SLC2A1 / GLUT1 as biomarker for high-risk epithelial dysplasia

Abstract

Provided herein are methods of stratifying oral lesions based on the risk of progressing into a malignant tumor, and methods of treating subjects involving selective removal of oral lesions identified as high risk. In some aspects, the methods provided herein include assessing risk based on expression of SLC2A1 on a lesion.

Description

[0001] Priority Statement This application claims priority to U.S. Provisional Application No. 63 / 527,223, filed July 17, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Government Support Statement This invention was completed with government support under DE029255 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0003] sequence list The accompanying text of the computer-readable sequence list, titled "UM-41943-601_SQL", was created on July 17, 2024, and is 1,960 bytes in size. It is hereby incorporated in its entirety by reference. Technical Field

[0004] This article provides a method for stratifying oral lesions based on the risk of progression to malignancy, and a method for treatment involving the removal of subjects with oral lesions identified as high-risk. In some aspects, the method presented in this article includes assessing risk based on the expression of SLC2A1 on the lesions. Background Technology

[0005] Human papillomavirus (HPV)-head and neck squamous cell carcinoma (HNC) has an extremely low 5-year overall survival rate. However, HNC is curable if diagnosed early. Oral epithelial dysplasia (OED) and oral lichen planus (OLP) are potential precursor lesions of HNC, potentially offering an opportunity for disease eradication. Current standard treatment for these lesions involves histological examination. However, histological grading has no prognostic value in predicting malignant transformation. Furthermore, current standards require frequent follow-up of every patient with OED / OLP, which is extremely challenging and impractical. Therefore, there is an urgent need to identify OED / OLP patients at high risk of progressing to HNC. Summary of the Invention

[0006] In some respects, this paper provides a method for stratifying oral lesions based on the risk of progression to malignancy. In some embodiments, this paper provides a method for stratifying oral lesions based on the risk of progression to malignancy, the method comprising: measuring the expression of solute carrier family 2 member 1 (SLC2A1) in the oral lesion; and identifying the oral lesion as high-risk when the expression level of SLC2A1 is equal to or higher than a threshold; or identifying the oral lesion as low-risk when the expression level of SLC2A1 is lower than the threshold.

[0007] In some embodiments, SLC2A1 expression in the oral lesion is measured by obtaining a biopsy sample from the oral lesion and measuring the expression of SLC2A1 in the biopsy. In some embodiments, SLC2A1 expression is measured by tissue staining. In some embodiments, SLC2A1 expression in the oral lesion is measured directly on the oral lesion within the subject's oral cavity. In some embodiments, SLC2A1 expression on the oral lesion is measured by contacting the oral lesion with an agent that binds SLC2A1, wherein the agent comprises a marker, and subsequently detecting the marker. In some embodiments, detecting the marker includes applying a light source to the oral lesion to activate the marker and visually detecting the marker. In some embodiments, the method further includes selectively eliminating the oral lesion when it is identified as high-risk, or monitoring the oral lesion when it is identified as low-risk.

[0008] In some embodiments, this document provides a method for stratifying oral lesions based on the risk of progression to malignancy. The method includes: measuring the expression of solute carrier family 2 member 1 (SLC2A1) and an additional risk marker in multiple oral lesions within a region of interest in a subject's oral cavity; and identifying oral lesions as high-risk based on: the interaction score between SLC2A1 and the additional marker and / or the Wasserstein distance between the oral lesion and adjacent oral lesions. For example, in some embodiments, the method includes identifying an oral lesion as high-risk when its SLC2A1-additional marker interaction score is higher than a threshold interaction score. In some embodiments, the method includes identifying an oral lesion as high-risk when it expresses SLC2A1 and the Wasserstein distance between the oral lesion and an adjacent oral lesion expressing the additional risk marker is equal to or less than a threshold Wasserstein distance.

[0009] In some embodiments, the SLC2A1-additional biomarker interaction score of oral lesions is calculated by multiplying the expression of SLC2A1 in the oral lesion by the expression of the additional risk biomarker in the oral lesion, and dividing the result by the number of cells expressing both SLC2A1 and the additional risk biomarker within the region of interest. In some embodiments, the expression of SLC2A1 and the expression of the additional risk biomarker are measured directly on the plurality of oral lesions in the subject's oral cavity. In some embodiments, the expression of SLC2A1 and the expression of the additional risk biomarker are measured by contacting the plurality of oral lesions with an agent binding to SLC2A1 and an agent binding to the additional risk biomarker, wherein each agent includes a marker, and the marker is subsequently detected. In some embodiments, detecting the marker includes applying a light source to the plurality of oral lesions to activate the marker and visually detecting the marker. In some embodiments, the method further includes selectively eliminating the oral lesion when it is identified as high-risk. In some embodiments, the additional risk markers are nuclear expression of epidermal growth factor receptor (EGFR), SRY-Box transcription factor 2 (SOX-2), or Yes1-associated transcriptional regulator (YAP-1).

[0010] In some aspects, this article provides methods for treating subjects at risk of head and neck cancer. In some embodiments, this article provides a method for treating subjects at risk of head and neck cancer (HNC), the method comprising: measuring the expression of solute carrier family 2 member 1 (SLC2A1) in oral lesions of the subject; identifying oral lesions as high-risk or low-risk based on SLC2A1 expression levels, and selectively eliminating high-risk oral lesions or monitoring low-risk oral lesions. In some embodiments, the method comprises: identifying the oral lesion as high-risk when the SLC2A1 expression level is equal to or higher than a threshold level; or identifying the oral lesion as low-risk when the SLC2A1 expression level is lower than a threshold level. In some embodiments, the method comprises: selectively eliminating the oral lesion when it is identified as high-risk; or monitoring the oral lesion when it is identified as low-risk. In some embodiments, the expression of SLC2A1 in the oral lesion is measured by obtaining a biopsy from the oral lesion and measuring the expression of SLC2A1 in the biopsy. In some embodiments, SLC2A1 expression is measured by tissue staining. In some embodiments, SLC2A1 expression is measured directly on oral lesions within the subject's oral cavity. For example, in some embodiments, SLC2A1 expression on the oral lesion is measured by contacting the oral lesion with an agent that binds SLC2A1, wherein the agent comprises a marker, and subsequently detecting the marker. In some embodiments, detecting the marker includes applying a light source to the oral lesion to activate the marker, and visually detecting the marker.

[0011] In some embodiments, this document provides a method for treating a subject at risk of head and neck cancer (HNC), the method comprising: directly measuring the expression of a solute carrier family 2 member 1 (SLC2A1) in each of a plurality of oral lesions in the subject's oral cavity; identifying an oral lesion as high-risk when the expression level of SLC2A1 in the oral lesion is equal to or higher than a threshold level, and / or identifying an oral lesion as low-risk when the expression level of SLC2A1 in the oral lesion is lower than a threshold level; and selectively eliminating high-risk oral lesions in the subject's oral cavity. In some embodiments, the method further comprises monitoring low-risk oral lesions.

[0012] In some embodiments, SLC2A1 expression is measured by contacting the plurality of oral lesions with an agent that binds SLC2A1, wherein the agent comprises a marker, and subsequently detecting the marker. In some embodiments, detecting the marker includes applying a light source to the oral lesions to activate the marker, and visually detecting the marker.

[0013] In some embodiments, this document provides a method for treating a subject at risk of head and neck cancer (HNC), the method comprising: measuring the expression of solute carrier family 2 member 1 (SLC2A1) and an additional risk marker in multiple oral lesions within a region of interest in the subject's oral cavity; identifying oral lesions as high-risk based on an interaction score between SLC2A1 and the additional marker and / or a Wasserstein distance between the oral lesion and adjacent oral lesions; and selectively eliminating high-risk oral lesions in the subject's oral cavity. In some embodiments, an oral lesion is identified as high-risk when the SLC2A1-additional marker interaction score of the oral lesion is higher than a threshold interaction score; and / or when the oral lesion expresses SLC2A1 and the Wasserstein distance between the oral lesion and an adjacent oral lesion expressing the additional risk marker is equal to or less than a threshold Wasserstein distance.

[0014] In some embodiments, the SLC2A1-additional biomarker interaction score of oral lesions is calculated by multiplying the expression of SLC2A1 in the oral lesion by the expression of the additional risk biomarker in the oral lesion, and dividing the result by the number of cells expressing both SLC2A1 and the additional risk biomarker within the region of interest. In some embodiments, the expression of SLC2A1 and the expression of the additional risk biomarker are measured directly on the plurality of oral lesions in the subject's oral cavity. In some embodiments, the expression of SLC2A1 and the expression of the additional risk biomarker are measured by contacting the plurality of oral lesions with an agent binding to SLC2A1 and an agent binding to the additional risk biomarker, wherein each agent includes a marker, and the marker is subsequently detected. In some embodiments, detecting the marker includes applying a light source to the plurality of oral lesions to activate the marker and visually detecting the marker.

[0015] In some aspects, this document provides a method comprising directly measuring the expression of a solute carrier family 2 member 1 (SLC2A1) in at least one oral lesion within the oral cavity of a subject, wherein the expression of SLC2A1 is measured by contacting the at least one oral lesion with an agent that binds to SLC2A1, wherein the agent comprises a marker, and subsequently detecting the marker on the at least one oral lesion. In some embodiments, detecting the marker comprises applying a light source to the oral lesion to activate the marker, and visually detecting the marker. Attached Figure Description

[0016] Figure 1A-1CThis study demonstrates significant alterations in immune cell markers during the malignant transformation of OED. A longitudinal series of paired OED / HNC specimens were stained using the MSI combination shown. Each slide was reviewed by a pathologist, and 6–13 regions of interest were manually selected for high-resolution scanning. The frequencies of the selected immune cell subsets were quantified using Perkin Elmer's inFORM. To address inter-patient heterogeneity, a nonparametric ANOVA using the aligned rank transform was performed to examine the significance of changes between OED and HNC. This was accomplished using the ARtool package in R.

[0017] Figure 2A-2B The IFN-I signature is shown to be fundamental to HNC immunogenicity. (Figure 2A) UMAP analysis was performed on the transcriptomes of 38,862 TILs from HPV-HNC and 20,703 TILs from HPV+ HNC. Clusters showing significant changes between the two diseases are highlighted in red. (Figure 2B) TILs from 520 HNC samples in the TCGA dataset were deconvolved, and the relationship between STING levels and major subset frequencies was assessed.

[0018] Figures 3A-3B Markers of glycolytic genes in hypoxia-induced HNCs were shown. HNC cells were exposed to normoxic and hypoxic (1% pO2) conditions for 24 hours. The mRNA levels of glycolytic genes were compared using a two-tailed t-test.

[0019] Figures 4A-4B The upregulation of glycolytic markers in malignant transformation is shown. (Figure 4A) The expression levels of glycolytic marker genes were compared between 44 normal mucosa samples and 520 HNC samples. (Figure 4B) TIL composition in 520 HNC patients was deconvolved using FARDEEP. Marginal correlations between major TIL subsets and SLC2A1 levels were analyzed.

[0020] Figure 5A-5KHistological findings of OED / HNC in immune-active Sox2-driven GEMM are shown. (Fig. 5A) Incidence of OED and HNC in K5-CreER;Sox2+ / +, K5-CreER;Sox2+ / -, or K5-CreER control mice treated with or without DMBA after local tamoxifen induction are shown. Ten K5-CreER;Sox2+ / + mice were treated with DMBA alone (n=5) or with a loading alone (n=5). K5-CreER;Sox2+ / - mice were treated with DMBA alone (n=8) or with a loading alone (n=8). Ten K5-CreER control mice were treated with DMBA alone (n=5) or with a loading alone (n=5). Tongue and buccal mucosa were harvested and histologically examined. (Fig. 5B) Wide-field endoscopy with white light illumination shows typical tongue tumors. (Fig. 5C) GFP-expressing tumor cells were observed in live animals using a first-generation emphyseal monoaxial laser confocal microscopy (arrow). (Figs. 5D-5E) Representative H.&E images of two cases of human primary oral squamous cell carcinoma are shown. MEER (Fig. 5F) and MOC2-E6 / E7 (Fig. 5G) tumor cells were subcutaneously implanted into C57BL / 6 mice. (Figs. 5H-5I) Four weeks after induction, the oral mucosa of tamoxifen-induced K5-CreER;Rosa26Sox2+ / + mice was harvested. (Figs. 5J-5K) Similar histological features of OED between human and mouse buccal mucosa (near the corner of the mouth) are shown.

[0021] Figures 6A-6C show the upregulation of Slc2a1 by OED conversion. (Figure 6A) Four weeks after induction, buccal mucosa from induced Sox2-GEMM mice and littermate control mice was harvested and IHC staining was performed on the markers shown. (Figure 6B) buccal mucosa from control mice and Sox2-induced mice was harvested for qPCR analysis. (Figure 6C) Slc2a1 staining was performed on control, OED, and HNC samples from Sox2-GEMM.

[0022] Figures 7A-7HHigh-risk immune markers were shown to appear before the histological appearance of HNC. K5-CreER;Sox2+ / + GEMM buccal mucosa was harvested throughout the entire time span of malignant transformation. CD45+ viable intralesional immune cells were purified by FACS prior to scRNA-Seq. (Figs. 7A-7C) A total of 8,960 transcriptomes were analyzed over this time span. UMAP revealed different immune lineages determined by differential expression and unique gene markers in the immune marker matrix gene set. Functional annotation was based on the marker genes with the highest expression levels and most significant differential expression. (Fig. 7D) PHATE trajectory analysis was performed on the myeloid cell population revealed by UMAP. The distribution patterns of myeloid cells at different time points are shown. (Figs. 7E-H) The most significantly differentially expressed genes associated with OED transformation were mapped onto the feature map. p<.0001).

[0023] Figure 8 This study demonstrates how YAP1 activation drives HNC. Constitutive active form of Yap1 (Yap5sa) was induced by applying tamoxifen to the oral mucosa for five days. Six weeks after induction, buccal mucosa samples from K5-CreER-;Yap5sa+ / - controls and K5-CreER+;Yap5sa+ / - controls were harvested and stained with designated IHC markers.

[0024] Figures 9A-9B This study demonstrates how YAP1-driven transformation reshapes the local immune landscape. K5-CreER-;Yap5sa+ / - controls and K5-CreER+;Yap5sa+ / - mice were orally tamoxifen-applied (n=4 per group). Six weeks after induction, buccal mucosa was harvested for CD45+EpCAM assays. - Cells were sorted by FACS and single-cell RNA-Seq was performed. (Figure 9A) Immune subsets were clustered using the most expressed genes and anchored genomes for pedigree typing. (Figure 9B) The composition of each subset is shown.

[0025] Figures 10A-10CThis study demonstrates that confocal laser endoscopy provides an optical biopsy tool for capturing cellular features at high risk of HNC (hepatic squamous cell carcinoma). (Fig. 10A) Imaging was performed by placing the laser confocal endoscopy microscope in contact with the oral mucosa. The mucosa was excited at λ=488nm. (Figs. 10B-10C) C57BL / 6J mice were fed 50 μg / ml 4-NQO drinking water for 16 weeks, followed by 8 weeks of normal water. Normal tongue (Fig. 10B) and squamous cell carcinoma (Fig. 10C) were locally treated with a FITC-labeled EGFR-targeting QRHKPRE (SEQ ID NO: 1) peptide for 5 minutes to generate contrast. Excess peptide was rinsed off with water. Confocal images were acquired in vivo at 10 Hz.

[0026] Figure 11A-11B The study showed that IL-R1 blockade improved outcomes in high-risk oral lesions. Figure 11A Showing the effect of induction K5- CreER;Sox2 + / + In mice, intraperitoneal injection of 200 μg of anti-Il1r1 one week after induction improved their survival rate. Figure 11B Show K5-CreER;Sox2 + / + ;Il1r1 - / - Tumor evaluation in a composite strain, which consists of... K5-CreER; Sox2 + / + GEMM and Il1r1 - / - Generated by crossbreeding mice. K5-CreER;Sox2 + / + ;Il1r1 + / + Compared with littermates, K5- CreER;Sox2 + / + ;Il1r1 - / - The tumor area was significantly reduced in mice.

[0027] Figures 12A-12S demonstrate that blocking IL1 signaling can improve outcomes in high-risk oral lesions. Figure 12A shows tumor growth after injection of MOC2 tumor cells (Moc2-gW cells) or MOC2-Sox2 (Sox2-positive cells). Figure 12B shows the percentage of CD8+CD3+ T cells in the gW and Sox2 groups as analyzed by flow cytometry. Figure 12C shows the frequency of CD45+ cells within the CD8+CD3+ T cell population. Figure 12D shows the percentage of CD8+CD11c+ dendritic cells in the MOC2-gW and MOC2-Sox2 groups as analyzed by flow cytometry. Figure 12E shows the frequency of CD45+ cells within the CD8+Cd11c+ cell population in the MOC2-Gw and MOC2-Sox2 groups. Figure 12F shows the flow cytometry analysis of FSCA+ and Gr1+ cells in the MOC2-gW and MOC2-Sox2 groups. Figure 12G shows the frequency of CD11b+ cells in Gr1+ MDSCs. Figure 12H shows a heatmap analysis of Sox2, IL1a, and Csf2 expression in EV and Sox2 mice. Figure 12I shows the fold change in IL1a mRNA after administration of gW or Sox2 using the squamous cell line UMSC22b. Figure 12J shows the survival probability of K5-Cre / Sox2+ / + mice hybridized with IL1r1+ / + or IL1R1- / -. Figure 12K shows the cellular expression of TCRγ / δ and CD11b. Figure 12L shows the frequency of CD45+ cells within the Cd11b-TCRγ / δ+ cell population. Figure 12M shows the expression of Glut1 and CD206 cells. Figure 12N shows the frequency of Cd11b+ cells within the Glut1+CD206+ cell population. Figure 12O shows tissue staining of K5-Cre;Sox2+ / +;IL1r+ / + and K5-Cre;Sox2+ / +;IL1r- / - mice, and Figure 12P quantifies the tumor surface area of ​​K5-Cre;Sox2+ / +;IL1r+ / + and K5-Cre;Sox2+ / +;IL1r- / - mice. Figure 12Q shows the survival probability of mice treated with anti-IL1r or PBS (control) after tumor induction. Figure 12R shows the results of cell population assessment by flow cytometry after treatment with PBS or anti-IL1r1. Figure 12S shows the frequency of PDL1+GR1+CD11B+ MDSCs in different treatment groups.

[0028] definition While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the embodiments described herein, this document describes some preferred methods, compositions, apparatuses, and materials. However, before describing the materials and methods of the invention, it should be understood that the invention is not limited to the specific molecules, compositions, methods, or schemes described herein, as these can vary based on routine experimentation and optimization. It should also be understood that the terminology used in the description is merely for describing specific schemes or embodiments and is not intended to limit the scope of the embodiments described herein.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of conflict, this specification (including the definitions) shall prevail. Therefore, the following definitions shall apply in the context of the embodiments described herein.

[0030] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural references. Thus, for example, reference to “compound” means one or more compounds and their equivalents known to those skilled in the art, etc.

[0031] As used herein, the term “and / or” includes any and all combinations of the listed items, including any single item listed. For example, “A, B and / or C” covers A, B, C, AB, AC, BC, and ABC, where each item should be considered as being described individually by the expression “A, B and / or C”.

[0032] As used herein, the term "comprising" and its variations indicate the presence of one or more described features, elements, or method steps, but do not exclude the presence of additional features, elements, or method steps. Conversely, the term "consisting of" and its variations indicate the presence of one or more mentioned features, elements, or method steps, excluding any unmentioned features, elements, or method steps, except for generally relevant impurities. The phrase "substantially consisting of" indicates the presence of one or more mentioned features, elements, or method steps, and any additional features, elements, or method steps that do not substantially affect the fundamental properties of the composition, system, or method. Many embodiments herein are described using the open "comprising" language. Such embodiments encompass multiple closed "consisting of" and / or "substantially consisting of" embodiments, which may also be claimed or described using such language.

[0033] As used herein, the terms "administration" and "administering" refer to the act of introducing a substance, such as a drug, prodrug, or other agent or therapeutic agent, into a subject or into cells, tissues, and organs, whether in vivo or in vitro. Generally, any route of administration may be used, including, for example, parenteral (e.g., intravenous), oral, local, subcutaneous, intraperitoneal, intra-arterial, inhalation, vaginal, rectal, nasal, intracerebrospinal fluid, infusion, or intracavitary perfusion. Exemplary routes of administration to humans may include parenteral (e.g., intravenous, subcutaneous, etc.), oral, etc.

[0034] As used herein, the terms “about” and “approximately” are intended to cover general statistical variations that a person skilled in the art will understand to apply to the relevant context. In some implementations, unless otherwise stated or explicitly indicated from the context (unless the value exceeds 100% of the possible value), the terms “about” or “approximately” refer to a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower in either direction (greater or less) of the reference value.

[0035] As used herein, the terms "co-administration" and "co-administering" refer to administering at least two agents or therapies to a subject. In some embodiments, co-administration of two or more agents or therapies is simultaneous (e.g., in a single formulation / composition or in a separate formulation / composition). In other embodiments, the first agent / therapy is administered before the second agent / therapy. Those skilled in the art will understand that the formulations and / or routes of administration of the various agents or therapies used can vary. Those skilled in the art can readily determine the appropriate dose for co-administration. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered at a lower dose than would be appropriate for its individual administration. Therefore, co-administration is particularly desirable in embodiments where co-administration of agents or therapies would reduce the necessary dose of a potentially harmful (e.g., toxic) agent, and / or when co-administration of two or more agents results in the subject becoming sensitive to the beneficial effects of one agent via co-administration of another agent.

[0036] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier (inert or active) that makes the composition particularly suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo. As used herein, the term "pharmaceuticalally acceptable carrier" means any standard pharmaceutical carrier, including but not limited to phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic agents and absorption retarders, disintegrants (e.g., potato starch or sodium glycolate starch), etc. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), the entire contents of which are incorporated herein by reference.

[0037] As used herein, the term "pharmaceuticalally acceptable" refers to a composition that, when administered to a subject, will substantially not produce adverse reactions (e.g., toxicity, anaphylaxis, or immunological reactions).

[0038] As used herein, the term "selectively" refers to the selective removal and / or selective elimination of high-risk oral lesions, indicating that high-risk oral lesions are targets for removal from the subject. The term "selectively" does not necessarily mean that high-risk oral lesions are exclusively removed from the subject. In some implementations, portions of non-high-risk oral lesions may also be removed during the selective removal of high-risk oral lesions.

[0039] As used herein, the term "subject" broadly refers to any animal, including both human and non-human animals. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0040] As used herein, the term "treatment" (also known as "treat" or "treating") refers to achieving a desired pharmacological and / or physiological effect against a specific disease, ailment, or condition. Preferably, the effect is therapeutic, meaning it partially or completely relieves, improves, reduces, inhibits, delays, reduces the severity, and / or decreases the frequency, incidence, or severity of one or more symptoms, features, and / or causes of a specific disease, ailment, and / or condition. Subjects receiving such treatment may be subjects who do not exhibit signs of the relevant disease, ailment, and / or condition, and / or subjects who exhibit only early signs of the disease, ailment, and / or condition. Alternatively or additionally, subjects receiving such treatment may be subjects exhibiting one or more confirmed signs of the relevant disease, ailment, and / or condition. In some embodiments, the subject receiving treatment may be a subject who has been diagnosed with the relevant disease, ailment, and / or condition. In some embodiments, the subject being treated may be known to have one or more susceptibility factors that are statistically associated with an increased risk of developing a related disease, ailment, and / or condition. In some embodiments, “treatment” refers to preventing a benign lesion (e.g., OEP, OLP) from progressing to a malignant tumor (e.g., head and neck cancer (HNC)) in the subject. For example, “treatment” encompasses selectively removing a high-risk lesion in a subject to prevent that lesion from transforming into a malignant tumor in the subject. As used herein, the term “prevention” refers to preventive steps taken to reduce the likelihood of a subject (e.g., a high-risk subject) developing or having a specific disease, ailment, or condition. Prevention does not require reducing the likelihood of a subject having the disease, ailment, or condition to zero; rather, if these steps reduce the risk of the disease, ailment, or condition in a population, then these steps prevent the disease, ailment, or condition in an individual subject within the scope and meaning of this document. For example, in some embodiments, “treatment” refers to taking steps to reduce the likelihood of a subject developing HNC by selectively removing a high-risk lesion in the subject, thereby effectively “preventing” the subject’s cancer. Detailed Implementation

[0041] The diagnosis of oral epithelial dysplasia is based on a histopathological grading system, which has no prognostic value in identifying which lesions have a high risk of transforming into malignant tumors. The method presented in this paper is theoretically based, at least in part, on the finding that the expression of solute carrier family 2 member 1 (SLC2A1, also known as GLUT1) provides a basis for effectively stratifying oral lesions based on the risk of progression to head and neck cancer. Therefore, the method presented addresses the long-standing need for early, accurate assessment of the risk of precancerous lesions for rapid treatment before progression to head and neck cancer, where survival is only 5 years. Furthermore, the method presented facilitates the selective removal of high-risk lesions, thereby avoiding extensive and invasive surgical treatment of subjects to remove lesions with a very low actual risk of transformation, which can significantly impair quality of life in the process.

[0042] In some respects, this document provides methods for evaluating one or more oral lesions. The term "oral lesion" as used herein has a very broad meaning and includes any lesion found in the oral cavity of a subject. In some embodiments, the lesion is found to be located in the oral mucosal layer (lining). This mucosal layer is called the mucosa. The mucosa covers the inner cheek, inner lip, gingiva, tongue, and the roof and floor of the oral cavity. Oral lesions include, for example, oral epithelial dysplasia (OED), oral lichen planus (OLP), oral leukoplakia, oral submucosal fibrosis (OSMF), proliferative verrucous leukoplakia (PVL), and oral erythroplakia. In some embodiments, the lesion is oral epithelial dysplasia (OED). In some embodiments, the lesion is oral lichen planus (OLP).

[0043] In some embodiments, this document provides a method for stratifying one or more oral lesions based on the risk of progression to malignancy. The terms “malignant,” “malignant tumor,” “cancer,” and “cancerous” are used interchangeably herein. The terms “progression” or “transformation” are used interchangeably herein and refer to the process by which a benign lesion becomes malignant (e.g., cancerous). In some embodiments, this document provides a method for stratifying oral lesions based on the risk of progression to malignancy, including measuring the expression of SLC2A1 in the oral lesion and identifying the risk of progression to malignancy based on the expression of SLC2A1 in the lesion. In some embodiments, the method includes identifying oral lesions into a range from high risk to low risk, where high risk means a high probability of progression to malignancy, and low risk means a low probability of progression to malignancy (e.g., a high probability of remaining benign). For example, in some embodiments, the method includes identifying oral lesions as high risk, intermediate to high risk, intermediate risk, intermediate to low risk, or low risk based on the expression of SLC2A1 on the oral lesion. In some embodiments, risk-based stratification of oral lesions includes assigning a risk score to the lesion, as will be described in further detail below.

[0044] In some implementations, other factors are considered when determining how a lesion is classified. For example, in some implementations, the expression of one or more biomarkers other than SLC2A1 in the lesion is measured and used to assess the risk of transformation. These one or more biomarkers are referred to herein as “risk biomarkers” or “additional risk biomarkers.” Alternatively, in some implementations, one or more additional risk biomarkers in the lesion are measured, and the expression of SLC2A1, along with the expression of one or more additional risk biomarkers, is used to identify the risk of the lesion progressing to malignancy. Suitable measurable biomarkers (e.g., additional risk biomarkers) include, for example, nuclear expression of epidermal growth factor receptor (EGFR), SRY-Box transcription factor 2 (SOX-2), Yes1-associated transcriptional regulator (YAP-1), expression of phosphorylated YAP-1, and / or type I interferon (IFN-I) target genes, including CXC motif chemokine ligand 10 (CXCL10), interferon-induced transmembrane protein 3 (IFITM3), ISG15 ubiquitin-like modifier (ISG15), major histocompatibility complex II DRα (HLA-DRA), and major histocompatibility complex II DRβ1 (HLA-DRB1).

[0045] In some embodiments, one or more cell types in oral lesions are measured / quantified and used alone or in combination with the expression level of SCL2A1 in the lesion to assess risk. For example, in some embodiments, myeloid cells in oral lesions are measured. In some embodiments, increased expression of the above-mentioned markers on myeloid cells in the lesion indicates a higher risk of progression to malignancy, which may occur before the appearance of invasive HNC histological features. In some embodiments, lymphocytes (e.g., cytotoxic T lymphocytes) in the lesion are measured / quantified. In some embodiments, macrophages in the lesion are measured / quantified. In some embodiments, regulatory T cells (Tregs), B cells, or regulatory B cells (Bregs) in the lesion are measured / quantified. In some embodiments, a decrease in lymphocytes in the lesion (e.g., a decrease in cytotoxic T lymphocytes, including CD8+ T cells, TBET+CD8+ T cells, CXCR3+CD8+ T cells, natural killer (NK) cells, and γδ-T cells), and / or an increase in the expression of CD206+ macrophages and CD33+PD-L1+ myeloid cells, both indicate high risk. In some embodiments, myeloid cells within the lesion exhibit high IL1 and low IFN-1 markers, indicating a high risk of progression to malignancy. For example, in some embodiments, a decrease in CD8+CD3+ T cells or their activation (e.g., a decrease in CD45+CD8+CD3+ T cells), a decrease in CD8+CD11c+ cells (e.g., a decrease in type 1 conventional dendritic cells cDC1) or their activation (e.g., a decrease in CD45+CD8+CD11c+ cells), an increase in FSC-A+GR1+ cells, an increase in myeloid-derived suppressor cells (e.g., an increase in CD11b+GR1+ cells), a decrease in Cd11b-TCRγ / δ+ cells or their activation (e.g., a decrease in Cd11b-TCRγ / δ+ CD45+ cells), a decrease in Cd11b+ TCRγ / δ+ cells, or a decrease in Glut1+CD206+ macrophages or their activation (e.g., a decrease in Glut1+CD206+ Cd11b+ cells) all indicate a high risk of progression to malignancy. In some embodiments, lesions are selectively eliminated when a high risk of progression to malignancy is identified. In some embodiments, cell types comprising the aforementioned cell types are measured and used to assess the risk of progression to malignancy as described above, without measuring SLC2A1. In some embodiments, cell types comprising the aforementioned cell types are measured and combined with SLC2A1 to assess the risk of progression to malignancy. In some embodiments, increased expression of PDL1+GR1+CD11b+ myeloid suppressor cells indicates a high risk of progression to malignancy. In some embodiments, the expression of markers on specific cell types within the lesion is assessed and used for risk stratification.For example, in some implementations, the expression of markers on epithelial cells, immune cells, and / or myeloid cells in the lesion is measured and used to assess the risk of transformation.

[0046] In some implementations, methods for stratifying one or more oral lesions based on the risk of progression to malignancy include evaluating the expression of one or more cell types in the oral lesions or samples obtained from the oral lesions. In some embodiments, a decrease in CD8+CD3+ T cells or their activation (e.g., a decrease in CD45+CD8+CD3+ T cells), a decrease in CD8+CD11c+ cells (e.g., a decrease in type 1 conventional dendritic cells cDC1) or their activation (e.g., a decrease in CD45+CD8+CD11c+ cells), an increase in FSC-A+GR1+ cells, an increase in myeloid-derived suppressor cells (e.g., an increase in CD11b+GR1+ cells), a decrease in Cd11b-TCRγ / δ+ cells or their activation (e.g., a decrease in Cd11b-TCRγ / δ+ CD45+ cells), a decrease in Cd11b+ TCRγ / δ+ cells, or a decrease in Glut1+CD206+ macrophages or their activation (e.g., a decrease in Glut1+CD206+ Cd11b+ cells) all indicate a high risk of progression to malignancy. In some embodiments, when a high risk of progression to malignancy is identified, the lesion is selectively eliminated.

[0047] In some embodiments, the method includes identifying the oral lesion as high-risk when the expression level of SLC2A1 is equal to or higher than a threshold; or identifying the oral lesion as low-risk when the expression level of SLC2A1 is lower than a threshold. In some embodiments, the method includes identifying the oral lesion as high-risk when the amount or percentage of a given cell type (including the aforementioned cell types) is equal to or higher than a threshold; or identifying the oral lesion as low-risk when the amount or percentage of a given cell type is lower than a threshold. In some embodiments, the method includes identifying the oral lesion as high-risk when the amount or percentage of a given cell type (including the aforementioned cell types) is lower than a threshold; or identifying the oral lesion as low-risk when the amount or percentage of a given cell type is equal to or higher than a threshold. The terms “threshold,” “critical value,” and “control” are used interchangeably herein and refer to a value at or above which a specific risk (e.g., high risk) of a lesion is determined, and a different risk (e.g., low risk) of a lesion is determined below which a different risk (e.g., low risk) of a lesion is determined. In some embodiments, the method includes assigning a risk score to the lesion based on the expression of one or more markers and / or cell types in the lesion. In some implementations, the risk score is specified based on: SLC2A1 expression, a combination of SLC2A1 expression and the expression of additional potential biomarkers, or a combination of SLC2A1 expression and the amount of one or more cell types in the lesion, or the amount or percentage of one or more cell types in the lesion. In some implementations, when determining the risk score of a lesion, the expression of SLC2A1 and the expression of additional biomarkers or cell types are assigned equal values ​​(e.g., equal weights). In some implementations, when determining the risk score of a lesion, one or more factors (e.g., biomarkers, cell types) receive greater weight than another factor. In other words, in some implementations, each distinct factor is not assigned equal weight when determining the overall risk score of a lesion.

[0048] In some embodiments, the expression of solute carrier family 2 member 1 (SLC2A1) and the expression of additional risk markers are measured in multiple oral lesions. In some embodiments, an oral lesion is identified as high-risk when the expression level of SLC2A1 in the lesion is equal to or higher than a threshold for SLC2A1 and / or the expression level of an additional risk marker in the lesion is equal to or higher than a threshold for the additional risk marker. In some embodiments, an oral lesion is identified as low-risk when the expression level of SLC2A1 in the oral lesion is less than a threshold and the expression level of an additional risk marker in the oral lesion is less than a threshold.

[0049] In some embodiments, an oral lesion is identified as high-risk when the SLC2A1-additional biomarker interaction score is higher than a threshold interaction score; and / or when the oral lesion expresses SLC2A1 (or the expression level of SLC2A1 in the oral lesion is equal to or higher than the SLC2A1 threshold), and the Wasserstein distance between the oral lesion and an adjacent oral lesion expressing the additional risk biomarker is equal to or less than the threshold Wasserstein distance. In some embodiments, the interaction score (e.g., the SLC2A1-additional biomarker interaction score) is calculated by multiplying the expression of SLC2A1 in the oral lesion by the expression of the additional risk biomarker in the oral lesion, and dividing the result by the number of cells expressing both SLC2A1 and the additional risk biomarker within the region of interest. In some embodiments, an SLC2A1-additional biomarker interaction score higher than the threshold interaction score indicates that the lesion is high-risk. In some implementations, an oral lesion is identified as high-risk when it expresses SLC2A1 (or the expression level of SLC2A1 in an oral lesion is equal to or higher than the SLC2A1 threshold), and the Wasserstein distance between the oral lesion and a neighboring oral lesion expressing the additional risk marker is equal to or less than the threshold Wasserstein distance. The formula for calculating the Wasserstein distance is as follows:

[0050] Where P and Q are the empirical spatial distributions of the two cell types, and (,) represents the marginal distribution of all (X,Y) joint distributions of P and Q. In some embodiments, a reduced Wasserstein distance between cells expressing SLC2A1 and cells expressing additional risk markers indicates an increased risk of malignancy in cells expressing SLC2A1 and / or cells expressing additional risk markers (e.g., EGFR). In some embodiments, the additional risk marker is EGFR, YAP-1, or SOX-2.

[0051] In some embodiments, the expression of SLC2A1 and / or additional risk markers in oral lesions is measured by obtaining biopsy samples from oral lesions and measuring the expression in the biopsies. In some embodiments, expression is measured at the protein level. In some embodiments, expression is measured by tissue staining. For example, in some embodiments, expression is measured by immunohistochemistry. In some embodiments, expression is measured by other immunoassays, including immunofluorescence, FISH, multiplexing, etc. In some embodiments, expression is measured at the nucleic acid level (e.g., mRNA). For example, in some embodiments, expression is measured at the nucleic acid level using PCR-based techniques (e.g., qPCR, FISH, and spatial transcriptomics, etc.).

[0052] In some embodiments, SLC2A1 expression and / or the expression of additional risk markers are measured at the cell type level, and the expression at the cell type level is used to determine the risk of the lesion progressing to malignancy. For example, in some embodiments, SLC2A1 expression is measured on myeloid cells within the lesion. In some embodiments, the amount of Glut1+CD206+CD11b+ macrophages within the lesion is measured. In some embodiments, myeloid cells are identified in a biopsy by immunostaining, and SLC2A1-positive myeloid cells are measured. In some embodiments, myeloid cells are identified by the expression of one or more cell surface markers (such as CD33, CD68, or both) associated with myeloid cell lineage, and SLC2A1-positive myeloid cells are measured. In some embodiments, the presence of SLC2A1-positive myeloid cells in the lesion indicates that the lesion is of high risk. In some embodiments, an amount of Glut1+CD206+CD11b+ macrophages below a threshold indicates that the lesion is of high risk.

[0053] In some embodiments, the expression of SLC2A1 and / or the expression of additional risk markers in oral lesions is measured directly in the oral cavity of the subject. In other words, in some embodiments, instead of obtaining a biopsy, the expression of SLC2A1 (and / or the expression of additional risk markers) in oral lesions within the subject's oral cavity is measured. In some embodiments, the expression of SLC2A1 in oral lesions is measured by contacting the oral lesion with an agent that binds to SLC2A1. In some embodiments, the expression of additional risk markers in oral lesions is measured by contacting the oral lesion with an agent that binds to additional risk markers. In some embodiments, the agent includes a marker (also referred to as a "detectable marker") such that the agent binds to lesions expressing a given marker (e.g., SLC2A1, YAP-1, SOX-2, EGFR, etc.), and the lesion can subsequently be identified by detecting the marker. In some embodiments, the marker includes a fluorescent marker. Any suitable fluorescent marker can be used. Exemplary suitable fluorescent markers include fluorescein, coumarin, rhodamine, anthocyanins, and their derivatives. In some embodiments, the marker includes a near-infrared dye. In some implementations, the label includes fluorescein isothiocyanate (FITC).

[0054] In some embodiments, the agent binding to SLC2A1 is a peptide. In some embodiments, the peptide binds to a portion of the extracellular domain of SLC2A1. In some embodiments, the peptide comprises 5-50 amino acids. In some embodiments, the peptide comprises 5-40 amino acids, 5-35 amino acids, 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, or 5-10 amino acids. In some embodiments, the peptide is conjugated to a detectable tag via a linker. The linker can be of any suitable length and amino acid combination to facilitate linker-peptide attachment without impairing the peptide's ability to bind to SLC2A1.

[0055] In some embodiments, detecting the marker involves applying a light source to the oral lesion to excite the marker. For example, in some embodiments, detecting the marker involves applying a light source (e.g., a laser) with an appropriate excitation wavelength to the oral lesion to excite the marker and allow its detection. In some embodiments, the marker may be visually detected. In some embodiments, the marker may be detected by the naked eye (e.g., without the need for detection equipment such as a microscope). Therefore, the method provided herein facilitates the simple and economical identification of high-risk oral lesions without the need for expensive medical equipment or biopsies, thereby providing rapid and convenient point-of-care diagnosis and treatment of high-risk lesions in subjects.

[0056] In some embodiments, the methods provided herein further include selectively eliminating oral lesions when they are identified as high-risk. In some embodiments, the methods provided herein further include administering an inhibitor of IL-1α signaling to a subject. In some embodiments, the inhibitor of IL-1α signaling may delay (e.g., prevent, inhibit, slow) the continued progression of high-risk lesions. In some embodiments, the inhibitor of IL-1α signaling includes an anti-IL-1R antibody. In some embodiments, the inhibitor of IL-1α signaling (e.g., an anti-IL-1R antibody) is administered systemically to the subject. In some embodiments, the inhibitor of IL-1α signaling (e.g., an anti-IL-1R antibody) is administered to high-risk oral lesions, such as by intralesional injection. In some embodiments, the methods provided herein further include monitoring oral lesions when they are not identified as high-risk. For example, when oral lesions are identified as low-risk (or low to intermediate risk, or intermediate risk), these methods may include monitoring oral lesions.

[0057] In some aspects, this document provides methods for treating subjects at risk of developing head and neck cancer (HNC). In some embodiments, a patient is identified as being at risk of HNC due to the presence of one or more oral lesions in the subject's oral cavity. In some embodiments, this document provides methods for treating subjects at risk of developing head and neck cancer (HNC), comprising: stratifying oral lesions based on the risk of progression to malignancy as described above; and selectively eliminating an oral lesion when it is identified as high-risk. In some embodiments, the methods provided herein further include monitoring oral lesions when they are not identified as high-risk. For example, these methods may include monitoring oral lesions when they are identified as low-risk (or low to intermediate risk, or intermediate risk).

[0058] In some embodiments, selective elimination of oral lesions includes removing the oral lesions from the subject. For example, lesions (e.g., high-risk lesions) can be removed by incision or other surgical resection techniques. In some embodiments, selective elimination of oral lesions includes damaging / destroying the lesion tissue, such as through ablation, thermotherapy, radiation therapy, chemotherapy, cryotherapy, etc. In some embodiments, "monitoring" oral lesions includes repeating the methods described herein after an appropriate duration to reassess the risk of the lesion transforming into a malignant tumor. In some embodiments, "monitoring" oral lesions includes repeating the methods described herein after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer than 1 year to reassess the risk of the lesion transforming into a malignant tumor.

[0059] In some embodiments, a method of treating a subject at risk of head and neck cancer (HNC) includes assessing the risk of a single lesion transforming into a malignant tumor and selectively eliminating or monitoring the lesion based on that risk assessment. In some embodiments, a method of treating a subject at risk of head and neck cancer includes simultaneously assessing the risk of multiple oral lesions transforming into malignant tumors and selectively eliminating or monitoring each of the assessed multiple oral lesions.

[0060] In some aspects, this document provides a method for directly measuring SLC2A1 expression in at least one oral lesion within the oral cavity of a subject (as opposed to measuring SLC2A1 expression in a biopsy). In some embodiments, SLC2A1 expression is measured by contacting at least one oral lesion with an agent that binds to SLC2A1. In some embodiments, the agent comprises a marker (e.g., a detectable marker). In some embodiments, SLC2A1 expression on an oral lesion is measured by contacting the oral lesion with an agent that binds to SLC2A1. In some embodiments, the agent comprises a marker (also referred to as a “detectable marker”) such that the agent binds to the lesion expressing SLC2A1, and the lesion can then be identified by detecting the marker. In some embodiments, the marker comprises a fluorescent marker. Any suitable fluorescent marker can be used. Exemplary suitable fluorescent markers include fluorescein, coumarin, rhodamine, anthocyanins, and derivatives thereof. In some embodiments, the marker comprises fluorescein isothiocyanate (FITC).

[0061] In some embodiments, the agent binding to SLC2A1 is a peptide. In some embodiments, the peptide comprises 5-50 amino acids. In some embodiments, the peptide comprises 5-40 amino acids, 5-35 amino acids, 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, or 5-10 amino acids. In some embodiments, the peptide is conjugated to a detectable tag via a linker. The linker can be of any suitable length and amino acid combination to facilitate linker-peptide attachment without impairing the peptide's ability to bind to SLC2A1.

[0062] In some embodiments, detecting the marker involves applying a light source to the oral lesion to excite the marker. For example, in some embodiments, detecting the marker involves applying a light source (e.g., a laser) with an appropriate excitation wavelength to the oral lesion to excite the marker and allow its detection. In some embodiments, the marker can be visually detected. In some embodiments, the marker can be detected by the naked eye (e.g., without the need for detection equipment such as a microscope).

[0063] Example Example 1 Oral leukoplakia and oral lichen planus (OLP) are the most common oral mucosal lesions, and approximately 284 million people worldwide require long-term follow-up for head and neck squamous cell carcinoma (HNC) screening. Current methods, such as autofluorescence imaging, tissue reflectance, cytology, and circulating tumor DNA, may help differentiate precancerous lesions—oral epithelial dysplasia (OED)—from HNC. However, from a histological perspective, this distinction has never posed a diagnostic challenge. In contrast, the main challenge in caring for these patients is that most OEDs and OLPs remain benign, and surgical resection of benign lesions is impractical due to the reduced overall quality of life associated with invasive and aggressive procedures. Furthermore, it is impossible to maintain high-frequency follow-up for every patient to determine whether previously benign OEDs / OLPs have progressed to malignancy, and to date, consistent characteristics have not been established to differentiate between high-risk OEDs and low-risk lesions. In fact, standard histological grading cannot predict the risk of transformation.

[0064] Although most lesions remain benign, some mild OEDs can progress to HNC within a year. Once diagnosed, the 5-year overall survival rate for HNC is extremely low. Therefore, clinical examination is an important component of HNC risk assessment.

[0065] During standard clinical examination of OED / OLP, biopsy should be performed if erythematous color changes and induration are present. These features indicate inflammatory and mechanical changes in the tumor microenvironment early in the development of HNC. This paper constructs a model to reproduce these immune and mechanical changes during HNC development. Two driver oncogenes commonly associated with HNC were selected. The first gene is SOX2, located at a hotspot at 3q26.3 and amplified in ~21% of HNCs. SOX2 excludes cytotoxic T lymphocytes (CTLs) from the tumor microenvironment (TME) by inhibiting type I interferon-1 (IFN-I)-mediated cancer immunoassay. The second gene is YAP1, a signaling hub for sensing extracellular stiffness. YAP1 is frequently activated by genomic amplification at the 11q22 locus, loss-of-function mutations in the YAP1-repressing FAT1 gene, and environmental mechanical cues. More than two-thirds of HNCs show evidence of YAP1 pathway activation. Therefore, two genetically engineered mouse models (GEMMs) were established, driven by oral epithelial cell-specific activation of Sox2 and Yap1, respectively. Both GEMMs exhibited highly faithful histological transformation of normal oral mucosa to OED and then to HNC. Furthermore, using paired longitudinal specimens, reproducible immunoprofiling changes were shown in high-risk OEDs after transformation. The results presented in this paper collectively demonstrate that induction of the SLC2A1 (aka GLUT1)-glycolysis pathway and loss of the IFN-I marker establish a robust tumorigenic microenvironment that precedes the appearance of HNC histological features.

[0066] To complement on-slide biopsy findings, laser confocal microscopy (CLE) was developed. CLE, approved by the U.S. Food and Drug Administration (FDA), enables the monitoring of submucosal molecular targets at microscopic resolution and aids in the detection of precancerous epithelial lesions. By combining tissue and clinical evaluations, the detection of truly high-risk oEDs can be achieved. In summary, the results presented in this article demonstrate that immunological, metabolic, and biomechanical markers can inform on-slide and optical biopsy techniques, thereby enabling accurate detection of high-risk oEDs.

[0067] result Based on HPV status, HNC can be classified into two distinct diseases with different causes. HPV+ HNC responds better to chemotherapy. Therefore, HPV+ HNC provides a model for identifying mechanisms of immunosuppressive activity.

[0068] The proliferation of suppressive myeloid cells and the depletion of CTLs are hallmarks of OED transformation.

[0069] Transformed omental edema (OED) in the longitudinal cohort was stained using a multispectral imaging (MSI) platform. Six to 13 regions of interest were selected from each slide for high-quality scanning and subsequent analysis. Malignant transformation of OED was associated with extensive infiltration of myeloid cells labeled CD33+PD-L1+ (Fig. 1A) and CD68+PD-L1+ (data not shown). Atypical epithelial cells and tumor islands were surrounded by effector CD8+ CTLs of varying densities. Notably, the frequencies of two functional Th1 subsets producing IFN-γ—T-BET+CD8+ and CXCR3+CD8+ CTLs—significantly decreased after malignant transformation (Figs. 1B-1C).

[0070] The absence of the IFN-I marker is associated with the depletion of CTLs in HNCs. UMAP analysis was performed on single-cell HNCs using an enhanced workflow that stabilizes immune lineage typing. HPV+HNCs showed a significant reduction in cDC1 (cluster 25), CXCL10-overexpressing macrophages (clusters 10, 19, and 20), and γδT cells (cluster 4) (Fig. 2A). Clusters 10, 19, and 20 expressed high levels of IFN-I target genes, such as CXCL10, IFITM3, ISG15, HLA-DRA, and HLA-DRB1. IFN-I and its target genes play a role in antigen-presenting cell (APC) maturation and CTL chemotaxis. Induction of IFN-I markers depends on the proper activation of pattern recognition receptors (PRRs), which are central to the host immune system's detection of "non-self" substances. Unstable cancer genomes and treatments lead to DNA damage, which acts as a danger signal to activate the PRR. STING is an important cytoplasmic DNA-sensing PRR that promotes anti-tumor immunity. STING translates cytoplasmic DNA signaling into IFN-I induction and promotes APC maturation. TIL profiles of 520 HNC samples were analyzed using a deconvolution method. STING expression levels were positively correlated with M1 macrophages, CD8+ CTLs, and γδT cells (Figure 2B). The STING-CTL correlation was further validated using a human HNC tissue microarray composed of the cores of 264 samples.

[0071] Malignant transformation of oral mucosa upregulates glycolysis markers To recognize transformed cells, APCs migrate to the lesion, process antigens, synthesize high levels of Th1 cytokines de novo, and cross-initiate CTLs. CD8+ CTLs rapidly replicate their genome, bind to synapses, and deliver cytotoxic payloads to transformed cells. These processes are powered by extracellular glucose, and a lack of metabolic support leads to CTL depletion. Hypoxia is a key feature of HNCs, which exhibit a median oxygen partial pressure (pO2) of 9 mmHg, compared to 40–60 mmHg in paired normal mucosa. In HNCs, hypoxia shifts the dominant metabolic pathway from oxidative phosphorylation to glycolysis, which depletes glucose reserves in the TME. Indeed, hypoxia upregulates glycolytic markers in HNC cells, including SLC2A1, hexokinase 1 (HK1), BCL2 interactor protein 3-like protein (BNIP3L), and pyruvate dehydrogenase kinase 1 (PDK1). Figures 3A-3B Then, 520 HNC cases were compared with 44 normal mucosa cases, and the transformation was found to increase glycolytic markers, including SLC2A1, PDK1, and PKM (Figure 4A). Deconvolution of TILs revealed a negative correlation between SLC2A1 levels and anti-tumor TIL subsets (such as CTLs, memory T cells, and M1 macrophages) (Figure 4B).

[0072] SOX2-driven OED progression is characterized by IFN-I marker loss and upregulation of glycolysis. To identify markers of high-risk oral edema (OED), better models are needed to rigorously interpret changes in epithelial and immune markers over time. SOX2 is frequently amplified in squamous cell carcinoma (including HNC). To longitudinally monitor SOX2-driven OED transformation, K5-CreER was hybridized to Rosa26-Sox2-enhanced green fluorescent protein (EGFP), followed by topical application of tamoxifen to the oral mucosa to construct a Sox2-GEMM model in which oral epithelial cells express Sox2-EGFP. Sox2-GEMM or K5-CreER control mice were treated with the carcinogen DMBA or a loading medium. Histological examination of the buccal mucosa and tongue was performed to determine the penetrance of OED / HNC. The K5-CreER+;Sox2+ / - strain without DMBA showed approximately 60-80% HNC penetrance at 160 days post-induction, while the K5-CreER+;Sox2+ / + strain showed 100% spontaneous penetrance at approximately 4 weeks post-induction (Fig. 5A). Some tumors were coarsely visible (Fig. 5B), and most tumors formed by the K5-CreER+;Sox2+ / + strain were detectable by microscopy at four weeks post-induction. A microendoscope was designed to enter the mouse oral cavity for direct laser confocal imaging and to display EGFP-expressing Sox2+ transformed epithelial cells in live animals (Fig. 5C).

[0073] While implantable HNC models are valuable for characterizing HNC TME, they fail to reproduce the complexity of the matrix, cytological and structural details, and importantly, the temporal progression from OED to HNC (Figs. 5F-5G). Sox2 drives the spontaneous development of infiltrating HNC. The histology of Sox2-driven cancers is highly similar to that of human disease, including high-grade cytological features, keratin bead formation, matrix establishment, and immune cell infiltration (Figs. 5H-5I). Notably, this model shows all the histological features of OED prior to HNC (Figs. 5J-5K). These tumors are Sox2 positive with a high proliferation index (Ki-67). The staining patterns of p53 and p63 are similar to those of human HNC (Fig. 6A). Sox2-GEMM shows elevated levels of Slc2a1 mRNA after transformation (Fig. 6B). Normal mucosa was also stained with a Slc2a1-specific antibody after transformation to OED at 2 weeks post-induction and to HNC at 5 weeks post-induction. In normal mucosa, Slc2a1 staining is negative or shows only a weak cytoplasmic staining pattern. However, in transformed OED, Slc2a1 shows a distinct cell membrane staining pattern in the lesion area, with a clear boundary from the adjacent relatively normal epithelium lacking cell membrane staining. HNC, on the other hand, shows a diffuse, strong cell membrane staining pattern (Fig. 6C).

[0074] Immunological markers appeared before the development of HNC histology. To test whether early immune alterations could identify high-risk oral edema (OED), single-cell (sc)RNA-Seq of intralesional immune cells was performed on transformed benign mucosa. Normal oral mucosa contains Th1, Th2, and Th17 cells, as well as balanced M1 macrophages and suppressive myeloid cells with high expression of the IFN-I marker. Sox2-induced transformation triggered myeloid cell expansion and simultaneous CTL loss (Fig. 7A-7C), which has also been observed in human samples (Fig. 1). Transformation-driven myeloid clusters exhibited a distinct compensatory suppressive signature. For example, their subsets expressed Pd-l1. Functional suppression also involved Galectin-9+, Cox2+, or Tgfb1+ clusters that were negative for Pd-l1. As OED progressed, the suppressive signature became more diverse (Fig. 7B-7C). Notably, at the two-week time point, no signs of invasive disease were observed, only OED. However, key features of local immunosuppression were established at this early stage. Therefore, high-risk immunomarkers appear before HNC histology, making them ideal biomarker candidates for early detection.

[0075] After isolating myeloid cell clusters, their differentiation trajectories were plotted using the affinity transfer embedding-based thermal diffusion potential algorithm (PHATE) (92). Myeloid expansion was primarily attributed to de novo recruitment (discontinuous clusters), in addition to in situ proliferation (continuous clusters) (Fig. 7D). Differentially expressed genes from myeloid cells derived from normal and transformed oral mucosa were then characterized. Resting myeloid cells from normal mucosa were distributed in the southwest and northeast corners of the PHATE plot (Fig. 7D, left inset). Transformation led to proliferation and recruitment of myeloid cells, which exhibited unique markers including high levels of Slc2a1 and Pd-l1, while IFN-I markers such as Stat1 and Isg15 were lost (Fig. 7E-7H).

[0076] Yap1-driven GEMM reveals key histological, IHC, and immunological features of HPV-HNC. Hardening is an early clinical sign of OED transformation, indicating altered lesion mechanical properties. To mimic this process, another GEMM was constructed by hybridizing K5-CreER with Rosa26-Yap5sa to generate a K5-CreER+;Yap5sa+ / - composite strain. Phosphorylation of Yap1 can retain the protein in the cytosol and promote its degradation. The Yap5sa mutant contains amino acid alterations that prevent Yap1 from phosphorylating. Constitutive active Yap5sa expression in the oral epithelium was induced by topical application of tamoxifen to the oral mucosa. Active Yap1 is a potent driver of OED transformation; 100% of K5-CreER+;Yap5sa+ / - mice (n=13) developed microscopically visible keratotic HNCs within 6 weeks after induction. The Yap1-GEMM highly reproduced the histological features of human HNCs. These lesions consist of sheet-like and nest-like squamous tumor cells with abundant eosinophilic cytoplasm, high cellular and nuclear atypia, keratin bead formation, and high mitotic counts. Importantly, Yap1-GEMM exhibits stromal complexity, immune infiltration, and structure similar to human HNC.

[0077] Under IHC staining, approximately 30%-60% of tumor cells showed significant Yap1 nuclear staining, suggesting that Yap1 signaling has been activated. In contrast to Sox2-GEMM, this type of tumor was negative for Sox2 expression. The p63 staining pattern was similar to that in human HNC. Notably, p16 expression was absent in the tumor islands compared to the adjacent relatively normal epithelium. Figure 8 This key feature is unique to HPV-HNC, in contrast to HPV+ tumors with elevated p16 expression levels.

[0078] To characterize the immune markers associated with Yap1-driven OED conversion, scRNA-Seq was performed on the buccal mucosa where most lesions occurred. After filtering, 6,375 high-quality transcriptomes were obtained. Major immune lineages were identified by labeling the most differentially expressed genes and ~70 anchored genomes (Fig. 9A) (58,59). Similar to human OED conversion, myeloid cells expressing high levels of TnF and IFN-I marker genes (cluster 1) were significantly reduced, while Tox+ exhausted T cells (cluster 2) and CD206+ M2 macrophages (clusters 5 and 9) expanded (Fig. 9B).

[0079] Targeted laser confocal microscopy (CLE) is a real-time optical biopsy tool that can reveal details of mucosal epithelial cells during hematoma nucleation (HNC). Currently, the diagnosis and treatment of OED / OLP rely on slide diagnostics and clinical examination. General tissue autofluorescence and reflectance cannot provide the molecular information abundant in high-risk OED / OLP. The ADA does not recommend tissue autofluorescence or reflectance as an adjunct diagnostic tool. To improve the clinical ability to monitor high-risk molecular markers, this paper develops a robust CLE method that utilizes a miniature scanner and actuators to probe tissue at microscopic resolution by deflecting laser beams laterally and axially. These instruments are fiber-coupled and miniaturized to the millimeter scale for minimally invasive access to typically small oral lesions (Figure 10A). Both anteroposterior and lateral imaging geometries are provided to maintain adequate contact between the optics and the tissue surface, resulting in high-quality images. High-speed scanning allows for real-time image acquisition while minimizing motion artifacts.

[0080] Epidermal growth factor receptor (EGFR) overexpression was observed in >90% of HNCs, showing potential for early oncogenicity detection. The peptide QRHKPRE showed high affinity binding to the extracellular domain of EGFR. C57BL / 6 wild-type mice were fed 50 μg / ml of drinking water for 16 weeks, followed by 8 weeks of normal water. Mucosa from normal mice without tongue lesions was sprayed with the FITC-QRHKPRE peptide and subjected to CLE. Normal mucosa showed a low background fluorescence signal compared to the overall picture, with only scattered positive cell boundaries (Fig. 10B). In contrast, histologically validated HNCs showed a lattice-like imaging pattern after binding to the FITC-labeled EGFR-targeting peptide. Keratin did not bind to the EGFR-targeting imaging peptide, and its signal was suppressed in the background (Fig. 10C). Therefore, CLE provides an optical biopsy tool that can complement markers on a slide for longitudinal tracking of high-risk features.

[0081] Example 2 Since the penetrance of the buccal site is 100%, Sox2 and Yap1-driven genetically engineered mouse models (as used in Example 1) can be used to establish optical biopsy features for high-risk OEDs. First, baseline readings for different fluorescence channels can be established. For example, Cy5-labeled EGFR-targeting peptides and IRDye800-labeled SLC2A1-targeting peptides can be sprayed onto the oral mucosa of both types of GEMMs. After incubation for 5 minutes, excess peptides can be rinsed off. Then, tamoxifen application to the oral mucosa can induce K5-CreER+;EGFP-Sox2+ / + and K5-CreER+;Yap5sa+ / - GEMMs. Following induction, bilateral buccal mucosa were examined weekly until all mice were euthanized after six weeks. Both EGFR and SLC2A1 are plasma membrane proteins that provide signals to identify cell boundaries in imaging analysis. The number of EGFR+ and SLC2A1+ cells can be quantified, and their mean intensity determined. For each region of interest (ROI), the EGFR-SLC2A1 interaction score can be calculated. For example, the EGFR-SLC2A1 interaction score can be calculated using the following formula:

[0082] in, n The number of double-positive cells, I EGFR and I SLC2A1 The fluorescence intensities of EGFR and SLC2A1 are respectively. It can be determined whether the intensity of a single biomarker or interaction score increases over time, and thus such scores can be used to assess the transformation risk of a given lesion. Furthermore, the spatial distance between EGFR and SLC2A1 positive features may decrease with mucosal transformation. Therefore, the spatial distance between EGFR and SLC2A1 positive features may also be a factor in determining the transformation risk of a given lesion. In some implementations, the Wasserstein distance (W value) between EGFR+ and SLC2A1+ cells can be used to assess their spatial relationship. The formula for calculating the Wasserstein distance is as follows:

[0083] Here, P and Q are the empirical spatial distributions of the two cell types, and (,) represents the joint distribution of all (X,Y) marginal distributions of P and Q. It can be determined whether the W value decreases over time during OED transformation.

[0084] Example 3 Blocking early IL-1 signaling can delay the onset of Sox2-driven HNSCC.

[0085] To better evaluate the role of this IL-1α amplified signaling circuit in Sox2-driven conversion, intraperitoneal injection of 200 μg of anti-IL1r1 was administered one week post-induction to treat the induced IL-1α signaling pathway. K5-CreER;Sox2 + / + Mice. Il1r1 blockade significantly prolonged the survival of these mice. Figure 11A RNA-Seq was performed on the buccal mucosa of both groups. The most significantly upregulated pathways in the anti-Il1r1 treatment group were IFN-γ response, allogeneic transplant rejection, and IFN-α response, suggesting enhanced Th1 immunity. Then, K5-CreER;Sox2 + / + GEMM and Il1r1 - / - Mouse hybridization to generate K5-CreER;Sox2 + / + ;Il1r1 - / - Composite strain. Due to the rapid tumor development in this model, most buccal cancers are microscopic cancers. The regions of invasive tumor islands were investigated, and compared with... K5-CreER;Sox2 + / + ;Il1r1 + / + Compared with littermates, K5-CreER;Sox2 + / + ;Il1r1 - / - Mice showed a significant reduction in tumor area ( Figure 11B Flow cytometry analysis showed that... Il1r1 The absence of [a specific substance] significantly reduced the infiltrated MDSCs in Sox2-driven HNSCCs.

[0086] Additional experiments were conducted to elucidate the role of IL1 signaling in oral lesions with malignant tumors. The results shown in Figure 12 indicate that blocking IL1 signaling can improve outcomes in high-risk oral lesions. Myeloid cells within the lesions exhibited a novel marker of high IL1 and low IFN-I, suggesting an increased risk of malignant transformation. Genetic and pharmacological methods significantly reduced the level of Glut1+CD206+ macrophages within the lesions and delayed tumor onset. Figure 12A shows tumor growth after injection of MOC2 tumor cells (Moc2-gW cells) or MOC2-Sox2 (Sox2-positive) cells. Tumor volume is shown. Figure 12B shows the percentage of CD8+CD3+ T cells in the gW and Sox2 groups, as analyzed by flow cytometry. Figure 12C shows the frequency of CD45+ cells within the CD8+CD3+ T cell population. Figure 12C shows that the frequency of CD45+ cells in the Cd8+CD3+ T cell population was lower in the MOC2-Sox2 group compared to the MOC2-gW group. Figure 12D shows the percentage of CD8+CD11c+ dendritic cells in both the MOC2-gW and MOC2-Sox2 groups as analyzed by flow cytometry. Figure 12E shows the frequency of CD45+ cells in the CD8+Cd11c+ cell population in both the MOC2-Gw and MOC2-Sox groups. As shown in Figure 12E, the percentage of CD45+ cells in the Cd8+Cd11c+ cell population was lower in the MOC2-Sox2 group compared to the MOC2-gW group. Figure 12F shows the flow cytometry analysis of FSCA+ and Gr1+ cells in both the MOC2-gW and MOC2-Sox2 groups. Figure 12G shows the frequency of CD11b+ cells in Gr1+ MDSCs. As shown in Figure 12G, the frequency of CD11b+Gr1+ MDSCs was significantly higher in the MOC2-Sox2 group compared to the MOC2-gW group. Figure 12H shows a heatmap analysis of Sox2, IL1a, and Csf2 expression in EV and Sox2 mice. Figure 12I shows the fold change in IL1a mRNA after administration of the squamous cell line UMSC22b to gW or Sox2 mice. The fold change in IL1a mRNA was significantly higher in Sox2 mice compared to gW mice. To further investigate the role of ILR1 signaling, K5-Cre / Sox2+ / + mice were crossed with IL1r1+ / + or IL1R1- / - mice. The survival probability over 75 days is shown in Figure 12J. Compared with K5-Cre; Sox2+ / +; IL1r+ / + mice, K5-Cre; Sox2+ / +; IL1r- / - mice had an increased survival rate and a longer survival duration, indicating that IL1r signaling plays a role in tumor growth and progression / malignant transformation.To further evaluate cell types in these groups, flow cytometry was performed on samples obtained from the K5-Cre; Sox2+ / +; IL1r+ / + and K5-Cre; Sox2+ / +; IL1r- / - groups. Figure 12K shows the cellular expression of TCRγ / δ and CD11b. As shown in Figure 12K, a unique Cd11b-TCRγ / δ+ cell population was identified in the IL1r+ / + group, which was not prominent in the IL1r- / - group. Both groups had distinct Cd11b+TCRγ / δ- cell populations. Figure 12L shows the frequency of CD45+ cells within the Cd11b-TCRγ / δ+ cell population. The frequency of CD45+ cells was significantly higher in the IL1+ / + group compared to the IL1- / - group. Figure 12M shows the expression of Glut1 and CD206 cells. Figure 12N shows the frequency of Cd11b+ cells within the Glut1+CD206+ cell population. As shown in the figure, the IL1- / - group had fewer Glut1+CD206+Cd11b+ macrophages compared to the IL1r+ / + group. Tissue staining was performed to evaluate tissues from K5-Cre; Sox2+ / +;IL1r+ / + and K5-Cre; Sox2+ / +;IL1r- / - mice. The mean tumor surface area was quantified in Figure 12P. As shown in Figure 12P, the IL1r- / - group had a significantly smaller tumor surface area compared to the IL1r+ / + group.

[0087] To further investigate the role of IL1 signaling in oral lesions and malignant tumors, tumor growth was induced in mice, followed by administration of PBS (control) or anti-IL1r1 antibody. As shown in Figure 12Q, the anti-IL1r1 treatment group had higher survival rates and longer survival durations compared to the control. Flow cytometry was performed to assess cell populations after treatment with PBS or anti-IL1r1. The results are shown in Figure 12R. The anti-IL1r1 group had a significantly reduced number of PDL1+GR1+ cells compared to the PBS-treated group. Figure 12S shows the frequency of PDL1+GR1+CD11B+ MDSCs in the different treatment groups. Anti-IL1r1 mice had fewer PDL1+GR1+CD11B+ MDSCs compared to the control.

Claims

1. A method of stratifying an oral lesion based on risk of progression to a malignant tumor, the method comprising: a) measuring expression of solute carrier family 2 member 1 (SLC2A1) in an oral lesion; and b) identifying the oral lesion as high risk when the level of expression of SLC2A1 is at or above a threshold value; or identifying the oral lesion as low risk when the level of expression of SLC2A1 is below a threshold value.

2. The method of claim 1, wherein, The expression of SLC2A1 in the oral lesion is measured by obtaining a biopsy sample from the oral lesion and measuring expression of SLC2A1 in the biopsy.

3. The method of claim 2, wherein, The expression of SLC2A1 is measured by histological staining.

4. The method of claim 2, wherein, The expression of SLC2A1 in the oral lesion is measured directly on the oral lesion in the subject's mouth.

5. The method of claim 4, wherein, The expression of SLC2A1 on the oral lesion is measured by contacting the oral lesion with an agent that binds SLC2A1, wherein the agent comprises a label, and subsequently detecting the label.

6. The method of claim 5, wherein, Detecting the label comprises applying a light source to the oral lesion, thereby activating the label, and visually detecting the label.

7. The method of any one of claims 1-6, further comprising: The oral lesion is selectively eliminated when the oral lesion is identified as high risk; or the oral lesion is monitored when the oral lesion is identified as low risk.

8. A method of stratifying an oral lesion based on risk of progression to a malignant tumor, the method comprising: a) measuring expression of solute carrier family 2 member 1 (SLC2A1) and expression of an additional risk marker in a plurality of oral lesions within a region of interest of a subject's mouth; and b) identifying an oral lesion as high risk when: i) the SLC2A1-additional marker interaction score of the oral lesion is above a threshold interaction score; and / or ii) the oral lesion expresses SLC2A1 and the Waddell distance between the oral lesion and an adjacent oral lesion expressing the additional risk marker is at or below a threshold Waddell distance.

9. The method of claim 8, wherein, The SLC2A1-additional marker interaction score of an oral lesion is calculated by multiplying the expression of SLC2A1 in the oral lesion by the expression of the additional risk marker in the oral lesion and dividing the result by the number of cells within the region of interest that express both SLC2A1 and the additional risk marker.

10. The method of claim 8 or claim 9, wherein, The expression of SLC2A1 and the expression of the additional risk marker are measured directly on the plurality of oral lesions in the subject's mouth.

11. The method of claim 10, wherein, The expression of SLC2A1 and the expression of the additional risk marker are measured by contacting the plurality of oral lesions with an agent that binds SLC2A1 and an agent that binds the additional risk marker, wherein each agent comprises a label, and subsequently detecting the labels.

12. The method of claim 11, wherein, Detecting the labels comprises applying a light source to the plurality of oral lesions, thereby activating the labels, and visually detecting the labels.

13. The method of any of claims 8-12, further comprising: The oral lesion is selectively eliminated when the oral lesion is identified as high risk.

14. The method of any one of claims 8-13, wherein, The additional risk marker is nuclear expression of epidermal growth factor receptor (EGFR), SRY-Box Transcription Factor 2 (SOX-2), or Yes1 Associated Transcriptional Regulator (YAP-1).

15. A method of treating a subject at risk of developing head and neck cancer (HNC), the method comprising: a) measuring expression of solute carrier family 2 member 1 (SLC2A1) in an oral lesion of the subject; b) identifying the oral lesion as high risk when the level of expression of SLC2A1 is at or above a threshold level; or as low risk when the level of expression of SLC2A1 is below a threshold; and c) selectively eliminating the oral lesion when the oral lesion is identified as high risk; or monitoring the oral lesion when the oral lesion is identified as low risk. The expression of SLC2A1 in the oral lesion is measured by obtaining a biopsy from the oral lesion and measuring expression of SLC2A1 in the biopsy.

16. The method of claim 15, wherein, The expression of SLC2A1 is measured by histological staining.

17. The method of claim 16, wherein, The expression of SLC2A1 in the oral lesion is measured directly on the oral lesion in the subject's mouth.

18. The method of claim 15, wherein, The expression of SLC2A1 on the oral lesion is measured by contacting the oral lesion with an agent that binds SLC2A1, wherein the agent comprises a label, and subsequently detecting the label.

19. The method of claim 18, wherein, Detecting the label comprises applying a light source to the oral lesion, thereby activating the label, and visually detecting the label.

20. The method of claim 19, wherein, 21. A method of treating a subject at risk of developing head and neck cancer (HNC), the method comprising: a) measuring expression of solute carrier family 2 member 1 (SLC2A1) in each of a plurality of oral lesions directly in the subject's mouth; b) identifying an oral lesion as high risk when the level of expression of SLC2A1 in the oral lesion is at or above a threshold level; and / or as low risk when the level of expression of SLC2A1 in the oral lesion is below a threshold level; and c) selectively eliminating high risk oral lesions of the subject's mouth.

22. The method of claim 21, further comprising monitoring low risk oral lesions. The expression of SLC2A1 is measured by contacting the plurality of oral lesions with an agent that binds SLC2A1, wherein the agent comprises a label, and subsequently detecting the label.

23. The method of claim 21 or claim 22, wherein, Detecting the label comprises applying a light source to the oral lesion, thereby activating the label, and visually detecting the label.

24. The method of claim 23, wherein, 25. A method of treating a subject at risk of developing head and neck cancer (HNC), the method comprising: a) measuring expression of solute carrier family 2 member 1 (SLC2A1) and expression of an additional risk marker in a plurality of oral lesions within a region of interest of the subject's mouth; b) identifying an oral lesion as high risk when: i) the SLC2A1-additional marker interaction score for the oral lesion is above a threshold interaction score; and / or ii) the SLC2A1-additional marker interaction score for the oral lesion is above a threshold interaction score and the expression of the additional risk marker in the oral lesion is at or above a threshold level. ii) the oral lesion expresses SLC2A1, and the Wassertain distance between the oral lesion and an adjacent oral lesion expressing the additional risk marker is equal to or less than a threshold Wassertain distance; and c) selectively eliminating a high-risk oral lesion of the oral cavity of the subject.

26. The method of claim 25, wherein, The SLC2A1-additional marker interaction score of an oral lesion is calculated by multiplying the expression of SLC2A1 in the oral lesion with the expression of the additional risk marker in the oral lesion, and dividing the result by the number of cells within the region of interest that express both SLC2A1 and the additional risk marker.

27. The method of claim 25 or claim 26, wherein, The expression of SLC2A1 and the expression of the additional risk marker are measured directly on the plurality of oral lesions within the oral cavity of the subject.

28. The method of claim 27, wherein, The expression of SLC2A1 and the expression of the additional risk marker are measured by contacting the plurality of oral lesions with an agent that binds SLC2A1 and an agent that binds the additional risk marker, wherein each agent comprises a label, and subsequently detecting the labels.

29. The method of claim 28, wherein, Detecting the labels comprises applying a light source to the plurality of oral lesions, thereby activating the labels, and visually detecting the labels.

30. A method comprising measuring expression of solute carrier family 2 member 1 (SLC2A1) in at least one oral lesion directly within the oral cavity of a subject, wherein, The expression of SLC2A1 is measured by contacting the at least one oral lesion with an agent that binds SLC2A1, wherein the agent comprises a label, and subsequently detecting the label on the at least one oral lesion.

31. The method of claim 30, wherein, Detecting the label comprises applying a light source to the oral lesion, thereby activating the label, and visually detecting the label.

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