Methods for treating ocular cancers

Abstract

Disclosed herein are methods of treating ocular cancers in a subject.

Description

[0001] Docket No. 11348-063WO1

[0002] METHODS FOR TREATING OCULAR CANCERS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 701,778, filed October 1, 2024, which is incorporated by reference herein in its entirety.

[0005] BACKGROUND

[0006] Ocular surface squamous neoplasia (OSSN) is a diagnosis that encompasses a spectrum of squamous cell neoplasms affecting the conjunctiva and cornea. It is the third most common ocular tumor and most common non-pigmented tumor on the orbital surface. OSSN is, at times, difficult to differentiate from other UV exposure-associated ocular surface lesions, such as pterygia, pannus, and pinguecula. Additionally, these lesions may co-exist, further complicating the identification of OSSN. If not caught early, this lesion can lead to loss of vision, loss of the eye, metastasis or death. Early detection, therefore, is critical in ensuring favorable outcomes.

[0007] Another common entity on the ocular surface is conjunctival melanosis, which can be benign and associated with complexion or have atypia (primary acquired melanosis, PAM), which is the leading cause of conjunctival melanoma (CM). Determination of whether the melanosis has atypia and may progress is critical. CM is an extremely aggressive disease with local and distant metastatic risks of up to 50% and a mortality rate of roughly 25% at 10 years. Therefore, early detection and management is essential.

[0008] Histopathologic evaluation following incisional or excisional biopsy is the current gold standard for the definitive diagnosis and staging of ocular surface cancers. Unfortunately, biopsies are invasive, may lead to tumor seeding, and may result in false negative results, particularly when the tumor is subtle. Additionally, assessing ophthalmic specimens is challenging for general pathologists who do not regularly work with conjunctival biopsies, and there are a limited number of ocular pathologists available to conduct such evaluations. Accordingly, there is a need for a non-invasive test that provides excellent diagnostic power to identify ocular surface tumors. Docket No. 11348-063WO1

[0009] SUMMARY

[0010] In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to miRNA and assays involving miRNAs.

[0011] Thus, in one example, disclosed herein is a method of treating a subject having an Ocular Surface Squamous Neoplasia (OSSN) is provided, including obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, 9-5p miRNA, or any combination thereof; determining that the subject has OSSN when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of topical 5-fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, retinoic acid, anti-vascular endothelial growth factor, or any combination thereof.

[0012] In a further example, disclosed herein is a method for determining the responsiveness of an ocular surface squamous neoplasia (OSSN) to a treatment of OSSN is provided, including obtaining a biological sample derived from a subject having ocular surface squamous neoplasia; quantifying an expression level of one or more biomarkers that are associated with responsiveness of OSSN to the treatment of OSSN in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9-5p miRNA; and determining the OSSN as responsive to the treatment of OSSN if the expression level of one or more of the biomarkers 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9-5p miRNA is differentially expressed in the biological sample derived from the subject compared to the reference control.

[0013] In one example, disclosed herein is a method of treating a subject having an ocular surface melanoma is provided, including obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, 1307-5p miRNA, or any combination thereof; determining that the subj ect has ocular surface melanoma when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of topical 5-fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, retinoic acid, anti-vascular endothelial growth Docket No. 11348-063WO1 factor, or any combination thereof.

[0014] In another example, disclosed herein is a method for determining the responsiveness of an ocular surface melanoma to a treatment of ocular surface melanoma is provided, including obtaining a biological sample derived from a subject having ocular surface melanoma; quantifying an expression level of one or more biomarkers that are associated with responsiveness of ocular surface melanoma to the treatment of ocular surface melanoma in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA; and determining the ocular surface melanoma as responsive to the treatment of ocular surface melanoma if the expression level of one or more of the biomarkers 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, 1307-5p miRNA is differentially expressed in the biological sample derived from the subject compared to the reference control.

[0015] Using nucleic acid preparations extracted from tear samples of patients with OSSN, ocular surface melanoma, PAM or pterygium, the various molecular compartments of the tear film were profiled, and it is shown that micro RNAs (miRNA) constitute the most stable RNA moiety and that the miRNA concentration in a typical tear sample volume was amenable for molecular assay development. With this profiling, a miRNA assay is developed to detect cancer on the eye surface. It is accomplished utilizing differential signatures identified via unbiased next generation sequencing. A panel of specific miRNA probes, which provides 94% to 99% diagnostic power to differentiate OSSN from pterygia via a simple and rapid qPCR reaction. The assay can be used for melanocytic lesions as well derived from patients with primary acquired melanosis (PAM), PAM with severe atypia, and conjunctival melanoma. Additionally, a panel of specific miRNA probes, which provides 94% to 99% diagnostic power to differentiate ocular surface melanoma from PAM via a simple and rapid qPCR reaction.

[0016] All practicing ophthalmologists and optometrists can utilize this assay to evaluate conjunctival lesions suspicious for OSSN or ocular surface melanoma. This assay allows for the non-invasive diagnosis of cancer from a drop of the patient’s tears, which are easily acquired. This approach thereby could reduce invasive biopsies and minimize patient discomfort. Docket No. 11348-063WO1

[0017] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0020] FIGS. 1A-1E show quality control of tear RNA isolation and sequencing. FIG. 1A shows a bioanalyzer result of OSSN tear RNA sample. FIG. IB shows bioanalyzer results of pterygium tear RNA sample. FIG. 1C shows PCA plot of control, pterygium, and OSSN tear miRNA sequencing results. FIG. ID shows a Venn diagram of miRNA genes differentially expressed unique to each sample cohort and overlapping gene expressions. FIG. IE shows differentially expressed directions of miRNAs in two group comparisons.

[0021] FIGS. 2A-2D show differential expression and downstream ontology analysis of miRNA sequencing. FIG. 2A shows a violin plot of OSSN verse pterygium visualizing the up and down regulated genes in the two-way comparison. FIG. 2B shows a violin plot of OSSN verse control samples visualizing the differentially expressed genes between the two cohorts. FIG. 2C shows a dot plot of upregulated gene ontologies in OSSN verse pterygium. FIG. 2D shows a dot plot of upregulated KEGG pathways in OSSN verse pterygium.

[0022] FIGS. 3 A-3C show the target gene downstream analysis. FIG. 3 A shows a dot plot of KEGG upregulated pathways in target gene predictions of OSSN verse pterygium from differentially expressed miRNAs. FIG. 3B shows biological process gene ontology analysis of target prediction genes for up and down regulated genes in OSSN. FIG. 3C shows molecular function gene ontology analysis of target prediction genes for up and down regulated genes in OSSN.

[0023] FIGS. 4A-4D show a miRNA sequencing differential expression and downstream analysis results of ocular surface melanoma, PAM, and PAM with severe atypia. FIG. 4A shows a volcano plot of melanoma verse PAM differentially expressed genes. FIG. 4B shows a volcano plot of melanoma verse PAM with serve atypia differentially expressed genes. FIG. Docket No. 11348-063WO1

[0024] 4C shows a scatter plot of gene ontology (GO) of melanoma miRNA signature. FIG. 4D shows KEGG pathway analysis scatter plot of melanoma miRNA signature.

[0025] FIG. 5 shows a miRNA RT-qPCR results for preliminary diagnostic panel of OSSN. Relative expression and ROC curve plots for the four miRNAs with an AUC > 0.7 for the RT-qPCR results.

[0026] FIG. 6 shows a ROC curve of multiple miRNA gene panel ability to predict OSSN versus pterygium. ROC curve analysis combining miRNAs: 143-3p, 3615, 4728-3p, and 9- 5p ability to predict OSSN versus pterygium together versus the individual gene ROC curves. Multiple variable AUC = 0.9383, p-value = 0.0017.

[0027] FIGS. 7A and 7B show the workflow and overlap of differentially expressed miRNAs. FIG. 7A shows the analysis pipeline starting with removal of duplicated miRNAs, followed by differential expression analysis using thresholds of P < 0.05 and |logFC| < 2. This resulted in identification of 2 differentially expressed miRNAs in Tumor vs Control and 8 differentially expressed miRNAs in Tumor vs PAM, which were subsequently used for target gene mapping and downstream analysis. FIG. 7B shows a Venn diagram illustrating the distribution of differentially expressed miRNAs among PAM vs Control (59 unique), Tumor vs Control (118 unique), and Tumor vs PAM (6 unique). Shared miRNAs include 24 between PAM vs Control and Tumor vs Control, 3 between PAM vs Control and Tumor vs PAM, 2 between Tumor vs Control and Tumor vs PAM, and 1 miRNA common to all three comparisons.

[0028] FIGS. 8A and 8B show volcano plots of differentially expressed miRNAs. FIG. 8A shows the comparison of Melanoma vs PAM, where hsa-miR-200c-3p is significantly downregulated, and hsa-miR-145-5p is significantly upregulated along with several other miRNAs including hsa-miR-142-3p_R-l, hsa-miR-223-3p_R+l, hsa-miR-147b-3p, and hsa- miR-1307-5p. FIG. 8B shows the comparison of Melanoma vs Control, where hsa-miR-200c- 3p is significantly downregulated and hsa-miR-145-5p is significantly upregulated, along with other differentially expressed miRNAs such as hsa-miR-223-3p_R+l, hsa-miR-147b- 3p, hsa-miR-1307-5p, and hsa-miR-142-3p_R-l. The x-axis represents log fold change, and the y-axis represents the negative loglO of the P value.

[0029] FIG. 9 shows hierarchical clustering and heatmap analysis of differentially expressed miRNAs. The expression profiles of hsa-miR-145-5p and hsa-miR-200c-3p across Control, PAM, and Tumor groups. The heatmap illustrates distinct expression patterns, with hsa-miR- 145-5p upregulated and hsa-miR-200c-3p downregulated in Tumor compared to PAM and Docket No. 11348-063WO1

[0030] Control. Samples are clustered based on similarity in expression, and expression levels are represented on a gradient scale from low (gray) to high (black). Group classification (Control, PAM, Tumor) is indicated at the top.

[0031] FIG. 10 shows functional enrichment analysis of target genes of differentially expressed miRNAs. The network plot illustrates enriched Gene Ontology (GO) biological processes, where node size corresponds to the number of genes, node color intensity indicates adjusted P value, and edges represent functional similarity between terms. Key enriched processes include response to hypoxia, response to decreased oxygen levels, ameboidal-type cell migration, positive regulation of MAPK cascade, regulation of cell-substrate adhesion, axon development, axonogenesis, cell growth, regulation of cell growth, and response to wounding. The accompanying table summarizes significantly enriched GO terms with gene counts, highlighting processes such as developmental growth involved in morphogenesis, cell growth, and regulation of cell growth. Genes contributing to these pathways include well- known eye and cancer-related genes (TP53, CDKN2A, PPP1R15A, ZC3H12A, MITF, BRCA2, MYC), additional cancer-associated genes (TP53BP1, BCOR, SMARCB1), and unique or novel cancer genes (SF3B4, TMEM43, CCNE1).

[0032] FIG. 11 shows categorized gene groups identified from target analysis. Genes are classified into Cancer & Melanoma & Eye genes, including TP53, CDKN2A, PPP1R15A, ZC3H12A, MITF, BRCA2, MYC, PRKCA, VHL, and MYBBP1A, and unique genes including SF3B4, TMEM43, and CCNE1. The table in the figure provides the official symbol, full name, and functional summary of each gene, highlighting roles in tumor suppression, oncogenesis, DNA repair, cell cycle regulation, and stress response.

[0033] DETAILED DESCRIPTION

[0034] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the Docket No. 11348-063WO1 aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0035] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0036] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0037] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0038] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Docket No. 11348-063WO1

[0039] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0040] Definitions

[0041] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0042] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes-1from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. 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. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0043] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: Docket No. 11348-063WO1

[0044] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0045] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more increase so long as the increase is statistically significant.

[0046] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0047] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0048] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0049] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be Docket No. 11348-063WO1 reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0050] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0051] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, or stabilize a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0052] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0053] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0054] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, Docket No. 11348-063WO1 depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0055] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0056] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0057] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. Docket No. 11348-063WO1

[0058] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0060] The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

[0061] The term “amplification” refers to the production of one or more copies of a genetic fragment or target sequence, specifically the “amplicon”. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product. "

[0062] The term “subject” preferably refers to a human in need of treatment with an anticancer agent or treatment for any purpose, and more preferably a human in need of such a Docket No. 11348-063WO1 treatment to treat cancer, or a precancerous condition or lesion. However, the term “patient” can also refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others, that are in need of treatment with an anticancer agent or treatment.

[0063] “Ocular Surface Squamous Neoplasia” (OSSN) refers to a spectrum of epithelial tumors that arise from the conjunctiva and cornea. The condition encompasses mild epithelial dysplasia, carcinoma in situ, and invasive squamous cell carcinoma. It is recognized as the most common non-pigmented malignancy of the ocular surface. Clinically, OSSN may resemble non-neoplastic lesions associated with ultraviolet light exposure, including pterygium, pinguecula, and pannus, complicating diagnosis. If untreated, OSSN has the potential to progress, leading to significant ocular morbidity, vision loss, or metastasis.

[0064] As used herein, “conjunctival melanosis” refers to increased pigmentation of the conjunctiva, which may be benign or associated with complexion. “Primary acquired melanosis” (PAM) is a melanocytic proliferation that develops later in life and, when accompanied by atypia, demonstrates abnormal cellular features with potential for malignant transformation. PAM with atypia is considered the primary precursor lesion for conjunctival melanoma. Conjunctival melanoma is a malignant tumor of melanocytes located in the conjunctiva and represents a life-threatening neoplasm due to its potential for local invasion, recurrence, and distant metastasis.

[0065] “Sebaceous gland carcinoma” is an aggressive malignancy originating from the sebaceous glands of the eyelid or adnexal tissues. The tumor may extend to the conjunctival or corneal surface, mimicking benign inflammatory or degenerative lesions, leading to delayed recognition. Early diagnosis is critical due to the tumor’s high rate of recurrence and metastatic potential.

[0066] “Conjunctival lymphoma” is a malignant lymphoproliferative disorder affecting the conjunctival tissue. It frequently presents as a painless, salmon-colored patch on the bulbar conjunctiva. Although often localized, conjunctival lymphoma may be associated with systemic disease, necessitating further clinical evaluation and management.

[0067] “Retinoblastoma” refers to a malignant intraocular tumor of childhood arising from retinal progenitor cells. It represents the most common primary intraocular malignancy in children. The disease may present with leukocoria or strabismus and carries a high risk of mortality if untreated. Advances in focal therapy and chemotherapy have improved outcomes, but enucleation and systemic therapy remain necessary in advanced cases. Docket No. 11348-063WO1

[0068] “Uveal melanoma” is the most common primary intraocular malignancy in adults. The tumor arises from melanocytes of the uveal tract, including the iris, ciliary body, or choroid. It is characterized by local invasion and a high risk of hematogenous spread, particularly to the liver. Despite effective local control of the primary tumor, prognosis is limited by metastatic disease.

[0069] As used herein, “tears” refers to a complex mixture of proteins, lipids, nucleic acids, and metabolites that coat the ocular surface. The molecular components in the tear film reflect the physiologic condition of the underlying tissues, and lesions such as tumors shed biologic materials into the tear film which can be assayed non-invasively. Differential molecular compositions of tear fluid due to damaged, diseased, or neoplastic ocular tissue can be leveraged to elucidate valuable diagnostic information for the evaluation of ocular surface cancers.

[0070] Methods

[0071] Method of Treating a Subject Having an Ocular Surface Squamous Neoplasia (OSSN)

[0072] The present disclosure, in one aspect, provides for a method of treating a subject having an OSSN comprising obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, 9-5p miRNA, or any combination thereof; determining that the subject has OSSN when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of 5 -fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, programmed cell death- 1 inhibitors, retinoic acid, anti -vascular endothelial growth factor, or any combination thereof.

[0073] In one aspect, disclosed herein is a method of treating a subject having an OSSN comprising obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the one or more biomarkers comprise 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, and 9-5p miRNA; determining that the subject has OSSN when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of 5- fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, programmed cell death- 1 inhibitors, retinoic acid, anti-vascular endothelial growth factor, or any combination thereof. Docket No. 11348-063WO1

[0074] In some embodiments, instead of treatment with a therapeutic, the OSSN is removed by surgical excision.

[0075] A gene or miRNA is “differentially expressed” when its measured expression level (e.g. transcript abundance) is significantly different between two or more groups (for example, disease vs control, or OSSN vs pterygium). Statistical methods such as t-tests, ANOVA, or more sophisticated RNA-seq differential analysis pipelines are used to assess whether the observed differences exceed technical and biological variability. Differential expression can be either up-regulation (higher expression in one group relative to the other) or down-regulation (lower expression). The concept is central in biomarker discovery: genes or miRNAs that are differentially expressed in disease relative to controls become candidate biomarkers. As used herein, miRNA sequencing with ANOVA and pairwise comparisons is used to identify miRNAs whose expression in tear samples is significantly different across OSSN, pterygium, and control cohorts (for example, 62 miRNAs by ANOVA, 72 miRNAs comparing OSSN vs pterygium), these are the differentially expressed miRNAs.

[0076] Also, as used herein, a “biomarker” refers to a molecule (e.g. RNA, protein, metabolite) whose level correlates with a disease state, prognosis, or therapeutic response. An expression biomarker or RNA biomarker is a transcript with differential expression associated with a phenotype (e.g. disease vs healthy) and thus serve as a diagnostic, prognostic, or predictive marker. A biomarker can satisfy criteria including reproducibility, specificity (disease vs non-disease), sensitivity, and ideally robustness across samples. In the current application, the biomarkers were validated by RT-qPCR and ROC / AUC values (e.g. > 0.7) were used as demonstration of discriminatory power (i.e. good biomarker performance). Methods of detection of miR biomarkers are known in the art.

[0077] In further examples, the biomarker comprises 143-3p miRNA.

[0078] In specific examples, the biomarker comprises 3615 miRNA.

[0079] In certain examples, the biomarker comprises 4728-3p miRNA.

[0080] In some examples, the biomarker comprises 9-5p miRNA.

[0081] In further examples, the 143-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0082] In certain examples, the 3615 miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0083] In specific examples, the 4728-3p miRNA is differentially expressed to be downregulated in comparison to the reference control. Docket No. 11348-063WO1

[0084] In some examples, the 9-5p miRNA is differentially expressed to be downregulated in comparison to the reference control

[0085] In further examples, the biological sample comprises tears.

[0086] In specific examples, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

[0087] In certain examples, the OSSN is a carcinoma.

[0088] In some examples, the subject is a human.

[0089] In some examples, the reference control is the expression level of the biomarker from a subject without OSSN (a non-diseased subject). In some embodiments, the reference control is the expression level of the biomarker from a healthy tissue from the subject (a non- cancerous cell from the subject). In some embodiments, the reference control is a healthy control. In some embodiments, the reference control is a non-cancerous control. In some embodiments, the reference control is from a pooled population of patient samples or biological samples.

[0090] In some examples, the expression of the biomarker is at least about 10% higher (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control. In some embodiments, the expression of the biomarker is at least about 10% lower (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control.

[0091] Also provided herein is a method of treating a subject as described herein, wherein the subject has a further types of tumors or ocular conditions. In some examples, the tumor is an ocular tumor. Ocular tumors include but are not limited to uveal melanoma, intraocular lymphoma, eyelid tumors, conjunctival tumors, lacrimal gland tumors, and retinoblastomas, for example. In some examples, the conjunctival tumor comprises a conjunctival melanoma. In some embodiments, the ocular condition is diabetic retinopathy. In some embodiments, the ocular condition is age-related macular degeneration. In some embodiments, the ocular condition is glaucoma. In some embodiments, the ocular condition is one or more of age- related macular degeneration, allergy, blepharitis, cataract, conjunctivitis, cellulitis, central Docket No. 11348-063WO1 serous retinopathy, chalazion, contact lens related injuries, corneal / conjunctival abrasions, dystrophy, erosion, laceration, ulceration, corneal graft rejection, cytomegalovirus retinitis, diabetic retinopathy, ocular cancer, eutrophication, graves' disease, histoplasmosis, glaucoma, infection, keratitis, keratoconus, maculopathy, neovascularization, ocular hypertension, optic neuritis, conjunctival macula, pterygium, retinitis pigmentosa, retinoblastoma, scleritis, trachoma, trichiasis, or uveitis.

[0092] In further examples, a method of treating a subject as described herein is provided, wherein the subject has melanosis. In some examples, melanosis of the eye includes but is not limited to ocular melanosis, ocular dermal melanosis, and conjunctival primary acquired melanosis (PAM), for example.

[0093] In further examples, a method of treating a subject as described herein is provided, wherein the subject has an eye nevus. In some examples, the eye nevus is precancerous. In further examples, the eye nevus turns into melanoma.

[0094] Method for Determining the Responsiveness of an Ocular Surface Squamous Neoplasia (OSSN) to a Treatment of OSSN

[0095] Also provided herein is a method for determining the responsiveness of an OSSN to a treatment of OSSN comprising obtaining a biological sample derived from a subject having ocular surface squamous neoplasia; quantifying an expression level of one or more biomarkers that are associated with responsiveness of OSSN to the treatment of OSSN in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9- 5p miRNA; and determining the OSSN as responsive to the treatment of OSSN if the expression level of one or more of the biomarkers 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9-5p miRNA is differentially expressed in the biological sample derived from the subject compared to the reference control.

[0096] In one aspect, disclosed herein is a method for determining the responsiveness of an OSSN to a treatment of OSSN comprising obtaining a biological sample derived from a subject having ocular surface squamous neoplasia; quantifying an expression level of one or more biomarkers that are associated with responsiveness of OSSN to the treatment of OSSN in the biological sample derived from the subject relative to a reference control, wherein the one or more biomarkers comprise 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, and 9-5p miRNA; and determining the OSSN as responsive to the treatment of OSSN if the expression level of the biomarkers 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, and 9-5p miRNA Docket No. 11348-063WO1 are differentially expressed in the biological sample derived from the subject compared to the reference control.

[0097] In some examples, the method further comprises administering the OSSN treatment to the subject based on the determination of the subject as having OSSN.

[0098] In further examples, the biomarker comprises 143-3p miRNA.

[0099] In specific examples, the biomarker comprises 3615 miRNA.

[0100] In certain examples, the biomarker comprises 4728-3p miRNA.

[0101] In some examples, the biomarker comprises 9-5p miRNA.

[0102] In further examples, the 143-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0103] In certain examples, the 3615 miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0104] In specific examples, the 4728-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0105] In some examples, the 9-5p miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0106] In further examples, the biological sample comprises tears.

[0107] In specific examples, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

[0108] In certain examples, the OSSN is a carcinoma.

[0109] In some examples, the subject is a human.

[0110] In some examples, the reference control is the expression level of the biomarker from a subject without OSSN (a non-diseased subject). In some embodiments, the reference control is the expression level of the biomarker from a healthy tissue from the subject (a non- cancerous cell from the subject). In some embodiments, the reference control is a healthy control. In some embodiments, the reference control is a non-cancerous control. In some embodiments, the reference control is from a pooled population of patient samples or biological samples.

[0111] In some examples, the expression of the biomarker is at least about 10% higher (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about Docket No. 11348-063WO1

[0112] 100% higher, and more) in the biological sample than the reference control. In some embodiments, the expression of the biomarker is at least about 10% lower (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control.

[0113] Using nucleic acid preparations extracted from tear samples of patients with OSSN or pterygium, the various molecular compartments of the tear film were profiled, and it is shown that micro RNAs (miRNA) constitute the most stable RNA moiety and that the miRNA concentration in a typical tear sample volume was amenable for molecular assay development. With this profiling, a miRNA assay is developed to detect cancer on the eye surface. It is accomplished utilizing differential signatures identified via unbiased next generation sequencing. In certain embodiments, a panel of specific miRNA probes provides diagnostic power within a range of about 94% to about 100% for distinguishing ocular surface squamous neoplasia (OSSN) from pterygia using a rapid qPCR reaction. For example, the diagnostic power may be about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the assay is also applicable to melanocytic lesions obtained from patients with primary acquired melanosis (PAM), PAM with severe atypia, and conjunctival melanoma. “Diagnostic power”, as used herein, refers to the ability of the assay to correctly classify a sample as OSSN or non-OSSN. It reflects a composite of sensitivity, specificity, and the area under the receiver operating characteristic curve. In some embodiments diagnostic power is reported as overall accuracy derived from a confusion matrix in binary classification of OSSN versus pterygium. In other embodiments diagnostic power is reported as AUC, with cutoffs selected using Youden J index or an equivalent method to balance sensitivity and specificity. For the tear-based miRNA assay described herein, the combined probe set yields approximately 94% to about 100% diagnostic power to distinguish OSSN from pterygium using a single, rapid qPCR. This performance was supported by sequencing discovery across OSSN, pterygium, and control cohorts, followed by RT-qPCR validation in which multiple individual miRNAs achieved AUC values greater than 0.7, and the multi-marker combination improved discrimination to the 94% to about 100% range.

[0114] The signature miRNA panel comprises a defined set of miRNA probes configured for quantitative detection in tear samples. Probes target differentially expressed miRNAs discovered by sequencing and selected for robust RT-qPCR performance. In some Docket No. 11348-063WO1 embodiments the panel includes 2 to 10 probes chosen from candidates with significant differential expression between OSSN and pterygium and with individual AUC values above 0.7. In some embodiments the panel incorporates miR-143-3p, miR-3615, miR-4728-3p, miR-9-5p, or any combination thereof. The panel further includes two internal normalizers identified by NormFinder as stably expressed across control, pterygium, and OSSN tears. Primers and hydrolysis probes are supplied pre-mixed at validated concentrations for single- plex or multiplex qPCR. The assay reports normalized expression values calculated by the comparative Ct method with dual internal references, and a classification score computed from a locked logistic or equivalent linear classifier.

[0115] Method of Treating a Subject Having an Ocular Surface Melanoma

[0116] The present disclosure, in one aspect, provides for a method of treating a subject having an ocular surface melanoma comprising obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, 1307-5p miRNA, or any combination thereof; determining that the subject has ocular surface melanoma when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of 5 -fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, programmed cell death- 1 inhibitors, retinoic acid, anti -vascular endothelial growth factor, or any combination thereof.

[0117] In one aspect, disclosed herein is a method of treating a subject having an ocular surface melanoma comprising obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the one or more biomarkers comprise of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, and / or 1307- 5p miRNA; determining that the subject has ocular surface melanoma when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of 5 -fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, programmed cell death-1 inhibitors, retinoic acid, anti- vascular endothelial growth factor, or any combination thereof.

[0118] In one aspect, disclosed herein is a method of treating a subject having an ocular surface melanoma comprising obtaining a biological sample derived from the subject; Docket No. 11348-063WO1 quantifying an expression level of one or more biomarkers, wherein the one or more biomarkers comprise of 200c-3p miRNA and 145-5p miRNA; determining that the subject has ocular surface melanoma when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; administering to the subject a therapeutically effective amount of 5-fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, programmed cell death- 1 inhibitors, retinoic acid, anti -vascular endothelial growth factor, or any combination thereof.

[0119] In some embodiments, instead of treatment with a therapeutic, the melanoma is removed by surgical excision.

[0120] A gene or miRNA is “differentially expressed” when its measured expression level (e.g. transcript abundance) is significantly different between two or more groups (for example, disease vs control, or ocular surface melanoma vs PAM). Statistical methods such as t-tests, ANOVA, or more sophisticated RNA-seq differential analysis pipelines are used to assess whether the observed differences exceed technical and biological variability. Differential expression can be either up-regulation (higher expression in one group relative to the other) or down-regulation (lower expression). The concept is central in biomarker discovery: genes or miRNAs that are differentially expressed in disease relative to controls become candidate biomarkers. As used herein, miRNA sequencing with ANOVA and pairwise comparisons is used to identify miRNAs whose expression in tear samples is significantly different across ocular surface melanoma, PAM, and control cohorts (for example, PAM vs Control (59 unique miRNAs), Tumor vs Control (118 unique miRNAs), and Tumor vs PAM (6 unique) miRNAs), these are the differentially expressed miRNAs.

[0121] Also, as used herein, a “biomarker” refers to a molecule (e.g. RNA, protein, metabolite) whose level correlates with a disease state, prognosis, or therapeutic response. An expression biomarker or RNA biomarker is a transcript with differential expression associated with a phenotype (e.g. disease vs healthy) and thus serve as a diagnostic, prognostic, or predictive marker. As used herein, the biomarker refers to a miRNA (such as, for example, including but not limited to of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA), wherein the expression of the miRNA biomarkers in the biological sample (tears) is measured and used to distinguish ocular surface melanoma from controls or other ocular lesions. A biomarker satisfies criteria including reproducibility, specificity (disease vs non-disease), sensitivity, and ideally robustness across samples. In the current application, Docket No. 11348-063WO1 the biomarkers were validated by RT-qPCR and ROC / AUC values (e.g. > 0.7) were used as demonstration of discriminatory power (i.e. good biomarker performance).

[0122] In further examples, the biomarker comprises 200c-3p miRNA.

[0123] In some examples, the biomarker comprises 145-5p miRNA.

[0124] In certain examples, the biomarker comprises 142-3p_R-l miRNA.

[0125] In further examples, the biomarker comprises 223-3p_R+l miRNA.

[0126] In some examples, the biomarker comprises 147b-3p miRNA

[0127] In further examples, the biomarker comprises 1307-5p miRNA.

[0128] In certain examples, the biomarker comprises 483-3p_L-lR+2 miRNA.

[0129] In some examples, the biomarker comprises 122-5p_R+l miRNA.

[0130] In further examples, the 145-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0131] In some examples, the 142-3p_R-l miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0132] In further examples, the 223-3p_R+l miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0133] In certain examples, the 147b-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0134] In some examples, the 1307-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0135] In further examples, the 200c-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0136] In certain examples, the 483-3p_L-lR+2 miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0137] In some examples, the 122-5p_R+l miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0138] In further examples, the biological sample comprises tears.

[0139] In specific examples, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

[0140] In certain examples, the ocular surface melanoma is a uveal melanoma or conjunctival melanoma. Docket No. 11348-063WO1

[0141] In some examples, the subject is a human.

[0142] In some examples, the reference control is the expression level of the biomarker from a subject without ocular surface melanoma (a non-diseased subject). In some embodiments, the reference control is the expression level of the biomarker from a healthy tissue from the subject (a non-cancerous cell from the subject). In some embodiments, the reference control is a healthy control. In some embodiments, the reference control is a non-cancerous control. In some embodiments, the reference control is from a pooled population of patient samples or biological samples.

[0143] In some examples, the expression of the biomarker is at least about 10% higher (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control. In some embodiments, the expression of the biomarker is at least about 10% lower (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control.

[0144] Also provided herein is a method of treating a subject as described herein, wherein the subject has a further types of tumors or ocular conditions. In some examples, the tumor is an ocular tumor. Ocular tumors include but are not limited to uveal melanoma, intraocular lymphoma, eyelid tumors, conjunctival tumors, lacrimal gland tumors, and retinoblastomas, for example. In some examples, the conjunctival tumor comprises a conjunctival melanoma. In some embodiments, the ocular condition is diabetic retinopathy. In some embodiments, the ocular condition is age-related macular degeneration. In some embodiments, the ocular condition is glaucoma. In some embodiments, the ocular condition is one or more of age- related macular degeneration, allergy, blepharitis, cataract, conjunctivitis, cellulitis, central serous retinopathy, chalazion, contact lens related injuries, corneal / conjunctival abrasions, dystrophy, erosion, laceration, ulceration, corneal graft rejection, cytomegalovirus retinitis, diabetic retinopathy, ocular cancer, eutrophication, graves' disease, histoplasmosis, glaucoma, infection, keratitis, keratoconus, maculopathy, neovascularization, ocular hypertension, optic neuritis, conjunctival macula, pterygium, retinitis pigmentosa, retinoblastoma, scleritis, trachoma, trichiasis, or uveitis.

[0145] In further examples, a method of treating a subject as described herein is provided, wherein the subject has melanosis. In some examples, melanosis of the eye includes but is Docket No. 11348-063WO1 not limited to ocular melanosis, ocular dermal melanosis, and conjunctival primary acquired melanosis (PAM), for example.

[0146] In further examples, a method of treating a subject as described herein is provided, wherein the subject has an eye nevus. In some examples, the eye nevus is precancerous. In further examples, the eye nevus turns into melanoma.

[0147] Method for Determining the Responsiveness of an Ocular Surface Melanoma

[0148] Also provided herein is a method for determining the responsiveness of an ocular surface melanoma to a treatment of ocular surface melanoma comprising obtaining a biological sample derived from a subject having ocular surface melanoma; quantifying an expression level of one or more biomarkers that are associated with responsiveness of ocular surface melanoma to the treatment of ocular surface melanoma in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA; and determining the ocular surface melanoma as responsive to the treatment of ocular surface melanoma if the expression level of one or more of the of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA is differentially expressed in the biological sample derived from the subject compared to the reference control.

[0149] In one aspect, disclosed herein is a method for determining the responsiveness of an ocular surface melanoma to a treatment of ocular surface melanoma comprising obtaining a biological sample derived from a subject having ocular surface melanoma; quantifying an expression level of one or more biomarkers that are associated with responsiveness of ocular surface melanoma to the treatment of ocular surface melanoma in the biological sample derived from the subject relative to a reference control, wherein the one or more biomarkers comprise of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA; and determining the ocular surface melanoma as responsive to the treatment of ocular surface melanoma if the expression level of the biomarkers of 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA are differentially expressed in the biological sample derived from the subject compared to the reference control.

[0150] In some examples, the method further comprises administering the ocular surface Docket No. 11348-063WO1 melanoma treatment to the subject based on the determination of the subject as having ocular surface melanoma.

[0151] In further examples, the biomarker comprises 200c-3p miRNA.

[0152] In some examples, the biomarker comprises 145-5p miRNA.

[0153] In certain examples, the biomarker comprises 142-3p_R-l miRNA.

[0154] In further examples, the biomarker comprises 223-3p_R+l miRNA.

[0155] In some examples, the biomarker comprises 147b-3p miRNA

[0156] In further examples, the biomarker comprises 1307-5p miRNA.

[0157] In certain examples, the biomarker comprises 483-3p_L-lR+2 miRNA.

[0158] In some examples, the biomarker comprises 122-5p_R+l miRNA.

[0159] In further examples, the 145-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0160] In some examples, the 142-3p_R-l miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0161] In further examples, the 223-3p_R+l miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0162] In certain examples, the 147b-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0163] In some examples, the 1307-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

[0164] In further examples, the 200c-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0165] In certain examples, the 483-3p_L-lR+2 miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0166] In some examples, the 122-5p_R+l miRNA is differentially expressed to be downregulated in comparison to the reference control.

[0167] In further examples, the biological sample comprises tears.

[0168] In specific examples, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

[0169] In certain examples, the ocular surface melanoma is a uveal melanoma or conjunctival melanoma. Docket No. 11348-063WO1

[0170] In some examples, the subject is a human.

[0171] In some examples, the reference control is the expression level of the biomarker from a subject without ocular surface melanoma (a non-diseased subject). In some embodiments, the reference control is the expression level of the biomarker from a healthy tissue from the subject (a non-cancerous cell from the subject). In some embodiments, the reference control is a healthy control. In some embodiments, the reference control is a non-cancerous control. In some embodiments, the reference control is from a pooled population of patient samples or biological samples.

[0172] In some examples, the expression of the biomarker is at least about 10% higher (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control. In some embodiments, the expression of the biomarker is at least about 10% lower (for example, about 10% higher, about 20% higher, about 30% higher, about 40% higher, about 50% higher, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 100% higher, and more) in the biological sample than the reference control.

[0173] Using nucleic acid preparations extracted from tear samples of patients with ocular surface melanoma or PAM, the various molecular compartments of the tear film were profiled, and it is shown that micro RNAs (miRNA) constitute the most stable RNA moiety and that the miRNA concentration in a typical tear sample volume was amenable for molecular assay development. With this profiling, a miRNA assay is developed to detect cancer on the eye surface. It is accomplished utilizing differential signatures identified via unbiased next generation sequencing. In certain embodiments, a panel of specific miRNA probes provides diagnostic power within a range of about 94% to about 100% for distinguishing ocular surface melanoma from pterygia using a rapid qPCR reaction. For example, the diagnostic power may be about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the assay is also applicable to melanocytic lesions obtained from patients with primary acquired melanosis (PAM), PAM with severe atypia, and conjunctival melanoma. “Diagnostic power”, as used herein, refers to the ability of the assay to correctly classify a sample as ocular surface melanoma or nonocular surface melanoma. It reflects a composite of sensitivity, specificity, and the area under the receiver operating characteristic curve. In some embodiments diagnostic power is reported as overall accuracy derived from a confusion matrix in binary classification of ocular Docket No. 11348-063WO1 surface melanoma versus PAM. In other embodiments diagnostic power is reported as AUC, with cutoffs selected using Youden J index or an equivalent method to balance sensitivity and specificity. For the tear based miRNA assay described herein, the combined probe set yields approximately 94% to about 100% diagnostic power to distinguish ocular surface melanoma from PAM using a single, rapid qPCR. This performance was supported by sequencing discovery across ocular surface melanoma, PAM, and control cohorts, followed by RT-qPCR validation in which multiple individual miRNAs achieved AUC values greater than 0.7, and the multi-marker combination improved discrimination to the 94% to about 100% range.

[0174] The signature miRNA panel comprises a defined set of miRNA probes configured for quantitative detection in tear samples. Probes target differentially expressed miRNAs discovered by sequencing and selected for robust RT-qPCR performance. In some embodiments the panel includes 2 to 10 probes chosen from candidates with significant differential expression between ocular surface melanoma and PAM and with individual AUC values above 0.7. In some embodiments the panel incorporates of 200c-3p miRNA, 483- 3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223- 3p_R+l miRNA, 147b-3p miRNA, 1307-5p miRNA, or any combination thereof. The panel further includes two internal normalizers identified by NormFinder as stably expressed across control, PAM, and ocular surface melanoma tears. Primers and hydrolysis probes are supplied pre-mixed at validated concentrations for single-plex or multiplex qPCR. The assay reports normalized expression values calculated by the comparative Ct method with dual internal references, and a classification score computed from a locked logistic or equivalent linear classifier.

[0175] A tear sample can be obtained by a minimally invasive collection method suitable for nucleic acid analysis. Total RNA is isolated using a small-volume kit validated for extracellular and low input RNA. Reverse transcription is performed with stem-loop primers or universal adapters specific for miRNAs included in the panel. qPCR is run on a benchtop thermocycler using fast cycling chemistry. Data are normalized to the two internal reference miRNAs to generate delta Ct values, followed by computation of delta-delta Ct if a calibrator is used. Samples with an index at or above the decision threshold are classified as OSSN positive and samples below the threshold are classified as OSSN negative as shown in FIGS. 4C and 4D.

[0176] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and Docket No. 11348-063WO1 scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0177] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0178] EXAMPLES

[0179] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0180] Example 1. Tear Based Assay for Ocular Surface Carcinomas

[0181] Diagnostics are changing from the low throughput, tedious histology approach to rapid, specific molecular biomarker techniques that are changing the field. The potential for molecular markers in liquid biopsies is being explored at a fast pace and the future of diagnosing diseases is becoming more accurate, and early detection much more attainable. There are many liquid compartments in the body that have been explored for diagnostics such as blood, urine, and aqueous humor. The current invention explores the potential of tears as a liquid compartment to develop diagnostic tests for ocular cancers.

[0182] The clinical utility of diagnosing ocular surface lesions with tears would also be extremely beneficial as the current clinical standard requires a tissue biopsy and high-end imaging. Tissue biopsies risk tumor seeding and infection and either create an unnecessary risk for benign lesions or if cancerous, delay treatment options as the eye must heal. Other diagnosis technologies include optical coherence tomography (OCT), impression cytology and confocal microscopy. All of these techniques require highly trained personnel, and special equipment and are not consistently used in the clinic. A cost-effective, reliable diagnostic technique would revolutionize the management of ocular surface cancers.

[0183] Two of the most prevalent ocular lesions are OSSN and ocular surface melanoma. Both cancers have benign lesions, pterygium, and primary acquired melanosis (PAM), respectively, that require tissue biopsies for diagnosis. In the current invention two ocular cancers are exemplified in the tear diagnostic tests. Docket No. 11348-063WO1

[0184] There are many different avenues to explore in developing molecular-based diagnostic tests including tissue, blood, urine, and saliva. The tear film provides a promising avenue for ocular lesions. It has been previously shown that in addition to hydrating the surface of the eye, the tear film contains molecular information such as cell-free nucleotides and proteins that can be obtained. The proteomics of tears has been studied in ocular diseases, such as dry eye, for characterizing and diagnosing disease. Previous studies have shown the feasibility of isolating RNA from tears, yet the potential for differential diagnosis has not been explored. In the current invention molecular information from eye surface lesions is collected in tears to use as a diagnostic technique for ocular lesions. Using RNA for molecular diagnostics allows for use of the simple, cost-effective technique of RT-qPCR. The RNA species in tears were collected from patient samples and miRNAs were identified as the most prevalent species. A previous study has shown the feasibility for differential analysis miRNA signatures in Sjogren Syndrome and healthy individuals. Developing a less-invasive, sensitive classifying technology for diagnosing ocular surface cancerous lesions would greatly impact the treatment and outcome for patients.

[0185] Materials and Methods

[0186] RNA Isolation

[0187] RNA was isolated using the Qiagen miRNeasy Serum / Plasma Kit (Qiagen, Hilden, Germany). Prior to isolation, Trizol was spun off Schirmer strips by puncturing hole at the bottom of 0.5 mL Eppendorf tubes, placed in 1.5 mL tube and spun at 12,000 g for 4 minutes. Trizol from the same sample eye was pooled together and 2x volume 100% EtOH was added. RNA isolation with the Qiagen miRNeasy Serum / Plasma kit proceeded from step 7. RNA was eluted in 14 pL of RNase free waster. BioAnalyzer was used to determine RNA quality (FIGS. 1 A and IB). miRNA Sequencing

[0188] RNA from 20 control, 10 pterygium, 10 OSSN, 6 PAM without atypia, 6 PAM with severe atypia, and 8 melanoma samples were sent to LC Sciences (Houston, TX, USA) for miRNA sequencing. In brief, TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA USA) was used for single-end 50 base pair sequencing on an Illumina HiSeq 2500 machine. miRNA Sequencing Analysis

[0189] Provided by LC Sciences. In brief, raw reads were inputted into an LC proprietary program, ACGTIOl-miR (LC Sciences, Houston, Texas, USA) to filter reads and align using Docket No. 11348-063WO1 miRbase 22.0 by BLAST to determine miRNAs present in samples. Differential analysis was done using normalized deep-sequencing counts using both ANOVA and Student t test, for 3- way and 2-way comparisons, respectively. The significance threshold was set at 0.05. Target prediction of miRNAs was done to be able to perform downstream gene ontology and pathway analysis using DIANA-microT web server (Version 5.0). Downstream GO and pathway analysis using DAVID v6.8 Functional Analysis Tool using GOTERM DIRECT ontologies and KEGG pathway analysis. Figures are made with ggplot2 package in R Studio (Version 3.4.0). cDNA and RT-qPCR Reactions

[0190] TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to make cDNA of the tear RNA samples. Kit instructions were followed, using 2 pL of RNA per sample to complete the cDNA synthesis. RT-qPCR of the miRNA cDNA samples was done using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for the RT-qPCR were from TaqMan Advanced miRNA Assay resources with pre-validated primers.

[0191] RT-qPCR Analysis

[0192] For analysis of RT-qPCR samples, the geometric mean of the cycle threshold (CT) values for the two normalizing genes of each individual sample was taken. Then the difference between the average CT of each gene of interest to the geometric mean of the normalizing genes was calculated. The negative difference was raised to the second power (2A-difference). This was done for each sample. To compare samples and obtain the relative expression, the geometric mean of the 2A-difference gene of interest of the control samples. The pterygium and OSSN samples 2A-difference for each gene were divided by the geometric 2-difference of the controls. Relative expression values were plotted using GraphPad Prism 10. One-way ANOVA tests were conducted using GraphPad Prism.

[0193] Results miRNA Sequencing of OSSN, Pterygium and Control Samples Gives Insight into Potential Biomarkers for Diagnostic Panel

[0194] To identify potential biomarkers and differentially expressed genes that could be used for a diagnostic panel, miRNA sequencing was completed on tear samples. Sequencing was completed on 10 control, 10 pterygia, and 10 OSSN samples (FIG. 1C). A total of 62 differentially expressed genes were identified using ANOVA analysis between the three Docket No. 11348-063WO1 sample cohorts. Comparing OSSN to pterygium, 72 differentially expressed genes were identified, 5 were overexpressed, and 67 were down-regulated (FIGS. ID, 2A and 2B). Downstream analysis of gene ontology and KEGG pathway was performed to understand the biology of the miRNAs detected in the tear samples. For the ANOVA comparison results, the top gene ontology terms were signal transduction, membrane, and protein binding for the three gene ontology components, biological process, cellular component, and molecular function. The top differentially expressed pathway was pathways in cancer (FIGS. IE, 2C and 2D). Target prediction analysis identified pathways in cancer as the top upregulated pathway and regulation of gene expression as the top biological process (FIGS. 3A, 3B and 3C). miRNA Sequencing of Ocular Surface Melanoma Compared to PAM Samples to Identify Differential Markers

[0195] The miRNA sequencing analysis identified 94 differentially expressed genes between control, PAM without atypia, PAM with severe atypia, and melanoma tear samples through ANOVA analysis. Individual unpaired T-tests were run between each combination of sample cohorts. Comparing Melanoma to PAM without atypia and PAM with severe atypia, 20 and 19 differentially expressed miRNAs were found respectively (FIGS. 4A and 4B). Target prediction analysis was performed to predict the genes regulated by the differentially expressed miRNAs. The top enriched gene ontology was protein binding (FIG. 4C). KEGG pathway analysis revealed pathways in cancer is the most dysregulated pathway in the multiway comparison. Viral infections were detected in pathway enrichment analysis, including Herpes simplex virus 1 and HPV infections. The most enriched signaling pathways were PI3K-Akt and MAPK signaling (FIG. 4D).

[0196] Determining RT-qPCR OSSN Diagnostic Panel

[0197] For the panel, the focus was on the differentially expressed genes between pterygium and OSSN samples as the goal was to distinguish between these two lesions. Six normalizing and 10 differentially expressed genes were preliminarily tested. The 6 normalizing genes were identified using NormFinder to determine the most consistently expressed miRNAs based on the miRNA sequencing. The normalizing genes were validated, and two were identified as consistently expressed in all tested tear samples. From the 10 differentially expressed genes, 4 upregulated and 2 downregulated genes were successfully measured by RT-qPCR. Of the Docket No. 11348-063WO1

[0198] 6 miRNAs that continued to be validated, 4 had AUCs > 0.7 and 3 of those had a significant p-value (FIG. 5).

[0199] The ROC curve analysis in FIG. 5 was conducted comparing control samples to OSSN. ROC curve analysis was also completed for pterygium versus OSSN, which did not result in any individual gene ROC having an AUC > 0.7 or a significant p-value, but in a multiple variable ROC curve with the four miRNAs of interest, the ROC curve resulted in an AUC of 0.9383 and a p-value of 0.0017 (FIG. 6).

[0200] Discussion

[0201] OSSN and ocular surface melanoma are two of the most prevalent eye surface cancers that cause serious vision impairments, risk requiring removal of the affected eye, and death. Current diagnostic techniques require painful tissue biopsies that risk seeding tumor, infection, unnecessary harm if the suspicious lesion is benign, and require highly trained specialists to confirm disease pathology. The tear film functions as a molecular reservoir for diagnostic tests and revolutionizes the field of ocular oncology. The tear film holds molecular information that is harnessed for differential diagnostics. Through miRNA sequencing, biomarkers for OSSN and melanoma in the tear film were identified to be used for diagnostic RT-qPCR panels. RNA as a molecular species is ideal for a diagnostic test as it translates into a rapid, inexpensive RT-qPCR test. RNA was explored as a modality to identify biomarkers. Based on Bioanalyzer results, miRNAs were identified as the most abundant species to explore for differential expression. miRNA sequencing identified differentially expressed miRNA genes between control, benign, and cancerous tears. For OSSN versus pterygium, 62 genes were identified as differentially expressed between the two groups that are used in combination to develop a diagnostic panel. There are predominantly more downregulated miRNAs in the cancer samples than in benign samples. This is consistent with studies showing that miRNAs are typically downregulated in cancer since the normal function of miRNAs is to control RNA expression to regulate cell cycle progression and unregulated growth.

[0202] Through miRNA sequencing, many differentially expressed miRNAs were identified that give insight into the biology occurring on the surface of the eye and in the tear film. Upregulated gene ontologies and pathways were identified for the miRNAs and target prediction demonstrated which genes were being regulated in the tear film. Pathways in cancer was the most upregulated in OSSN and melanoma differentially expressed miRNAs. This confirmed the hypothesis that cancer-specific markers are present in tears. miRNAs Docket No. 11348-063WO1 regulate gene expression and play an important role in cancer progression. The biological information gained from miRNA sequencing provides insight into cancerous lesions and is utilized to identify actionable targets for precise treatment.

[0203] In validation testing, 6 differentially expressed genes were consistently measured by RT-qPCR along with two normalizing genes. Of this panel, 4 genes had AUCs > 0.7 in ROC curve which was set as the threshold based on previous literature on miRNA qPCR panels. Four miRNA biomarkers were identified that form the basis of a diagnostic panel for OSSN. The multiple variable ROC curve results demonstrate the power of having a panel of genes for a diagnostic test rather than relying on one biomarker. Combining the four miRNAs of interest into a predictive marker for differentiating cancer versus benign resulted in greater accuracy in distinguishing the two. The power of a multi-gene panel has been shown in prediction of uveal melanoma class to predict metastasis. Multi-gene diagnostic tests are powerful tools for diagnosing cancer such as prostate and neuroendocrine cancers, as well as for developing pan-cancer screening tools.

[0204] Biomarkers for melanoma versus PAM were also identified that are utilized to build a miRNA qPCR diagnostic panel. Having identified the biomarkers through miRNA sequencing from the differentially expressed genes, different miRNAs are now tested for effectiveness in a qPCR setting. This technique applies broadly to ocular lesions by collecting tears and running miRNA sequencing to identify biomarkers that are utilized in diagnostic panels. Ocular biopsies are challenging to obtain and cause unnecessary harm if the lesion does not require removal. A tear biopsy provides a less invasive diagnostic test and transforms the paradigm of ocular biopsies. The groundwork has been laid for identifying and establishing a test for distinguishing between ocular lesions as malignant or benign.

[0205] Other advantages which are obvious, and which are inherent to the invention, will be evident to one skilled in the art. Certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and within the scope of the claims. Since many embodiments are made of the invention without departing from the scope thereof, all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Claims

Docket No. 11348-063WO1CLAIMSWhat is claimed is:

1. A method of treating a subject having an Ocular Surface Squamous Neoplasia (OSSN), comprising: obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, 9-5p miRNA, or any combination thereof; determining that the subject has OSSN when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; and administering to the subject a therapeutically effective amount of topical 5- fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, retinoic acid, anti-vascular endothelial growth factor, or any combination thereof.

2. The method of claim 1, wherein the biomarker comprises 143-3p miRNA.

3. The method of any one of claiml or claim 2, wherein the biomarker comprises 3615 miRNA.

4. The method of any one of claims 1-3, wherein the biomarker comprises 4728-3p miRNA.

5. The method of any one of claims 1-4, wherein the biomarker comprises 9-5p miRNA.

6. The method of any one of claims 1-5, wherein the 143-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

7. The method of any one of claims 1-6, wherein the 3615 miRNA is differentially expressed to be upregulated in comparison to the reference control.

8. The method of any one of claims 1-7, wherein the 4728-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

9. The method of any one of claims 1-8, wherein the 9-5p miRNA is differentially expressed to be downregulated in comparison to the reference control.

10. The method of any one of claims 1-9, wherein the biological sample comprises tears.Docket No. 11348-063WO111. The method of any one of claims 1-10, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

12. The method of any one of claims 1-11, wherein the OSSN is a carcinoma.

13. The method of any one of claims 1-12, wherein the subject is a human.

14. A method for determining the responsiveness of an ocular surface squamous neoplasia (OSSN) to a treatment of OSSN, comprising: obtaining a biological sample derived from a subject having ocular surface squamous neoplasia; quantifying an expression level of one or more biomarkers that are associated with responsiveness of OSSN to the treatment of OSSN in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9-5p miRNA; and determining the OSSN as responsive to the treatment of OSSN if the expression level of one or more of the biomarkers 143-3p miRNA, 3615 miRNA, 4728-3p miRNA, or 9-5p miRNA is differentially expressed in the biological sample derived from the subject compared to the reference control.

15. The method of claim 14, further comprising administering the OSSN treatment to the subject based on the determination of the subject as having OSSN.

16. The method of any one of claims 14-15, wherein the biomarker comprises 143-3p miRNA.

17. The method of any one of claims 14-16, wherein the biomarker comprises 3615 miRNA.

18. The method of any one of claims 14-17, wherein the biomarker comprises 4728-3p miRNA.

19. The method of any one of claims 14-18, wherein the biomarker comprises 9-5p miRNA.Docket No. 11348-063WO120. The method of any one of claims 14-19, wherein the 143-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

21. The method of any one of claims 14-20, wherein the 3615 miRNA is differentially expressed to be upregulated in comparison to the reference control.

22. The method of any one of claims 14-21, wherein the 4728-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

23. The method of any one of claims 14-22, wherein the 9-5p miRNA is differentially expressed to be downregulated in comparison to the reference control.

24. The method of any one of claims 14-23, wherein the biological sample comprises tears.

25. The method of any one of claims 14-24, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

26. The method of any one of claims 14-25, wherein the OSSN is a carcinoma.

27. The method of any one of claims 14-26, wherein the subject is a human.

28. A method of treating a subject having an ocular surface melanoma, comprising: obtaining a biological sample derived from the subject; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, 1307-5p miRNA, or any combination thereof; determining that the subject has ocular surface melanoma when the level of the one or more biomarkers is differentially expressed in comparison to a reference control; and administering to the subject a therapeutically effective amount of topical 5- fluorouracil, interferon alpha-2b (IFNa-2b), mitomycin C, retinoic acid, anti-vascular endothelial growth factor, or any combination thereof.Docket No. 11348-063WO129. The method of claim 28, wherein the biomarker comprises 200c-3p miRNA.

30. The method of any one of claim 28 or claim 29, wherein the biomarker comprises 145-5p miRNA.

31. The method of any one of claims 28-30, wherein the biomarker comprises 142-3p_R- 1 miRNA.

32. The method of any one of claims 28-31, wherein the biomarker comprises 223- 3p_R+l miRNA.

33. The method of any one of claims 28-32, wherein the biomarker comprises 147b-3p miRNA.

34. The method of any one of claims 28-33, wherein the biomarker comprises 1307-5p miRNA.

35. The method of any one of claims 28-34, wherein the biomarker comprises 483-3p_L- 1R+2 miRNA.

36. The method of any one of claims 28-35, wherein the biomarker comprises 122- 5p_R+l miRNA.

37. The method of any one of claims 28-36, wherein the 145-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

38. The method of any one of claims 28-36, wherein the 142-3p_R-l miRNA is differentially expressed to be upregulated in comparison to the reference control.

39. The method of any one of claims 28-36, wherein the 223-3p_R+l miRNA is differentially expressed to be upregulated in comparison to the reference control.

40. The method of any one of claims 28-36, wherein the 147b-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

41. The method of any one of claims 28-36, wherein the 1307-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

42. The method of any one of claims 28-36, wherein the 200c-3p miRNA is differentiallyDocket No. 11348-063WO1 expressed to be downregulated in comparison to the reference control.

43. The method of any one of claims 28-36, wherein the 483-3p_L-lR+2 miRNA is differentially expressed to be downregulated in comparison to the reference control.

44. The method of any one of claims 28-36, wherein the 122-5p_R+l miRNA is differentially expressed to be downregulated in comparison to the reference control.

45. The method of any one of claims 28-44, wherein the biological sample comprises tears.

46. The method of any one of claims 28-45, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

47. The method of any one of claims 28-46, wherein the ocular surface melanoma is a uveal melanoma or conjunctival melanoma.

48. The method of any one of claims 28-47, wherein the subject is a human.

49. A method for determining the responsiveness of an ocular surface melanoma to a treatment of ocular surface melanoma, comprising: obtaining a biological sample derived from a subject having ocular surface melanoma; quantifying an expression level of one or more biomarkers that are associated with responsiveness of ocular surface melanoma to the treatment of ocular surface melanoma in the biological sample derived from the subject relative to a reference control, wherein the biomarker comprises one or more of 200c-3p miRNA, 483-3p_L- 1R+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223- 3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA; and determining the ocular surface melanoma as responsive to the treatment of ocular surface melanoma if the expression level of one or more of the biomarkers 200c-3p miRNA, 483-3p_L-lR+2 miRNA, 122-5p_R+l miRNA, 145-5p miRNA, 142-3p_R-l miRNA, 223-3p_R+l miRNA, 147b-3p miRNA, or 1307-5p miRNA is differentially expressed in the biological sample derived from the subject comparedDocket No. 11348-063WO1 to the reference control.

50. The method of claim 49, further comprising administering the ocular surface melanoma treatment to the subject based on the determination of the subject as having ocular surface melanoma.

51. The method of any one of claim 49 or claim 50, wherein the biomarker comprises 200c-3p miRNA.

52. The method of any one of claims 49-51, wherein the biomarker comprises 145-5p miRNA.

53. The method of any one of claims 49-52, wherein the biomarker comprises 142-3p_R- 1 miRNA.

54. The method of any one of claims 49-53, wherein the biomarker comprises 223- 3p_R+l miRNA.

55. The method of any one of claims 49-54, wherein the biomarker comprises 147b-3p miRNA.

56. The method of any one of claims 49-55, wherein the biomarker comprises 1307-5p miRNA.

57. The method of any one of claims 49-56, wherein the biomarker comprises 483-3p_L- 1R+2 miRNA.

58. The method of any one of claims 49-57, wherein the biomarker comprises 122- 5p_R+l miRNA.

59. The method of any one of claims 49-58, wherein the 145-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

60. The method of any one of claims 49-58, wherein the 142-3p_R-l miRNA is differentially expressed to be upregulated in comparison to the reference control.

61. The method of any one of claims 49-58, wherein the 223-3p_R+l miRNA is differentially expressed to be upregulated in comparison to the reference control.Docket No. 11348-063WO162. The method of any one of claims 49-58, wherein the 147b-3p miRNA is differentially expressed to be upregulated in comparison to the reference control.

63. The method of any one of claims 49-58, wherein the 1307-5p miRNA is differentially expressed to be upregulated in comparison to the reference control.

64. The method of any one of claims 49-58, wherein the 200c-3p miRNA is differentially expressed to be downregulated in comparison to the reference control.

65. The method of any one of claims 49-58, wherein the 483-3p_L-lR+2 miRNA is differentially expressed to be downregulated in comparison to the reference control.

66. The method of any one of claims 49-58, wherein the 122-5p_R+l miRNA is differentially expressed to be downregulated in comparison to the reference control.

67. The method of any one of claims 49-58, wherein the biological sample comprises tears.

68. The method of any one of claims 49-67, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, and probe array.

69. The method of any one of claims 49-68, wherein the ocular surface melanoma is a uveal melanoma or conjunctival melanoma.

70. The method of any one of claims 49-69, wherein the subject is a human.

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