal (Fig.9B). Age and race have no effect on the expression of DEFB103 (Figs.9C-D). [00162] Using Linked Omics (Vasaikar, S.V. et al., Nucleic Acids Res 46 D956-963 (2018)), we identified 38 genes that were strongly correlated with DEFB103 (Spearman’s rho≥ 0.70) (Figs.9E-F). Two late cornified envelope (LCE) proteins 3D and 3E, belonging to the “epidermal differentiation complex” (EDC), were strongly associated with DEFB103 (Fig.9F). However, while both were higher in HNSC compared to normal, only LCE3D was significantly higher (Fig.9G). Interestingly, most of these genes belong to the EDC, comprising over fifty genes encoding proteins involved in the terminal differentiation and cornification of keratinocytes, the primary cell type of the epidermis (Fig.9H). [00163] Overexpression of hBD-3 in carcinoma in-situ and oral cancer [00164] Overexpression of hBD-3 and under-expression of hBD-2 was further confirmed by us in a small cohort of 7 oral carcinoma-in-situ (CIS) cases, compared to the healthy oral epithelium (N=4), using immunofluorescence (IMF) (Jin et al., PLoS One.2010;5(6):e10993). To expand the CIS cohort and to further confirm results in our OSCC cohort, we conducted an IMF study of 54 lesions (24, non- cancerous, 27 CIS, and 3 OSCC) and observed a similar phenotype in the additional CIS and cancerous lesions but not in non-cancerous lesions (Figs.2C-D). Thus, overexpression of hBD-3, with diminished expression of hBD-2, is a “reproducible phenotype” in CIS biopsies that is apparently maintained in OSCC. Furthermore, we performed analytical flow cytometry analysis of digested biopsy samples from three additional representative OSCC biopsy tissues to show that in all three cases, the percentage of hBD-3 positive cells was higher than that of hBD-2 (Fig.2E). In summary, transcript, protein and now flow cytometry from three independent studies confirm the overexpression of hBD-3 and reduced expression of hBD-2 in CIS and OSCC lesions. [00165] Based on our more extensive IMF findings and corroboratory FACS analysis supporting the differential expression of hBD-3 vs. hBD-2 in oral CIS and in OSCC lesions, respectively, we hypothesized that by collecting epithelial cells from a suspicious lesion and comparing its hBD-3/hBD-2 ratio to cells collected from the same subject’s contralateral healthy oral mucosa, a reliable biomarker for OSCC could be established. [00166] Non-invasively collected (cytobrushed) mucosal cells can be used to collect cancer cells and to assay for the expression of human beta defensins [00167] To confirm that our cytobrush technique collected cancerous cells, our board-certified pathologist conducted cytological analysis of cytobrush samples (Figs.3A-B). We identified cancerous cells in the lesional site (Fig.3A) but not in the contralateral site (Fig.3B) of the same patient. Whole exome sequencing (xE whole exome, TEMPUS, Chicago) also confirmed the collection of cancer cells in the cytobrush samples but not in the contralateral site. Copy number gains of CCND1, FGF3, FGF4, and FGF19, along with TP53 mutation (p.R156G), all associated with OSCC, were found exclusively in oral cancerous lesions. [00168] We collected cytobrush samples from two healthy participants’ cheeks and tongues and, upon FACS analysis, discovered that ~ 40% of E-cadherin- positive epithelial cells are positive for defensin peptides (Fig.3C; representative data from one participant), indicating that both peptides are detectable in non- invasively collected healthy mucosal samples. Interpersonal variability of the percentage of cytobrushed cells expressing hBD-2 vs hBD-3 in healthy cheek and tongue was observed between subjects. Next, we collected cytobrush samples from the tumor and contralateral normal sites of OSCC subjects (N=4), respectively, before biopsy sampling. FACS analysis of each paired sample showed that there were significantly higher levels of hBD-3 (but not hBD-2) from OSCC vs. cells from respective contralateral sites obtained by cytobrush (Figs.3D-E). Additionally, the difference in the ratio of hBD-3 to hBD-2 in the OSCC site when compared to the ratio of hBD-3 to hBD-2 in the normal contralateral site was highly significant (p=0.013, Fig.3F) compared to the expression of lesional hBD-3 itself in tumor vs. contralateral hBD-3 (p=0.03, Fig.3D). These observations provided us with the rationale to test an hBD-3/hBD-2 ratio-based OSCC biomarker using non-invasively cytobrush-obtained cell samples. [00169] We coined the term Beta-Defensin-Index (BDI) and defined it as the ratio of hBD-3/hBD-2 in the lesional site over the ratio of hBD-3/hBD-2 in the contralateral site of the same patient (Figure 3A). We hypothesized that the BDI should be higher in subjects with an oral cancerous lesion compared to a BDI from subjects with a benign lesion. [00170] Because FACS is costly, time-consuming, requires expensive equipment and expertise to run, and demands analyzing intact cells, it is not a practical platform to efficiently assess the relevance and efficacy of BDI as a biomarker for CIS and early-stage OSCC in a clinical setting. Instead, we developed a BDI ELISA-based assay (see Method section) and tested it on a separate cohort of clinical samples. [00171] BDI proof-of-concept study [00172] For an initial proof-of-principal analysis, we identified four patients with OSCC lesions and four with benign oral lesions (detailed diagnoses of all the participants are shown in Table 3). The median value of the BDI (calculated as defined in Fig.4A) of the four cancerous lesions was significantly higher (p<0.02) than that of the four benign lesions (Fig.4B); i.e., supporting our initial proof-of-concept. Table 3: Detailed diagnosis and BDI values of oropharyngeal samples

[00173] Here we tested if BDI is higher in HPV 16-induced OPC. We collected cytobrush samples from tonsillar lesions, determined their BDI scores, and found that the median BDI for the HPV16-induced OPC lesions is significantly higher (p<0.03) than benign tissue (Fig.10A); i.e., supporting our original finding and our proof-of- concept. [00174] Before proceeding with BDI-based assays using mucosal epithelial cells, we wanted to see if hBD-2, hBD-3 and/or the ratio of hBD-3 to hBD-2 are significantly different in saliva samples from OSCC vs non-OSCC cohorts. We determined the salivary levels of both defensins in a pilot cohort of subjects (N=30, Cancerous =18, non-cancerous =12) and found that neither hBD-2, hBD-3 nor the ratio of hBD-3 to hBD-2 were significantly different between the groups (Fig.10B); suggesting that BDI scoring from mucosal epithelial cells, but not from saliva, could be useful in distinguishing OSCC from benign lesions. [00175] To determine the efficacy of the BDI platform, we tested it in four non- overlapping patient-based studies. The study design consisted of four investigator- blinded studies using a discovery phase cohort, followed by a validation phase of three cohorts (summarized in Fig.4C). We recruited a total of 92 subjects for the discovery and validation study (18 years of age or older) undergoing clinical evaluation for OSCC due to the presence of suspicious red or white oral lesions present for two weeks or greater in the same location that had not been previously biopsied (see Table 1 for inclusion and exclusion criteria). While we initially included oropharyngeal (OPC) lesions in the proof-of-concept phase and found that the BDI could differentiate OPC-related OSCC from non-OSCC lesions (Fig.10A), we decided to exclude OPC lesions from discovery and validation, as cytobrushing the oropharyngeal region is challenging without sedation, and our goal was to discover and validate the BDI using a noninvasive protocol that did not require sedation. [00176] Discovery phase study [00177] For the discovery phase study, we recruited 40 subjects (Table 4, Section S1B) from University Hospitals Medical Center, Department of Otolaryngology (Cleveland, OH).

Medical Center (UHCMC) IRB committee, was followed, including informed consent documentation (IRB#: 07-15-03C/CASE4315). To avoid inter-operator variability, the same clinician collected paired cytobrush samples from the lesional and contralateral sites of each subject, respectively, before the biopsy of each lesional site. The anatomical locations of each lesion within the oral cavity are described in Table 4 – Section S1B). Out of the 40 oral lesions, 25 were diagnosed by pathology review as being cancerous while 15 were benign (see Table 4). The median age of the two patient cohorts was 65 (range 39-91) and 56 (range 35-73), respectively (p>0.05). One laboratory operator generated BDI values to avoid inter-operator variability. The samples were intentionally blinded so that pathologist-determined diagnoses were revealed only after BDI scores were obtained for all the patients. As shown in Fig. 5A, the BDI of cancerous subjects (N=25) was significantly (p<0.0001) higher than that of non-cancerous subjects (N=15). Next, we investigated the diagnostic accuracy of BDI in differentiating cancerous vs non-cancerous lesions by receiver operating characteristic (ROC) curve analysis (Fig.5B). The area under the curve (AUC) value was found to be 0.99 (Fig.5B). One desirable characteristic of an adjunct test intended to be used in a primary care setting is having a high sensitivity to minimize the proportion of false-negative results to avoid missing patients requiring biopsy or referral and hence, we used the "MaxSe" (maximizes sensitivity) approach (Goksuluk, D.K.S. et al., The R Journal 8, 213-230 (2016)). We arrived at a BDI cut-off value of 1.25 and found 100% sensitivity with an acceptable specificity value of 80% (Fig.5C). These results support BDI as a promising “Index” for screening OSCC, and may serve as a triage tool before traditional biopsy. [00179] Validation study [00180] The validation study consisted of 3 non-overlapping cohorts; one internal and two external. All three sites used the same IRB-approved protocol described in the discovery study above. For the internal cohort (UHCMC Medical Center), multiple clinicians provided cytobrush samples, while one laboratory operator generated the BDI scores. For the external cohorts, cytobrush samples were collected by multiple clinicians, and BDI scores were generated by multiple laboratory operators in the Cincinnati site while in the West Virginia site, there was one lab operator. Diagnoses based on pathology review of respective lesions are shown in Table 4, Sections S1C, S1D and S1E. The blinded analysis confirmed that the BDI values of OSCC patients were also significantly higher (p<0.0001) in all subjects with OSCC lesions when compared to benign lesions (Fig.5D). We then calculated the sensitivity and specificity of the BDI values, based on the threshold value of 1.25 from the discovery cohort, and found a 97% sensitivity and 82 % specificity rate, which was in agreement with the results from the discovery study. Overall, for all the subjects (N=92), sensitivity/specificity and positive/negative predictive values with a confidence interval of 95% are shown in Table 5. Table 5: Sensitivity, specificity, positive and negative predictive values of BDI in OSCC detection Out of 92 subjects we had eight false positive (FP) and one false negative (FN) (Table 5, Table 4). The FPs and FN are highlighted in Table 5. Importantly, most of the FPs were diagnosed as being dysplasia/hyperkeratosis. [00181] Cohort Characteristics, BDI and Clinical Parameters [00182] Demographic information (e.g., age, gender, and smoking status) along with lesion location in the oral cavity and diagnosis based on pathology review is included for every subject in the proof-of-principle, discovery, and validation studies (Table 4 and Table 6). Table 6: Demographic and clinical parameters (stages) of 92 subjects [Discovery and Validation cohorts]

Since smokin DI values correlated with smoking status (e.g., never or non-smokers; former and current smokers). Not surprisingly, the BDI values of both current and former smokers were significantly higher than those of non-smokers (Fig.11A). Moreover, we found that male participants had significantly higher BDI values compared to female participants (Fig.11B) consistent with the literature, showing that males smoke more than females and OSCC is more prevalent in males (Chinwong, D. et al., J Addict 2018; Lee, Y-C. et al., Medicine 100, e27674 (2021)). [00183] The prevalence of OSCC based on anatomic sites of the oral cavity has been well documented (Vigneswaran, N. et al., Oral Maxillofac Surg Clin North Am 26, 123-141 (2014)). Our oral cancer cohort data showed that the tongue was the most frequent site for OSCC (36%) followed by the floor of mouth (FOM) (26%) (Fig. 11C). This is in agreement with published data of patients in the U.S., where the tongue is the most common intraoral site of OSCC and accounts for 25-40% of all OSCC, followed by FOM (15-20%) (Vigneswaran, N. et al., Oral Maxillofac Surg Clin North Am 26, 123-141 (2014); Chen, A.Y. et al., Dis Mon 47, 275-361 (2001)). When we compared BDI values based on oral anatomical locations, i.e., cheek, tongue and all other locations combined, for both benign and cancerous lesions, respectively, we found that anatomical location was not a factor affecting BDI scoring within benign and within OSCC lesions (Fig.11D). Interestingly, we found no correlation between BDI levels and severity of cancer stage; i.e., high BDI scores were not associated with late-stage OSCC (Fig.11E). [00184] Benchmarking of BDI against other OSCC biomarkers/platforms [00185] Several biomarkers have been studied to detect OSCC and a few OSCC detection platforms have been proposed, with some in clinical use as well. The BDI was benchmarked against salivary (protein, mRNA and miRNA-based) and, serum biomarkers (protein and miRNA signature based), as well as against visual aids _{and cytological and transcriptomic- based detection platforms. These are shown} in Fig.12A and Table 7. Table 7 – OSCC biomarkers and detection platforms used in benchmark analysis M_{arkers/Platform Markers/platform Samples Detection} IL‐8 Protein Saliva ELISA IL‐1Beta Protein Saliva ELISA DUSP Protein Saliva ELISA S100P Protein Saliva ELISA solCD44 Protein Saliva ELISA CPLANE1 mRNA Saliva qPCR NUS1+RCN1 mRNAs Saliva qPCR mir31 miRNA Saliva qPCR mir345 miRNA Saliva qPCR mir424 miRNA Saliva qPCR GAS6 Protein Serum ELISA miR‐125b‐5p + miRNAs Serum qPCR miR342‐ 3p C_DOT ^TM Microbial and human Saliva RNA‐seq meta‐transcriptomic +Bioinformatics signature ViziLite® Chemiluminescence Oral Visual cavity VELscope® Florescence Oral Visual Oral CDx® Cytology Exfoliate Computer‐assist d cells ed specimen LBC Cytology Exfoliate ^an P^aa^lyp^saⁱn^sicolao d cells u‐ staining POCOCT Cytology Exfoliate Cytology on a d cells chip BDI Index Exfoliate ELISA d cells [00186] Figs.12A-E shows that the BDI is the most sensitive (98%) in detecting OSCC when compared against all the other biomarkers (sensitivity ranges 32-93) (Fig.12B), as well as against all commercially available/in use detection platforms (sensitivity ranges 69-94) (Fig.12D). [00187] Development of a microfluidic platform to determine BDI-Proof of Principle [00188] We explored the possibility of translating our novel laboratory-based BDI- biomarker assay into a microfluidic test, which we refer to as Microfluidic Intact Cell Assay or (MICA). The MICA detection principle utilizes the trapping of mucosal cells in a microfluidic chip by incorporating arrays of micro-fabricated polydimethylsiloxane pillars of variable spacing ranging from 160 μm to 20 μm to capture the cells without the need for surface chemistry (Fig.6A). The trapped cells are then exposed to 0.1% Triton-x100 in PBS, to permeabilize cell membranes, followed by incorporation of anti-hBD-3 and anti-hBD-2 Alexa Fluor conjugated antibodies to detect respective antibodies and quantify the BDI. We envision MICA could one day, be used at the chairside in a clinic setting. MICA includes micropillar arrays embedded into the microchannel forming narrow openings along the flow direction, coupled with two 400-μm wide side passageways. MICA is designed such that large cell aggregates are retained upstream within the coarse openings and individual cells are retained downstream within the narrower openings. The side passageways are designed to prevent clogging of the near-inlet portion of the micropillar arrays. The MICA microchannel overall dimensions are 27 mm × 4 mm × 120 μm (length × width × height). A photograph of MICA is shown in Fig.6B. The microscopic phase-contrast image shown in Figs.6C-D represents a typical cell distribution in the MICA microchannel and confirms that cell aggregates were being filtered upstream while individual cells were retained downstream. [00189] To demonstrate the preliminary efficacy of this platform, we used cytobrush samples from cancerous lesions and corresponding contralateral sites and incubated fluorescent antibodies to epidermal growth factor receptor (EGFR) within respective troughs of the MICA chip. EGFR was selected as a proof-of-principle marker as it is overexpressed in up to 90% of all OSCC patients (Grandis, J.R. et al., Cancer Res 53, 3579-3584 (1993); Xu, M.J. et al., Cancer Metastasis Rev 36, 463- 473 (2017); Rubin Grandis, J. et al., J Natl Cancer Inst 90, 824-832 (1998)), and its activation results in hBD-3 expression (Feng. Z. et al., Infect Immun 82, 4458-4465 (2014); Muhammad, J.S. et al., Pathog Dis 74, (2016); Shuyi, Y. et al., Oral Surg Oral Med Oral Pathol Oral Radiol Endod 112, 616-625 (2011)). Consistent increases in fluorescence intensities emanating from OSCC cells compared to contralateral cells (Fig.13); i.e., demonstrating EGFR overexpression in OSCC, strongly suggested that the MICA platform could be used for BDI determinations. [00190] To test the translational potential of the BDI biomarker using the MICA platform, we collected cytobrush samples from OSCC lesional sites and contralateral sites of five OSCC patients and from five healthy subjects’ left and right cheek (or tongue). Cells, after mild detergent treatment to permeabilize their membranes, were incubated with fluorescent antibodies specific for either hBD-3 or hBD-2 and BDI scores were determined as described in the Methods section. Representative phase contrast, hBD-3, hBD-2, and DAPI-stained images are shown in Figs.7A-B. Significantly higher (p=0.03) BDI values in OSCC lesions compared to healthy were observed, as shown in Fig.7C. These results demonstrate that the BDI biomarker, using the MICA platform, can be a point-of-care device. EXAMPLE 2 [00191] In Fig.15, and in the description that follows, this Example illustrates an alternative implementation of the POC, LFA device 10 described above. In particular, this Example illustrates one configuration of a single lane or channel comprising a device 10 of the present disclosure. Although only the single lane or channel is shown in Fig.15, it will be appreciated that a device 10 of the present application can be constructed having two or more lanes or channels, each of which being constructed as shown in Fig.15 and described herein. Below, testing and development of a POC, LFA device comprising a two-lane fluorescence immunoassay (FIA) test strip to measure hBD-2 and hBD-3 levels in lesion and healthy site cytobrush samples is described. Construction and application of the device is focused on ease of use, accuracy, and clarity of results. The device is designed to quantitatively determine hBD-2 and hBD-3 levels in lesion and healthy site cytobrush lysate samples. [00192] Development and Analytical Validation [00193] Material selection for device development [00194] A validated and tested nitrocellulose membrane (Sartorius, Germany) is designed on which two test lines and one control line is printed. Pore size and membrane properties determine the sample flow rate and sensitivity, which is considered in selection. A conjugated pad comprises a glass fiber or polyester pad (Ahlstrom, Pennsylvania) that holds and releases a fluorescent label (e.g., Europium) Thermo Scientific), which is conjugated to anti-human hBD-2&3 detection antibodies (polyclonal, Thermo Scientific). Conjugation of Europium to detection antibody is done using a covalent linkage kit from Pierce Biotechnology by Thermo Scientific. Sample pad is a diagnostic-grade, hydrophilic spunbonded polyester membrane (Ahlstrom, Grade 6614) that is highly pure when received. Sample pad is inert and does not interact with cationic peptides. Absorbent pad is cellulose paper (Ahlstrom 222) placed at a first end of the nitrocellulose membrane to keep the flow consistent by absorbing excess reagents. Europium fluorescent beads are used for signal detection, which produce robust detectable signals that are read using portable readers (Hemex Health, Portland, OR). Europium beads are conjugated to detection antibodies as well as rabbit IgG (Thermo Scientific) for control band generation. Cytobrush (Rovers Orcellex Brush, Netherlands) cell collection device provides reliable, reproducible, easy-to-use, fast, comfortable, non-invasive cell collection of high cellularity (e.g., about 55,000 cells). [00195] Reagent preparation [00196] Capture antibodies are printed (KinBio, Rehovot, Israel) on the nitrocellulose membrane to form hBD-2 and hBD-3 test lines and a control line. Polyclonal antibodies naturally bind to different epitopes on the target, which allows the use the same antibody for both detection and capture. If monoclonal antibodies are used, detection antibodies are different than the capture antibodies to achieve capture and detection on distinct epitopes of the target molecules. Europium beads are conjugated with selected detection antibodies, and their stability is determined at different temperatures (e.g., 25°C, 37°C, and 45°C). Binding efficiency is confirmed by serial dilution of purified antigens in composite samples by measuring the line intensity to determine limit of detection, limit of quantitation, and dynamic range. [00197] Device assembly [00198] Capture antibodies are printed on the nitrocellulose membrane using a dispenser, followed by a drying step. The nitrocellulose membrane is assembled with the conjugate pad (e.g., impregnated with the fluorescent conjugate bound to the detection antibodies), sample pad, and absorbent pad onto a backing card (Lohmann, Florida) for stability (shown as bottom layer in Fig.15). [00199] Assay optimization [00200] Test concentrations of the capture and detection antibodies, buffer conditions, and sample volume to ensure >90% sensitivity and >90% specificity are carefully tested. Flow rate is optimized by adjusting the sample pad and absorbent pad properties. Nitrocellulose paper is selected by considering both flow rate and protein binding capacity, which affect the limit of detection (LOD). [00201] Limit of Detection (LOD) and Limit of Quantification (LOQ) [00202] The lowest concentration of the hBD-2 and hBD-3 analytes that can be detected, but not necessarily quantitated, is determined as an exact value. LOD is determined by repeatedly measuring blank samples and samples with very low concentrations. LOQ, in the low pg/ml range, is determined as the lowest concentration at which the analyte can not only be detected but also measured with acceptable accuracy and precision. [00203] Analytical testing [00204] To evaluate analytical sensitivity and specificity as part of the assay development process, the device is tested with known positive and negative samples to assess its performance. Cross-reactivity analysis using similar analytes is performed to ensure specificity. Since the device is a fluorescent assay, a fluorescence reader is used, which is calibrated with known standards to quantify the fluorescent intensity correlating to analytical concentration. [00205] Other considerations [00206] Simple visual and/or audible results can be added to make the device more accessible and to let any frontline healthcare worker operate the device. The Hemex Reader, for example, has connectivity to upload test results to medical records to report to a clinician directly. An internal control line confirms that the test and the reader work as intended. Shelf-stable reagents are used to maximize the shelf-life, considering the temperature, storage, and shipping conditions of resource limited settings. Cytobrush sample collection efficiency and sample desorption into the testing buffer is monitored. Regulatory-compliant protective substances in the cytobrush buffer are used to maximize shelf-life in room temperature storage. [00207] Validation to Ensure Accuracy and Precision [00208] Accuracy and precision of device is evaluated to ensure that the test is both reliable in terms of true results (accuracy) and reproducible in terms of consistent results (precision). Construction and testing of a device and assay that demonstrates a sensitivity and specificity greater than 90% to ensure accuracy, and CV <25% to ensure precision is intended. Potential failure modes of the tests which may lead to inconclusive or repeat tests are analyzed. Standardization of room temperature lysis of cytobrush samples is achieved. Testing and analytical validation of the device using cytobrush cell lysates from normal subjects is performed. Batch testing (e.g., 1,200 devices) is performed in multiple (e.g., three) batches (e.g., 400 devices per batch). Analytical performance is assessed by following the BEST (Biomarkers, EndpointS, and Other Tools Resource) definitions and the FDA “Bioanalytical Method Validation” guidelines, including, standard curve creation, sensitivity, specificity, precision, limit of detection, and limit of quantification. Statistical design and data analysis is based on published guidelines. A sample size of about 200 tests for an adequately powered (significance at 0.05, power at 90%) study design is used. Typical measurement for normal and lesion sites, and the clinically meaningful difference expected in a cancerous lesion is used. These procedures ensure that the device provides accurate results that are close to the true value (accuracy) and that repeated tests yield similar results (precision). [00209] Pre-commercial manufacturing, packaging, and stability [00210] Manufacturing, packaging, and accelerated stability testing is performed to determine shelf life. Moving from small-scale production to larger batch processes to ensure consistency is envisioned. For this purpose, three different pilot batches are produced for mixed testing. Rigorous quality control measures by following ISO 13485 standards to ensure each batch meets the required standards are developed and implemented. As part of packaging, each device is accompanied with a reader and a mobile app for fluorescence detection, and clear instructions for use. [00211] Preparation of samples with known concentrations [00212] Lysate samples are prepared with known concentrations of hBD-2 and hBD-3 to cover the expected range of concentrations in real-world samples. For example, cytobrush samples from healthy (non-cancerous) are collected in 2 ml lysis buffer, hBD-3 is 6.8 ± 6 pg/ml, and hBD-2 is 16 ± 12.6 pg/ml, which are concentrated 20-fold during centrifugation steps. Concentration samples for hBD-2 and hBD-3 are prepared as follows: 5 pg/ml; 50 pg/ml; 100 pg/ml; 200 pg/ml; 500 pg/ml; 1000 pg/ml; and 4000 pg/ml. [00213] Standard curve creation and BDI cutoff [00214] The series of known concentration samples are used to run multiple tests (n=5 per sample for 7 distinct concentrations), and the fluorescence intensity is plotted against the known concentrations to create a standard curve to determine unknown concentrations from fluorescence readings. A BDI cutoff with known concentration samples that are in the positive range of oral cancer samples is used. [00215] Test accuracy [00216] Sensitivity and specificity of the device based on BDI cutoff is determined by testing samples known to be positive (e.g., a determined hBD-3 concentration greater than a determined hBD-2 concentration, e.g., high hBD-3 concentration and relatively low hBD-2 concentration). A highly sensitive correctly identifies most or all positive samples, with the aim being to achieve >90% sensitivity using the BDI-FIA test using the pre-determined BDI cutoff. Specificity is determined by testing samples that are known to be in the healthy range in terms of hBD-2 and hBD-3 intensities and BDI ratio. A specific test is used identify samples that do not have the analyte at sufficiently high concentrations, to achieve >90% specificity. In this task, a selected number of positive (e.g., high BDI) and negative (e.g., low BDI) samples are used to determine test performance. Healthy cytobrush clinical samples spiked with known concentrations of hBD-2 and hBD-3 target analytes are used to perform sensitivity and specificity analyses. [00217] Comparison with reference method [00218] Parallel tests using a well-established ELISA assay for detecting hBD-2 and hBD-3 levels and quantifying the BDI ratio are run. Pearson-correlation coefficient is performed to evaluate how well the test assay quantifies hBD-2 and hBD-3 levels to determine the BDI ratio. An appropriate number of samples are tested in parallel to determine BDI as determined by the device vs. BDI determined by ELISA. [00219] Test precision [00220] Repeatability (intra-assay precision) is evaluated by performing multiple tests on the same sample by the same operator under the same conditions within a short period of time (e.g., <4 hours). The coefficient of variation (CV) is calculated from the results. An appropriate number of tests are performed to determine CV. Reproducibility (Inter-assay precision) is evaluated by testing the same samples on different days, and by different operators, using different batches of the devices, on different fluorescence readers. CV is calculated to see how results vary under these differing conditions. The aim is to achieve <25% CV based on FDA “Bioanalytical Method Validation” guidelines. [00221] Multifactorial statistical analysis [00222] Mean, standard deviation, and CV are calculated for the replicate measurements to quantify precision. Data from the standard curve for linear regression is used to assess how well the fluorescence intensity correlates with analyte concentration. [00223] Sample matrix effects [00224] An evaluation of how different sample matrices might affect the performance of the test results is performed. This is done by spiking different matrices, i.e., cytobrush samples collected from different parts of the mouth (cheeks, tongue, floor of the mouth, lip, gingiva) with known amounts of the hBD-2 and hBD-3 analytes to see if there is any interference. Since the patient is his/her own control, if the samples are collected from comparable locations for lesion and healthy sites, the results are comparable. [00225] Robustness [00226] An evaluation of how small variations in the test procedure, including, temperature, humidity, cytobrush procedure, affect results is performed to ensure that the test performs well under less-than-ideal conditions. [00227] Other considerations [00228] Calibration coefficients are generated for each antibody. These calibration coefficients are the coefficient of a polynomial that normalizes the intensity of each test line with what was observed on ELISA. The calibration coefficients are encoded on a machine readable QR code that is on each test device or cartridge. This allows the Reader to automatically compensate for lot-to-lot variability. [00229] From the above description of the present disclosure, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes, and modifications are within the skill of those in the art and are intended to be covered by the appended claims. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Claims

CLAIMS The following is claimed: 1. A method for detecting oral cancer in a subject, the method comprising the steps of: (a) providing a point-of-care, lateral flow assay device comprising: a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein neither the healthy channel nor the suspect channel includes binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (e) characterizing the subject as having oral cancer based on the BDI; wherein at least step (c) is performed at about room temperature.

2. The method of claim 1, wherein step (d) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI.

3. The method of claim 1, wherein the suspect sample is obtained from a lesional site and the healthy sample is obtained from a corresponding contralateral normal site.

4. The method of claim 3, wherein the suspect sample is obtained from a lesional site comprising skin or mucosa.

5. The method of claim 1, wherein the suspect and healthy samples delivered to the first and second inlets at step (c) do not include intact epithelial cells.

6. The method of claim 1, wherein: at step (b), the healthy and suspect samples are obtained non- invasively by cytobrushing and comprise healthy and suspect cells, respectively; and following step (b), the healthy and suspect cells are lysed prior to step (c).

7. A point-of-care, lateral flow assay device for detecting oral cancer in a subject, the device comprising: a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein neither the healthy channel nor the suspect channel includes binding ligands that specifically bind to and immobilize intact epithelial cells

8. The device of claim 7, wherein each of the healthy and suspect channels is free of a filter.

9. The device of claim 7, wherein the first and second labeled binding ligands are antibodies.

10. The device of claim 9, wherein the antibodies are fluorescently-labeled antibodies.

11. A system for detection of oral cancer in a subject, comprising the device of claim 7 and a mobile device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI).

12. The system of claim 11, wherein the processor is operatively coupled to the camera and programmed to: acquire fluorescence intensity (FI) values for the suspect sample (L-lane) and the healthy sample (C-lane); generate a BDI value using the formula, [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane); and output the generated BDI value to a display device.

13. A method for detecting and treating oral cancer in a subject, the method comprising the steps of: (a) providing the device of claim 7; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; (e) characterizing the subject as having oral cancer if the BDI is greater than 1; (f) treating the oral squamous cell carcinoma with a therapy selected from the group consisting of surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy; wherein at least step (c) is performed at about room temperature.

14. A kit for detection of oral cancer in a subject, the kit comprising: a point-of-care, lateral flow device as recited in claim 7; and instructions for using the device for detection of oral cancer in the subject; optionally, means for obtaining healthy and suspect samples from the subject; optionally, separate cartridges for receiving healthy and suspect samples, each of which contains a cell lysis solution; optionally, means for delivering lysed suspect and healthy samples to the device; optionally, a mobile electronic device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI).

15. The kit of claim 14, wherein the instructions include the following steps: (a) obtain a healthy sample and a suspect sample from the subject; (b) deliver the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (c) determine the BDI based on detected levels of BD-2 and BD-3 by the device; and (d) characterize the subject as having oral cancer based on the BDI; wherein at least step (b) is performed at about room temperature.

16. The kit of claim 15, wherein step (c) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI.

17. The kit of claim 15, wherein the instructions further recite: at step (a), obtain the healthy and suspect samples non-invasively by cytobrushing so that the healthy and suspect samples comprise healthy and suspect cells, respectively; and following step (a), lyse the healthy and suspect cells prior to step (b).