Diagnosing and treatment uveal melanoma

Abstract

The present disclosure relates to methods for diagnosing and treating uveal melanoma.

Description

[0001] Atorney Ref. No. 25-0029-WO

[0002] DIAGNOSING AND TREATING UVEAL MELANOMA

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 764,853, filed February 28, 2025, the disclosure of which is incorporated by reference in its entirety.

[0005] FIELD OF THE DISCLOSURE

[0006] The present disclosure relates to methods for diagnosing and / or treating patients with uveal melanoma.

[0007] BACKGROUND

[0008] Uveal melanoma (UM) is the most common primary' cancer of the eye and has a propensity for fatal hematogenous metastasis to the liver. Clinical examination is the current gold standard for diagnosis of UM; however, it is sometimes clinically challenging to distinguish between a benign uveal melanocytic tumor and a malignant uveal melanoma, with an estimated incidence of 4,000-12,000 patients with indeterminate lesions in the U.S. alone. Once diagnosed clinically, UMs can be biopsied and categorized by gene expression profiling (GEP) into distinct molecular classes associated with differential metastatic risk, based on a combination of 15-gene expression profiling (15-GEP - class 1 (low risk) vs class 2 (high risk)) and PRAME gene expression status (PRAME- vs PRAME+).

[0009] Since tumor biopsy is not always feasible in small diagnostically challenging lesions (for example, uveal melanocytic tumors of indeterminate malignant potential; “UMTIMP ”), these lesions are typically treated with a “watch and waif’ approach such that patients would benefit from a minimally invasive method to monitor these lesions to determine as early as possible whether their tumor is biologically aggressive.

[0010] There is a need for early and more accurate identification of aggressive uveal melanomas, in particular using minimally invasive sampling and aqueous humor (AH) from the anterior chamber of the eye can be easily and safely obtained by an in office-based procedure that can be repeated periodically. Proteomic signatures in aqueous humor fluid could potentially allow early detection of uveal melanomas at high risk of malignant transformation, thus identifying patients who would benefit from a definitive tumor biopsy and earlier treatment as indicated.

[0011] SUMMARY

[0012] There is a need in the art for a more accurate and objective method of diagnosing and detecting aggressive uveal melanomas. This disclosure provides methods of analyzing one or more biomarkers and / or biomarker panels in a minimally invasive sample (e.g., ocular liquidAtorney Ref. No. 25-0029-WO

[0013] or aqueous humor sample). For example, the biomarker panel in the sample (for example, by assaying protein. DNA, RNA, miRNA, ctDNA) can be correlated with known tumor molecular profiles that accurately predict outcomes for patients with uveal melanoma with greater accuracy than previously developed tests.

[0014] In one aspect, this disclosure provides a method for diagnosing and treating a patient suspected of having uveal melanoma, the method comprising (a) obtaining a diagnosis identifying a risk of metastasis in a sample from the patient, wherein the diagnosis was obtained by: (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; (3) providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis based on the probability score generated in step (2); and (4) identifying that the uveal melanoma has a high risk of metastasis based on the probability score and diagnosing the uveal melanoma as having a high risk of metastasis; and (b) administering to the patient an aggressive treatment when the determination is made in the affirmative that the patient has uveal melanoma with a high risk of metastasis.

[0015] In another aspect, this disclosure provides a method of treating a patient with uveal melanoma, the method comprising administering an aggressive cancer treatment regimen to the patient, wherein the patient has uveal melanoma with a moderate risk or a high risk as generated by comparing the expression levels of 4 or more biomarkers selected from Tables 4-10 and Tables 13-14 from the uveal melanoma with the expression levels of the same 4 or more biomarkers from a predictive training set.

[0016] In yet another aspect, this disclosure provides a method for predicting risk of metastasis in a patient with uveal melanoma, the method comprising: (a) obtaining a sample from the patient; (b) determining the expression level of 4 or more biomarkers in a biomarker panel; wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (c) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and (d) providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis, based on the probability score generated in step (c).Atorney Ref. No. 25-0029-WO

[0017] In yet another aspect, this disclosure provides a method comprising determining the expression level of 4 or more biomarkers in a sample of a patient with uveal melanoma or suspected of having uveal melanoma by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof, wherein the 4 or more markers are selected from Tables 4-10 and Tables 13-14.

[0018] In yet another aspect, this disclosure provides a method of determining one or more treatment options for a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising: (a) identify ing a risk of metastasis in a sample from the patient, wherein the risk of metastasis was identified by: (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and (b) determining that the patient receive a low intensity treatment, a moderate intensity treatment, or a high intensity’ treatment when the determination is made that the patient has a uveal melanoma tumor with a low risk (Class 1 PRAME (-)), a moderate risk (Class 1 PRAME (+)), or a high risk (Class 2) of metastasis, respectively, based on the probability score.

[0019] In some embodiments, a low intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms as clinically indicated; and / or (b) surveillance imaging every 12 months for 5 years, or as clinically indicated.

[0020] In some embodiments, a moderate intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms; and / or (b) surveillance imaging every 6-12 months for 10 years, then as clinically indicated.

[0021] In some embodiments, a high intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms; (b) surveillance imaging every 3-6 months for 5 years, then every 6-12 months for years 6-10, then as clinically indicated.

[0022] In some embodiments, the method further comprises performing a resection of the uveal melanoma tumor when the determination is made in the affirmative that the patient has a uveal melanoma tumor with a moderate risk (Class 1 PRAME (+)) or a high risk (Class 2) of metastasis.Atorney Ref. No. 25-0029-WO

[0023] In certain embodiments, the method further comprises identifying that the uveal melanoma tumor is classified as having a low risk (Class 1 PRAME (-)), a moderate risk (Class 1 PRAME (+)), or a high risk (Class 2) based on the probability score in combination with one or more clinical or pathologic risk factors. In some embodiments, the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.

[0024] In some embodiments, the biomarker panel further comprises at least one control biomarker.

[0025] In certain embodiments, the method monitors treatment efficacy and / or guides treatment selection.

[0026] In certain embodiments of the methods disclosed herein, the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion. In certain embodiments of the methods disclosed herein, the sample is a minimally invasive sample. In certain embodiments of the methods disclosed herein, the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0027] In certain embodiments of the methods disclosed herein, the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof.

[0028] In certain embodiments of the methods disclosed herein, the probability score is between 0 and 1, and wherein a value of 1 indicates a higher probability of metastasis than a value of 0.

[0029] In certain embodiments of the methods disclosed herein, the probability score is a bimodal (low7risk or high risk) analysis, wherein a patient having a value of betw een 0 and 0.499 is designated as low risk and a patient having a value of betw een 0.500 and 1.00 is designated as high risk. In certain embodiments of the methods disclosed herein, the probability score is a tri-modal (low risk, moderate risk or high risk) analysis, wherein patients are designated as low risk, moderate risk, or high risk.

[0030] In certain embodiments of the methods disclosed herein, the biomarker panel further comprises at least one control biomarker.

[0031] In certain embodiments of the methods disclosed herein, the method further comprises identifying that the uveal melanoma is classified as having a high risk of metastasis based on the probability score in combination with one or more clinical or pathologic risk factors. InAtorney Ref. No. 25-0029-WO

[0032] certain embodiments of the methods disclosed herein, the one or more one risk factors are selected from tumor mutation status. GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.

[0033] In certain embodiments of the methods disclosed herein, the 4 or more biomarkers of the biomarker panel are:

[0034] a) ANXA1, ERBB3, FGFBP1, FOLR3, GPNMB, KLK14, LGALS1, LYPD3. MIA. NECTIN4. PPY. RSPO3, S100A11. TGFA. TXLNA. and VIM;

[0035] b) CASP7, CCER2, EDIL3, ENPP7, HLA-DRA, HM0X1 , ICAM2, LAMP2, MCEE, PFDN2, PRKAB1, PSRC1, SAT2, SSBP1, STC2, and TYRP1;

[0036] c) DKK1 , IL4R. LRP2, OFD 1 , SERPINA4, and VMO 1 ;

[0037] d) ABHD14B. ACE, BNIP2, CYTH3, FCRLB. GFRA1, KRT5, NHLRC3, REGIA, RGS8, SCPEP1, SPACA5 SPACA5B, TPBGL, and TSPAN15;

[0038] e) ATP1B3, BAMBI, CCL25, CD82, CLU, CNTN4, CPA2, EPHA4, FGF6, NT- proBNP, PMVK, SH2B3, TNFRSF13C, TNFRSF9, and ULBP2;

[0039] f) AGER, APOA4, C1GALT1C1, CEACAM20, CPM, CRLF1, FOLH1, GDF15, IL12B, PDCD1. SLITRK6. SUSD5, and TIMP4;

[0040] g) ARHGAP25, CA14, CCN2, CD274, CELSR2, CTRB1, FKBPL, GH1, M6PR, MATN2, MRPL58, PAGR1, PPBP, RLN1, STX4, TOR1AIP1, and VCAN; or h) ADAM23, AHSG, AXL, C4BPB, CCL27, CD33, CTSV, EIF4G3, NAGK, PBXIP1, RABI 1FIP3. RNASE10. SCGB3A2, SSH3. and WFIKKN2.

[0041] Other aspects, embodiments, and implementations will become apparent from the following detailed description and claims, with reference, where appropriate, to the accompanying drawings.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows expression profile of proteins selected from Olink Oncology I and II panels for aqueous proteins from uveal melanoma patients. The proteins were included in the PC Analysis based on passing the 0.01% False Discovery Rate. Class 1 GEP - low risk of metastasis, includes primarily Class 1 PRAME negative samples; Class 2 GEP - high risk of metastasis, includes primarily Class 2 PRAME positive; Intermediate / moderate risk group -mix of Class 1 and Class 2 GEP.

[0043] FIG.2 shows expression profile of proteins selected from Olink Inflammation I and II panels for aqueous proteins from uveal melanoma patients. The proteins were included in the PC Analysis based on passing the 0.01% False Discovery Rate. Class 1 GEP - low risk of metastasis, includes primarily Class 1 PRAME negative samples; Class 2 GEP - high risk ofAtorney Ref. No. 25-0029-WO

[0044] metastasis, includes primarily Class 2 PRAME positive; Intermediate / moderate risk group -mix of Class 1 and Class 2 GEP.

[0045] FIG. 3 shows expression profile of proteins selected from Olink combined Oncology and Inflammation panels for aqueous proteins from uveal melanoma patients. The proteins were included in the PC Analysis based on passing the 0.01% False Discovery Rate. Class 1 GEP - low risk of metastasis, includes primarily Class 1 PRAME negative samples; Class 2 GEP - high risk of metastasis, includes primarily Class 2 PRAME positive;

[0046] Intermediate / moderate risk group - mix of Class 1 and Class 2 GEP.

[0047] FIG. 4: Distribution of discriminant scores in an independent validation of the 16-protein algorithm in aqueous humor from uveal melanoma patients (N=71). Different thresholds can be established in the Weighted Subspace Random Forest (W) and Conditional Inference Random Forest (C) algorithms to define low- and high-risk lesions in patients with uveal melanoma. Class 1 - low risk of metastasis, includes Class 1 BAP 1 -negative samples; Class 2 - high risk of metastasis, includes all Class 2 and Class 1 BAPl-positive samples.

[0048] FIG. 5: Expression profile of 16 proteins in plasma and tear samples compared to aqueous humor (AH) samples assayed on Olink Target96 Oncology II panel. The distribution of the Normalized Protein Expression is matched for 16 proteins in all 3 sample sources. The AH samples are representative of clinical sample population included in the study.

[0049] FIG. 6: Schematic of an exemplary diagnostic workflow for patients with indeterminate lesions using aqueous humor samples.

[0050] DETAILED DESCRIPTION

[0051] There is a need in the art for a more accurate method of diagnosing and testing aggressive uveal melanoma, in particular using minimally invasive sampling. Disclosed herein is the development of a minimally invasive test that detects biomarkers that are used to identify uveal melanomas having high malignant potential, which allows earlier interventions and treatment decisions that may improve patient outcomes.

[0052] In one aspect, this disclosure provides a method for diagnosing and treating a patient suspected of having uveal melanoma, the method comprising (a) obtaining a diagnosis identifying a risk of metastasis in a sample from the patient, wherein the diagnosis was obtained by: (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; (3)Atorney Ref. No. 25-0029-WO

[0053] providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis based on the probability’ score generated in step (2); and (4) identifying that the uveal melanoma has a high risk of metastasis based on the probability score and diagnosing the uveal melanoma as having a high risk of metastasis; and (b) administering to the patient an aggressive treatment when the determination is made in the affirmative that the patient has uveal melanoma with a high risk of metastasis.

[0054] In another aspect, this disclosure provides a method of treating a patient with uveal melanoma, the method comprising administering an aggressive cancer treatment regimen to the patient, wherein the patient has uveal melanoma with a moderate risk or a high risk as generated by comparing the expression levels of 4 or more biomarkers selected from Tables 4-10 and Tables 13-14 from the uveal melanoma with the expression levels of the same 4 or more biomarkers from a predictive training set.

[0055] In yet another aspect, this disclosure provides a method for predicting risk of metastasis in a patient with uveal melanoma, the method comprising: (a) obtaining a sample from the patient; (b) determining the expression level of 4 or more biomarkers in a biomarker panel; wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (c) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and (d) providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis, based on the probability score generated in step (c).

[0056] In yet another aspect, this disclosure provides a method of determining one or more treatment options for a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising: (a) identifying a risk of metastasis in a sample from the patient, wherein the risk of metastasis was identified by: (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and Tables 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and (b) determining that the patient receive a low intensify treatment, a moderate intensify treatment, or a high intensity treatment when the determination is made that the patient has a uveal melanoma tumor with a low risk (Class 1 PRAME (-)). a moderate risk (Class 1 PRAME (+)), or a high risk (Class 2) of metastasis, respectively, based on the probability score.Atorney Ref. No. 25-0029-WO

[0057] In some embodiments, a low intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms as clinically indicated; and / or (b) surveillance imaging every 12 months for 5 years, or as clinically indicated.

[0058] In some embodiments, a moderate intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms; and / or (b) surveillance imaging every76-12 months for 10 years, then as clinically indicated.

[0059] In some embodiments, a high intensity treatment comprises one or more of: (a) imaging to evaluate signs or symptoms; (b) surveillance imaging every 3-6 months for 5 years, then every’ 6-12 months for years 6-10, then as clinically indicated.

[0060] In some embodiments, the method further comprises performing a resection of the uveal melanoma tumor when the determination is made in the affirmative that the patient has a uveal melanoma tumor with a moderate risk (Class 1 PRAME (+)) or a high risk (Class 2) of metastasis.

[0061] In certain embodiments, the method further comprises identifying that the uveal melanoma tumor is classified as having a low risk (Class 1 PRAME (-)), a moderate risk (Class 1 PRAME (+)), or a high risk (Class 2) based on the probability score in combination with one or more clinical or pathologic risk factors. In some embodiments, the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.

[0062] In some embodiments, the biomarker panel further comprises at least one control biomarker.

[0063] In certain embodiments, the method monitors treatment efficacy and / or guides treatment selection.

[0064] In certain embodiments of the methods disclosed herein, the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion. In certain embodiments of the methods disclosed herein, the sample is a minimally invasive sample. In certain embodiments of the methods disclosed herein, the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0065] In certain embodiments of the methods disclosed herein, the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof.Atorney Ref. No. 25-0029-WO

[0066] In certain embodiments of the methods disclosed herein, the probability score is between 0 and 1. and wherein a value of 1 indicates a higher probability of metastasis than a value of 0.

[0067] In certain embodiments of the methods disclosed herein, the probability score is a bimodal (low risk or high risk) analysis, wherein a patient having a value of between 0 and 0.499 is designated as low risk and a patient having a value of between 0.500 and 1.00 is designated as high risk. In certain embodiments of the methods disclosed herein, the probability score is a tri-modal (low risk, moderate risk or high risk) analysis, wherein patients are designated as low risk, moderate risk, or high risk.

[0068] In certain embodiments of the methods disclosed herein, the biomarker panel further comprises at least one control biomarker.

[0069] In certain embodiments of the methods disclosed herein, the method further comprises identifying that the uveal melanoma is classified as having a high risk of metastasis based on the probability score in combination with one or more clinical or pathologic risk factors. In certain embodiments of the methods disclosed herein, the one or more one risk factors are selected from tumor mutation status (e.g. BAP1, EIF1 AX, GNA11, GNAQ, PLCB4, or SF3B1), GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which the claimed invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the methods and kits disclosed or claimed herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the claimed invention will be apparent from the following detailed description.

[0071] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherw ise. For example, reference to "a nucleic acid" means one or more nucleic acids.

[0072] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather,Atorney Ref. No. 25-0029-WO

[0073] these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment disclosed or claimed herein.

[0074] As used herein, a "patient" refers to a subject having uveal melanoma or suspected as having uveal melanoma. In some embodiments, a patient suspected as having uveal melanoma can have a suspicious ocular lesion or a suspicious melanocytic ocular lesion. In some embodiments, a patient suspected as having uveal melanoma can have a uveal melanocytic lesion of indeterminate malignant potential. In some embodiments, a patient suspected as having uveal melanoma can have uveal melanoma in a pre-malignant state.

[0075] The term "sample" as used herein includes any biological specimen obtained from a patient. Samples can include, but are not limited to aqueous humor, whole blood, plasma, serum, red blood cells, white blood cells, lymph, saliva, urine, stool, sputum, tears, fine needle aspirate biopsy, a tissue sample, needle biopsy, sentinel lymph node biopsy, and cellular extracts thereof. In certain embodiments, the sample is obtained through minimally invasive sampling. For example, a minimally invasive sample can include a liquid biopsy, such as aqueous humor or ocular liquid or tears or cell sample. In one embodiment, the minimally invasive sample requires inserting an instrument through the skin or into a body opening. For example, to obtain aqueous humor, a small gauge needle is inserted into the front part of the eye and a small amount of fluid is extracted using a procedure called "anterior chamber paracentesis". In an embodiment, the sample is an aqueous humor sample. In certain embodiments, the sample is obtained from a patient having uveal melanoma or suspected as having uveal melanoma. For example, in some embodiments, a patient suspected as having uveal melanoma can have a suspicious ocular lesion or a suspicious melanocytic ocular lesion eye. In some embodiments, a patient suspected as having uveal melanoma can have an indeterminate uveal melanocytic lesion. In certain embodiments, a patient suspected as having uveal melanoma can have uveal melanoma in a pre-malignant state.

[0076] As used herein, a "biomarker" refers to a protein, a polypeptide, a gene or a nucleic acid, which is differentially present in a sample taken from a patient (having uveal melanoma or suspected as having uveal melanoma) as compared to a comparable sample taken from a control subject (e.g., a person with a negative diagnosis or normal or healthy subject). In some embodiments, the biomarkers are selected from genes or proteins as recited in Tables 4-10 and / or Tables 13-14.

[0077] In some embodiments, the biomarkers are proteins. Proteins can be detected using known methods. For example, proteins can be detected using biomarkers, ligands orAtorney Ref. No. 25-0029-WO

[0078] antibodies that specifically bind to a protein of interest. Detection of proteins can be accomplished using immunoassays (such as ELISA, Western blot, RIA assay, or protein arrays). In some embodiments, immunohistochemical staining may also be used. In some embodiments, an antibody microarray may also be used to measure the differential expression of one or more protein biomarkers. For example, a plurality of antibodies is arrayed and covalently attached to the surface of a microarray or biochip. In addition, a person skilled in the art will appreciate that a number of methods can be used to determine the amount of a protein product of a gene of the methods disclosed herein, including immunoassay, ELISA, Western blot, mass spectrometry, immunoprecipitation followed by SDS-PAGE, and immunocytochemistry.

[0079] In some embodiments, the biomarkers are nucleic acids. As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide," and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, cDNA, RNA, miRNA, derivatives thereof, or combinations thereof. In some embodiments, the biomarkers are DNA, cDNA, RNA, or miRNA.

[0080] The phrase "measuring the biomarker expression levels" or "determining the biomarker expression levels," as used herein, refers to determining or quantifying protein or RNA expressed by the gene or genes. The term "RNA" includes mRNA transcripts, and / or specific spliced variants of mRNA. The term "RNA product of the gene," as used herein, refers to RNA transcripts transcribed from the gene and / or specific spliced variants. In some embodiments, mRNA is converted to cDNA before the gene expression levels are measured. With respect to proteins, biomarker expression refers to proteins translated from the RNA transcripts transcribed from the gene. The term "protein product of the gene" refers to proteins translated from RNA products of the gene. A number of methods can be used to detect or quantify the level of RNA products of the gene or genes within a sample, including microarrays, Next Generation Sequencing (NGS), Real-Time PCR (RT-PCR; including quantitative RT-PCR), quantitative PCR (qPCR), nuclease protection assay s, RNA-sequencing (RNA-seq), and Northern blot analyses.

[0081] A person skilled in the art will appreciate that a number of detection agents can be used to determine gene expression. For example, to detect RNA products of the biomarkers, probes, primers, complementary' nucleotide sequences, or nucleotide sequences that hybridize to the RNA products can be used. In another example, to detect cDNA products of the biomarkers, probes, primers, complementary nucleotide sequences, or nucleotide sequences that hybridize to the cDNA products can be used. To detect protein products of theAtorney Ref. No. 25-0029-WO

[0082] biomarkers, ligands or antibodies that specifically bind to the protein products can be used.

[0083] As used herein, the term "hybridize" refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In one embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions that promote hybridization are know n to those skilled in the art.

[0084] As used herein, the terms "probe" and "primer" refer to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe and / or primer hybridizes to an RNA product of the gene or a complementary nucleic acid sequence. In another example, the probe and / or primer hybridizes to a cDNA product. The length of probe or primer depends on the hybridizing conditions and the sequences of the probe or primer and nucleic acid target sequence. In one embodiment, the probe or primer is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500, or more than 500 nucleotides in length. Probes and / or primers may include one or more label. Probes and / or primers may be commercially sourced from various providers (e.g., ThermoFisher Scientific). In certain embodiments, a label may be any substance capable of aiding a machine, detector, sensor, device, or enhanced or unenhanced human eye from differentiating a labeled composition from an unlabeled composition. Examples of labels include, but are not limited to: a radioactive isotope or chelate thereof, dye (fluorescent or non-fluorescent), stain, enzyme, or nonradioactive metal. Specific examples include, but are not limited to: fluorescein, biotin, digoxigenin, alkaline phosphates, biotin, streptavidin.3H.14C,32P,35S, or any other compound capable of emitting radiation, rhodamine, 4-(4'-dimethylamino-phenylazo)benzoic acid; 4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride); 5-((2-aminoethyl)-amino)-naphtalene-l -sulfonic acid; Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives; ethylene-diamine-tetra-acetic acid and derivatives thereof, or any other compound that may be differentially detected. The label may also include one or more fluorescent dyes. Examples of dyes include, but are not limited to: CAL-Fluor Red 610, CAL-Fluor Orange 560, dRUO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA. NED, dROX, PET, BHQ+, Gold540, and LIZ.

[0085] As used herein, the terms "differentially expressed" or "differential expression" refer to a difference in the level of expression of the proteins / genes that can be assayed by measuring the level of expression of the products of the genes (proteins), such as the difference in level of messenger RNA transcript expressed (or converted cDNA) or proteins expressed of the genes. In one embodiment, the difference can be statistically significant. The term "difference in the level of expression" refers to an increase or decrease in theAtorney Ref. No. 25-0029-WO

[0086] measurable expression level of a given gene as measured by the amount of messenger RNA transcript (or converted cDNA) and / or the amount of protein in a sample as compared with the measurable expression level of a given gene in a control, or control gene or genes in the same sample (for example, a non-recurrence sample). In another embodiment, the differential expression can be compared using the ratio of the level of expression of a given protein / gene or proteins / genes as compared with the expression level of the given protein / gene or proteins / genes of a control, wherein the ratio is not equal to 1.0. For example, an RNA, cDNA, or protein is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is greater than or less than 1.0. For example, aratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20, or more than 20, or a ratio less than 1, 0.8. 0.6, 0.4, 0.2, 0.1. 0.05. 0.001, or less than 0.0001. In yet another embodiment, the differential expression is measured using p-value. For instance, when using p-value, a biomarker is identified as being differentially expressed as between a first sample and a second sample when the p-value is less than 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.001.

[0087] The terms "increased expression" or "decreased expression," as used herein, refer to an expression level of one or more biomarkers (proteins and / or genes), or prognostic RNA transcripts, or their corresponding cDNAs, or their expression products that has been found to be differentially expressed in aggressive versus non-aggressive uveal melanoma. In some embodiments, specific gene expression patterns are associated with higher likelihood of the patient experiencing a poor clinical outcome (i.e. recurrence, metastasis, or both).

[0088] References herein to the "same" level of biomarker indicate that the level of biomarker measured in each sample is identical (i.e., when compared to the selected reference). References herein to a "similar" level of biomarker indicate that levels are not identical but the difference between them is not statistically significant (i.e., the levels have comparable quantities).

[0089] As used herein, the terms "control" and "standard" refer to a specific value that one can use to determine the value obtained from the sample. In one embodiment, a dataset may be obtained from samples from a group of subjects known to have a malignant uveal melanoma. The expression data of the proteins / genes in the dataset can be used to create a control (standard) value that is used in testing samples from new subjects. In such an embodiment, the "control" or "standard" is a predetermined value for each biomarker or set of biomarkers (e.g. a biomarker panel) obtained from subjects with a malignant uveal melanoma whose protein / gene expression values and tumor types are known. In some embodiments, aAtorney Ref. No. 25-0029-WO

[0090] control biomarker or set of control biomarkers can be used to normalize expression of the diagnostic biomarkers or discriminant biomarkers. For example, in some embodiments, one or more control biomarkers can be from a control population comprising healthy individuals, individuals with benign melanocytic ocular lesions, or a mixed population of individuals with or without uveal melanoma or without melanocytic ocular lesions.

[0091] As used herein, the term "normal" when used with respect to a sample population refers to an individual or group of individuals that does / do not have a particular disease or condition (e g., malignant or benign) and is also not suspected of having or being at risk for developing the disease or condition. The term "normal" is also used herein to qualify a biological specimen or sample (e.g., a biological fluid) isolated from a normal or healthy individual or subject (or group of such subjects), for example, a "normal control sample." The "normal" level of expression of a marker is the level of expression of the marker in cells in a similar environment or response situation, in a patient not afflicted with uveal melanoma. A normal level of expression of a marker may also refer to the level of expression of a "reference sample" (e.g.. a sample from a healthy subject not having the marker associated disease). A reference sample expression may be comprised of an expression level of one or more markers from a reference database. Alternatively, a "normal" level of expression of a marker is the level of expression of the marker in non-tumor cells in a similar environment or response situation from the same patient that the tumor is derived from.

[0092] In some embodiments, a "biomarker panel" can refer to a gene-expression profile, GEP, or gene-expression profile signature. As used herein, the terms "gene-expression profile," "GEP," or "gene-expression profile signature" refer to any combination of genes, the measured messenger RNA transcript expression levels, cDNA levels, or direct DNA / RNA expression levels, or immunohistochemistry levels of which can be used to distinguish between two biologically different corporal tissues and / or cells and / or cellular changes.

[0093] In some embodiments, a biomarker panel can refer to a tumor molecular profile, TMP, or expression profile. As used herein, the terms "tumor molecular profile," "TMP," or "expression profile" refer to any combination of proteins, the measured protein levels, or direct protein expression level, or immunohistochemistry levels of which can be used to distinguish between two biologically different corporal tissues and / or cells and / or cellular changes.

[0094] In certain embodiments, a biomarker panel is comprised of the expression levels of at least 100, 75, 50. 40. 30. 30, 20, 10, 9. 8, 7, 6. 5, or 4 discriminant biomarkers. In one embodiment, the biomarker panel is comprised of about 100 discriminant biomarkers. InAtorney Ref. No. 25-0029-WO

[0095] another embodiment, the biomarker panel is comprised of about 75 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 50 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 40 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 30 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 20 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 10 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 9 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 8 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 7 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 6 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 5 discriminant biomarkers. In another embodiment, the biomarker panel is comprised of about 4 discriminant biomarkers.

[0096] In certain embodiments, a biomarker panel is comprised of the protein levels of at least 100, 75, 50. 40. 30, 30, 20, 10, 9. 8, 7, 6, 5, or 4 discriminant proteins. In one embodiment, the biomarker panel is comprised of about 100 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 75 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 50 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 40 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 30 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 20 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 10 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 9 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 8 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 7 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 6 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 5 discriminant proteins. In another embodiment, the biomarker panel is comprised of about 4 discriminant proteins.

[0097] In certain embodiments, a biomarker panel comprises 4 or more biomarkers of the biomarkers disclosed in Table 4-10 and / or 13-14. In certain embodiments, the biomarker panel comprises ANXA1, ERBB3, FGFBP1, FOLR3, GPNMB, KLK14, LGALS1, LYPD3, MIA, NECTIN4. PPY. RSPO3, S100A11, TGFA, TXLNA, and VIM. In certain embodiments, the biomarker panel comprises CASP7, CCER2. EDIL3. ENPP7, HLA-DRA. HM0X1, ICAM2, LAMP2, MCEE, PFDN2, PRKAB1, PSRC1, SAT2, SSBP1, STC2, andAtorney Ref. No. 25-0029-WO

[0098] TYRP1. In certain embodiments, the biomarker panel comprises DKK1, IL4R, LRP2, OFD1, SERPINA4, and VMO1. In certain embodiments, the biomarker panel comprises ABHD14B, ACE, BNIP2, CYTH3, FCRLB, GFRA1, KRT5, NHLRC3, REGIA, RGS8, SCPEP1, SPACA5 SPACA5B, TPBGL, and TSPAN15. In certain embodiments, the biomarker panel comprises ATP1B3, BAMBI, CCL25, CD82, CLU, CNTN4, CPA2, EPHA4, FGF6, NT-proBNP, PMVK, SH2B3. TNFRSF13C, TNFRSF9, and ULBP2. In certain embodiments, the biomarker panel comprises AGER, APOA4, C1GALT1C1, CEACAM20, CPM, CRLF1, FOLH1, GDF15, IL12B, PDCD1, SLITRK6, SUSD5, and TIMP4. In certain embodiments, the biomarker panel comprises ARHGAP25, CAI 4, CCN2, CD274, CELSR2, CTRB1, FKBPL, GH1. M6PR. MATN2, MRPL58, PAGR1, PPBP, RLN1, STX4, TOR1AIP1, and VC AN. In certain embodiments, the biomarker panel comprises ADAM23, AHSG, AXL, C4BPB, CCL27, CD33, CTSV, EIF4G3, NAGK, PBXIP1, RABI 1FIP3, RNASE10, SCGB3A2, SSH3, and WFIKKN2.As used herein, the term "predictive training set" refers to a cohort of uveal melanomas with a know n risk profile (low risk, moderate risk, or high risk) or a known clinical outcome (i.e., malignant, intermediate risk or benign) and known genetic expression profile, used to define or establish all other uveal melanomas, based upon the genetic expression profile of each. Additionally, included in the predictive training set is the definition of "threshold points," which are points at which a classification of metastatic risk is determined, specific to each individual gene expression level.

[0099] Risk, as used herein, includes low risk, moderate risk, or high risk of metastasis according to any of the statistical methods disclosed herein. In one embodiment, risk of recurrence or metastasis for uveal melanoma can be classified from a low' risk to a high risk (for example, the uveal melanoma tumor has a graduated risk from low risk to high risk or high risk to low risk of metastasis, local recurrence, regional metastasis, or distant metastasis). In other embodiments, low risk refers to a 3-year relapse-free survival rate, a 3-year metastasis free survival rate, or a 3-year disease specific survival rate of greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%. 95%. or more than 95%, and high risk refers to a 3-year relapse-free survival rate, a 3-year metastasis free survival rate, or a 3-year disease specific survival rate of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 5%. Low risk, for example, can have a recurrence risk of less than 25%, 20%, 15%, 10%, 5%, or less than 5% risk of metastasis according to any of the statistical methods disclosed herein. Moderate risk, for example, can have a recurrence risk of 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or any number in between, risk ofAtorney Ref. No. 25-0029-WO

[0100] metastasis according to any of the statistical methods disclosed herein. High-risk, for example, can have a recurrence risk of 50. 75%. 80%. 85%. 90%. 95%. or higher than 95% risk of metastasis according to any of the statistical methods disclosed herein. In certain embodiments, a low risk uveal melanoma tumor has about a 0-10% risk for metastasis. In certain embodiments, a patient having a moderate risk uveal melanoma tumor has about a 10-49% risk for metastasis. In certain embodiments, a patient having a high risk uveal melanoma tumor has about a 50-100% risk for metastasis.

[0101] In certain embodiments, risk stratifications may be binned, for example a group with low risk may be selected based on recurrence risk of less than 25%, 20%, 15%, 10%, 5%, or less than 5%. In certain embodiments, a group with a moderate risk may be selected based on a risk of 75%. 70%. 65%. 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or any number in between. In certain embodiments, a group with a high risk may be selected based on a risk of 75%, 80%, 85%, 90%, 95%, or higher than 95%. These risk designations may comprise more than three groups or as few as two groups depending on the separation characteristics of the predictive algorithm. A person familiar with the art will be able to determine the optimal binning strategy depending on the distributions of risk probability scores developed by modeling.

[0102] As used herein, the terms "treatment," "treat," or "treating" refer to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the methods disclosed herein, treatment can refer to a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method of treating a disease is considered to be a treatment if there is a 5% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 5%. 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between 5% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0103] After a uveal melanoma tumor is identified as having a moderate risk or a high risk, a medical professional or team of medical professionals will recommend one or several treatment options. In determining a treatment plan, factors to consider include the type, location, and severity of the uveal melanoma, as well as the patient's overall physical health.

[0104] As used herein, the terms "treatment," "treat," or "treating" refer to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus,Atorney Ref. No. 25-0029-WO

[0105] in the methods disclosed herein, treatment can refer to a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%. 80%. 90%. or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method of treating a disease is considered to be a treatment if there is a 5% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between 5% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. After uveal melanoma is found and staged, a medical professional or team of medical professionals will recommend one or several treatment options. In determining a treatment plan, factors to consider include the type, location, and stage of the cancer, as well as the patient's overall physical health. Patients with uveal melanoma ty pically are managed by a health care team made up of doctors from different specialties, such as an ophthalmologist (in particular, an ophthalmologist who specializes in vitreoretinal surgery, oculoplastics or ocular oncology, a radiation oncologist, an ocular pathologist, and a medical oncologist). After a uveal melanoma is found and staged, a medical professional or team of medical professionals will typically recommend one or several treatment options including one or more of surgery, radiation, chemotherapy, and targeted therapy.

[0106] The NCCN Guidelines® define low risk uveal melanoma tumors as tumors that involve: (1) Class 1A (low risk) GEP result; (2) disomy of chromosome 3 (3) gain of chromosome 6p; (4) EIF1 AX mutation; (5) T1 based on AJCC staging.

[0107] The NCCN Guidelines® define moderate risk uveal melanoma as tumors that involve: (1) Class IB (moderate risk) GEP result; (2) SF3B1 mutation; (3) T2 and T3 based on AJCC staging.

[0108] The NCCN Guidelines® define high risk uveal melanoma tumors as tumors that involve: (1) Class 2 (high risk) GEP result; (2) monosomy of chromosome 3; (3) gain of chromosome 8q; (4) BAP1 mutation; (5) T4 based on AJCC staging.

[0109] In some embodiments, a low risk uveal melanoma tumor is Class 1 PRAME (-). In some embodiments, a moderate risk uveal melanoma tumor is Class 1 PRAME (+). In some embodiments, a high risk uveal melanoma tumor is Class 2.

[0110] In some embodiments, treatment of uveal melanoma can comprise screening for metastatic disease. For example, by imaging of the liver by ultrasound, magnetic resonance imaging (MRI) and liver function tests. In some embodiments, positron emission tomography -computed tomography (PET-CT) can also be used in some practices.Atorney Ref. No. 25-0029-WO

[0111] As used herein, the term "aggressive cancer treatment regimen" refers to a treatment regimen that is determined by a medical professional or team of medical professionals and can be specific to each patient. In certain embodiments, uveal melanoma predicted to have a high-risk of recurrence or a high-risk of metastasis, or a decreased chance of survival using the methods and kits disclosed herein, would be treated using an aggressive cancer treatment regimen. Whether a treatment is considered to be aggressive will generally depend on the cancer-type, the age of the patient, and other factors known to those of skill in the art. For example, in breast cancer, adjuvant chemotherapy is a common aggressive treatment given to complement the less aggressive standards of surgery and hormonal therapy. Those skilled in the art are familiar with various other aggressive and less aggressive treatments for each type of cancer. An aggressive cancer treatment regimen is defined by the National Comprehensive Cancer Network (NCCN), and has been defined in the NCCN Guidelines® as including one or more of: 1) imaging of the affected eye with color fundus photography and ultrasonography every 3-6 months for 5 years and every' 6-12 months thereafter for stable disease, 2) discussion and / or offering of Magnetic Resonance or ultrasound of the liver, 3) liver function tests may be considered as a part of follow-up, 4) additional imaging modalities may include chest / abdomen / pelvis CT with contrast, or dual energy subtraction chest x-ray, 5) additional risk factors for recurrence are juxtapapillaiy location and ciliary' body involvement, 6) intraocular recurrence may be treated with plaque brachytherapy or particle beam, enucleation or laser ablation, 7) for extraocular involvement may consider surgical resection, 8) for orbital involvement the treatment is surgical resection, 9) for distant metastatic disease - clinical trial is preferred OR consider systemic therapy, liver-directed therapy, or palliative care Guidelines for clinical practice are published in the National Comprehensive Cancer Network (NCCN Guidelines® Uveal Melanoma Version 1.2023, available on the World Wide Web at NCCN.org).

[0112] In certain embodiments, the methods disclosed herein can be used to guide treatments and / or monitor treatment efficacy. Those skilled in the art are familiar with various other treatments for each type of cancer. For example, some guidelines propose observation for uncertain diagnosis in patients with low risk. Observation may be appropriate for patients with uncertain diagnosis and / or fewer than three risk factors for lesion growth (Table 1). Patients with small choroidal lesions presumed to be nevi or indeterminate, can defer treatment until evidence of growth or features of malignancy develop (e.g., orange pigment, sub-retinal fluid, symptoms) is associated with a very low risk of metastasis, and even lower risk of death from uveal melanoma. For patients who meet the criteria for observation,Atorney Ref. No. 25-0029-WO

[0113] regular follow-up is recommended to periodically re-evaluate for growth or features of malignancy. Follow-up tests should include the same tests recommended for initial workup and diagnosis that would help clarify if there is progression and determine the natural history' of the indeterminate lesion. Initially, these patients should be re-evaluated every 2 to 6 months to determine rate of growth (if any) and to monitor for other changes indicative of malignancy. Close follow-up for 5 years is recommended to firmly establish whether or not there is any growth or progression; some lesions that initially seem stable may suddenly begin to grow and transform. The frequency of re-evaluation should depend on the index of suspicion, patient age, and medical frailty. For example, the presence of one to two risk factors for growth (Table 1), or evidence of changes w ould increase suspicion. If the size and features of a lesion appear static after 5 years of follow-up, the patient can be followed annually thereafter. Lesions that demonstrate growth or develop additional risk factors for growth (>3 total) should be managed as uveal melanoma, even if diagnosis is still uncertain.

[0114] In some embodiments, treatment of uveal melanoma comprises surgery (i.e. resection) to remove the primary tumor.

[0115] Additional therapeutic options may include, but are not limited to: 1) combination regimens such as: AD (doxorubicin, dacarbazine); AIM (doxorubicin, ifosfamide, mesna); MAID (mesna, doxorubicin, ifosfamide, dacarbazine); ifosfamide, epirubicin, mesna; gemcitabine and docetaxel; gemcitabine and vinorelbine; gemcitabine and dacarbazine; doxorubicin and olaratumab ; methotrexate and vinblastine; tamoxifen and sulindac; vincristine, dactinomycin, cylclophosphamide; vincristine, doxorubicin, cyclophosphamide; vincristine, doxorubicin, cyclophosphamide with ifosfamide and etoposide; vincristine, doxorubicin, ifosfamide; cyclophosphamide topotecan; or ifosfamide, doxorubicin; and / or 2) single agents, such as: cisplatin or other metallic compounds, 5-FU / capecitabine (Xeloda®), cetuximab (Erbitux®), cemiplimab (Libtayo®), pembrolizumab (MK-3475), panitumumab (Vectibix®), dacomitinib (PF-00299804), gefitinib (ZD1839, Iressa), doxorubicin, ifosfamide, epirubicin, gemcitabine, dacarbazine, temozolomide, vinorelbine, eribulin, trabectedin, pazopanib, imatinib, sunitinib, regorafenib, sorafenib, nilotinib, dasatinib, interferon, toremifene, methotrexate, irinotecan, topotecan, paclitaxel, nab-paclitaxel (abraxane), docetaxel, bevacizumab, temozolomide, sirolimus (Rapamune®), everolimus, temsirolimus, crizotinib, ceritinib, or palbociclib.

[0116] In some embodiments, treatment can include immunotherapy. Examples of immunotherapies (that can be used alone or in combination with any one or more of tumor resection if a tumor is determined to be resectable, radiation therapy, chemoradiation.Atorney Ref. No. 25-0029-WO

[0117] chemotherapy, regional limb therapy, palliative surgery, systemic therapy, additional immunotherapeutic, or inclusion in ongoing clinical trials), can include, for example, pembrolizumab (Keytruda®) and nivolumab (Opdivo®), Tebentafusp (immunotherapy agent that targets gplOO epitope presented by HLA-A*02:01 cells), cemiplimab (Libtayo®; a fully human monoclonal antibody to Programmed Death-1). PD-1 is a protein on T-cells that normally help keep T-cells from attacking other cells in the body. By blocking PD-1, these drugs can boost the immune response against cancer cells. CTLA-4 inhibitors (for example, ipilimumab (Y envoy®)) are another class of drugs that can boost the immune response. In some instances, cytokine therapy (such as, interferon-alpha and interleukin-2) can be used to boost the immune system. Examples of interferon and interleukin-based treatments can include, but are not limited to, aldesleukin (proleukin®), interferon alpha-2b (INTRON®), and pegylated interferon alpha- 2b (Sylvatron®; PEG-INTRON®, PEGASYS). In another embodiment, oncolytic virus therapy can be used. Along with killing the cells directly, the oncolytic viruses can also alert the immune system to attack the cancer cells. For example, talimogene laherparepvec (Imlygic®). also known as T-VEC, is an oncolytic virus that can be used to treat melanomas. Additional immunotherapies may include CV8102.

[0118] Additionally, targeted therapies may be used to treat patients with uveal melanoma. For example, targeted therapies can include, but are not limited to, vemurafenib (Zelboraf®), dabrafenib (Tafinlar®), trametinib (Mekinist®). CLL442, and cobimetinib (Cotellic®). These drugs target common genetic mutations, such as the BRAFV600 mutation, that may be found in a subset of uveal melanoma patients.

[0119] In certain embodiments, analysis of the biomarker panel (e.g., genetic expression profile and / or tumor molecular profile), and determination of outcome is carried out using radial basis machine and / or partial least squares analysis (PLS), partition tree analysis, logistic regression analysis (LRA), K-nearest neighbor, neural networks, ensemble learners, voting algorithms, or other algorithmic approach. These analysis techniques take into account the large number of samples required to generate a training set that will enable accurate prediction of outcomes as a result of cut-points established with an in-process training set or cut-points defined for non-algorithmic analysis, but that any number of linear and nonlinear approaches can produce a statistically significant and clinically significant result. As used herein, the term "Kaplan-Meier survival analysis" is understood in the art to be also known as the product limit estimator, which is used to estimate the survival function from lifetime data. In medical research, it is often used to measure the fraction of patients living for a certain amount of time after treatment. JMP GENOMICS®, R, Python librariesAtorney Ref. No. 25-0029-WO

[0120] including SciPy, SciKit, and numpy software or systems such as TensorFlow provides an interface for utilizing each of the predictive modeling methods disclosed herein, and should not limit the claims to methods performed only with JMP GENOMICS®, R, Python, or TensorFlow software.

[0121] Kits can include any combination of components that facilitates the performance of an assay. A kit that facilitates assessing the expression of the protein / gene or proteins / genes may include suitable nucleic acid-based and / or immunological reagents as well as suitable buffers, control reagents, and printed protocols. A "kit" is any article of manufacture (e.g., a package or container) comprising at least one reagent, e.g., a probe or primer set, for specifically detecting a marker or set of markers used in the methods disclosed herein. The article of manufacture may be promoted, distributed, sold, or offered for sale as a unit for performing the methods disclosed herein. The reagents included in such a kit comprise probes, primers, or antibodies for use in detecting one or more of the genes and / or gene sets that are useful for differentiating benign or malignant uveal melanoma. Kits that facilitate nucleic acid-based methods may further include one or more of the following: specific nucleic acids such as oligonucleotides, labeling reagents, enzymes including PCR amplification reagents such as Taq or Pfu, reverse transcriptase, or other, and / or reagents that facilitate hybridization. In addition, the kits disclosed herein may preferably contain instructions which describe a suitable detection assay. Such kits can be conveniently used, e.g., in clinical settings, to diagnose and evaluate patients having or suspected as having uveal melanoma.

[0122] EMBODIMENTS:

[0123] Embodiment 1 : A method for diagnosing and treating a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising:

[0124] (a) obtaining a diagnosis identifying a risk of metastasis in a sample from the patient, wherein the diagnosis was obtained by:

[0125] (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis;

[0126] (3) providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis based on the probability score generated in step (2); andAtorney Ref. No. 25-0029-WO

[0127] (4) identifying that the uveal melanoma has a high risk of metastasis based on the probability score and diagnosing the uveal melanoma as having a high risk of metastasis; and

[0128] (b) administering to the patient an aggressive treatment when the determination is made in the affirmative that the patient has uveal melanoma with a high risk of metastasis.

[0129] Embodiment 2: The method of embodiment 1, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.

[0130] Embodiment 3 : The method of either embodiment 1 or embodiment 2, wherein the sample is a minimally invasive sample.

[0131] Embodiment 4: The method of any one of embodiments 1-3. wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0132] Embodiment 5 : The method of any one of embodiments 1 -4, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative realtime PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof.

[0133] Embodiment 6: The method of any one of embodiments 1-5, wherein the probability score is between 0 and 1, and wherein a value of 1 indicates a higher probability of metastasis than a value of 0.

[0134] Embodiment 7: The method of any one of embodiments 1-5, wherein the probability' score is a bimodal (low-risk or high-risk) analysis, wherein a patient having a value of between 0 and 0.499 is designated as low-risk and a patient having a value of between 0.500 and 1.00 is designated as high-risk.

[0135] Embodiment 8: The method of any one of embodiments 1-5, wherein the probability' score is a tri-modal (low-risk, moderate-risk or high-risk) analysis, wherein patients are designated as low-risk, moderate-risk, or high-risk.

[0136] Embodiment 9: The method of any one of embodiments 1-8, wherein the biomarker panel further comprises at least one control biomarker.

[0137] Embodiment 10: The method of any one of embodiments 1-9 further comprising identifying that the uveal melanoma is classified as having a high-risk of metastasis based on the probability score in combination with one or more clinical or pathologic risk factors.Atorney Ref. No. 25-0029-WO

[0138] Embodiment 11: The method of embodiment 10, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness; optionally wherein the one or more one risk factors is PRAME status and the probability score in combination with PRAME status produces a bimodal classification of risk of malignancy or metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).

[0139] Embodiment 12: A method of treating a patient with uveal melanoma, the method comprising administering an aggressive cancer treatment regimen to the patient, wherein the patient has uveal melanoma with a moderate-risk or a high-risk as generated by comparing the expression levels of 4 or more biomarkers selected from Tables 4-10 and 13-14 from the uveal melanoma with the expression levels of the same 4 or more biomarkers from a predictive training set.

[0140] Embodiment 13: The method of embodiment 12, wherein the sample is from a patient with an indeterminate uveal melanocytic lesion or a suspicious ocular lesion.

[0141] Embodiment 14: The method of either embodiment 12 or embodiment 13, wherein the sample is a minimally invasive sample.

[0142] Embodiment 15: The method of any one of embodiments 12-14, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0143] Embodiment 16: The method of any one of embodiments 12-15, wherein the uveal melanoma is determined to have a low-risk, a moderate-risk, or a high-risk, wherein a patient having a low-risk uveal melanoma has about a 0-10% risk for metastasis, a patient having a moderate-risk uveal melanoma has about a 10-49% risk for metastasis, and a patient having a high-risk uveal melanoma has about a 50-100% risk for metastasis.

[0144] Embodiment 17: The method of any one of embodiments 12-16, wherein the 4 or more biomarkers further comprises at least one control biomarker.

[0145] Embodiment 18: The method of any one of embodiments 12-17 further comprising determining that the uveal melanoma has a low-risk, a moderate-risk, or a high-risk based on the expression levels of the 4 or more biomarkers in combination with one or more clinical or pathologic risk factors.

[0146] Embodiment 19: The method of embodiment 18, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness; optionally wherein the one or more one risk factors is PRAME status and the probability score in combination with PRAME statusAtorney Ref. No. 25-0029-WO

[0147] produces a bimodal classification of risk of malignancy or metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).

[0148] Embodiment 20: A method for predicting risk of metastasis in a patient with uveal melanoma, the method comprising:

[0149] (a) obtaining a sample from the patient;

[0150] (b) determining the expression level of 4 or more biomarkers in a biomarker panel: wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14;

[0151] (c) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and

[0152] (d) providing an indication as to whether the uveal melanoma has a low-risk to a high-risk of metastasis, based on the probability' score generated in step (c).

[0153] Embodiment 21 : The method of embodiment 20, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.

[0154] Embodiment 22: The method of either embodiment 20 or embodiment 21, wherein the sample is a minimally invasive sample.

[0155] Embodiment 23: The method of any one of embodiments 20-22, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0156] Embodiment 24: The method of any one of embodiments 20-23, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative realtime PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic Lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof.

[0157] Embodiment 25: The method of any one of embodiments 20-24, wherein the probability' score of metastasis is between 0 and 1, and wherein a value of 1 indicates a higher probability of metastasis than a value of 0.

[0158] Embodiment 26: The method of any one of embodiments 20-25, wherein the probability score is a bimodal (low-risk or high-risk) analysis, wherein a patient having a value of between 0 and 0.499 is designated as low-risk and a patient having a value of between 0.500 and 1.00 is designated as high-risk.Atorney Ref. No. 25-0029-WO

[0159] Embodiment 27: The method of any one of embodiments 20-25, wherein the probability' score is a tri-modal (low-risk, moderate-risk or high-risk) analysis, wherein patients are designated as low-risk, moderate-risk, or high-risk.

[0160] Embodiment 28: The method of any one of embodiments 20-27, further comprising identifying the uveal melanoma has a high-risk of metastasis based on the probability' score, and administering to the patient an aggressive tumor treatment.

[0161] Embodiment 29: The method of any one of embodiments 20-28, wherein the biomarker panel further comprises at least one control biomarker.

[0162] Embodiment 30: The method of any one of embodiments 20-29 further comprising identifying that the uveal melanoma has a high risk of metastasis based on the probability' score in combination with one or more clinical or pathologic risk factors.

[0163] Embodiment 31 : The method of embodiment 30, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness; optionally wherein the one or more one risk factors is PRAME status and the probability’ score in combination with PRAME status produces a bimodal classification of risk of malignancy or metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).

[0164] Embodiment 32. A method comprising determining the expression level of 4 or more biomarkers in a sample of a patient with uveal melanoma or suspected of having uveal melanoma by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry', quantitative Western blot, ELISA, or combinations thereof, wherein the 4 or more markers are selected from Tables 4-10 and 13-14.

[0165] Embodiment 33: The method of embodiment 32, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.

[0166] Embodiment 34: The method of either embodiment 32 or embodiment 33, wherein the sample is a minimally invasive sample.

[0167] Embodiment 35: The method of any one of embodiments 32-34, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0168] Embodiment 36: A method of determining one or more treatment options for a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising:Atorney Ref. No. 25-0029-WO

[0169] (a) identifying a risk of metastasis in a sample from the patient, wherein the risk of metastasis was identified by:

[0170] (1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14; (2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and

[0171] (b) determining that the patient receive a low intensity treatment, a moderate intensity treatment, or a high intensity treatment when the determination is made that the patient has a uveal melanoma tumor with a low-risk (Class 1 PRAME (-)). a moderate-risk (Class 1 PRAME (+)), or a high-risk (Class 2) of metastasis, respectively, based on the probability score.

[0172] Embodiment 37: The method of embodiment 36, wherein the low intensity treatment comprises one or more of:

[0173] (a) imaging to evaluate signs or symptoms as clinically indicated; and / or

[0174] (b) surveillance imaging every 12 months for 5 years, or as clinically indicated.

[0175] Embodiment 38: The method of embodiment 36, wherein the moderate intensity treatment comprises one or more of:

[0176] (a) imaging to evaluate signs or symptoms; and / or

[0177] (b) surveillance imaging every 6-12 months for 10 years, then as clinically indicated. Embodiment 39: The method of embodiment 36, wherein the high intensity treatment comprises one or more of:

[0178] (a) imaging to evaluate signs or symptoms; and / or

[0179] (b) surveillance imaging every 3-6 months for 5 years, then every 6-12 months for years 6-10, then as clinically indicated.

[0180] Embodiment 40: The method of embodiment 36, further comprising performing a resection of the uveal melanoma tumor when the determination is made in the affirmative that the patient has a uveal melanoma tumor with a moderate-risk (Class 1 PRAME (+)) or a high-risk (Class 2) of metastasis.

[0181] Embodiment 41: The method of embodiment 36, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCRAtorney Ref. No. 25-0029-WO

[0182] (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof. Embodiment 42: The method of embodiment 36, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.

[0183] Embodiment 43: The method of embodiment 36, wherein the sample is a minimally invasive sample.

[0184] Embodiment 44: The method of embodiment 36, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.

[0185] Embodiment 45: The method of embodiment 36 further comprising identifying that the uveal melanoma tumor is classified as having a low-risk (Class 1 PRAME (-)). a moderaterisk (Class 1 PRAME (+)), or a high-risk (Class 2) based on the probability7score in combination with one or more clinical or pathologic risk factors.

[0186] Embodiment 46: The method of embodiment 45, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness; optionally wherein the one or more one risk factors is PRAME status and the probability score in combination with PRAME status produces a bimodal classification of risk of malignancy or metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).

[0187] Embodiment 47: The method of embodiment 36, wherein the biomarker panel further comprises at least one control biomarker.

[0188] Embodiment 48: The method of any one of embodiments 36-47, wherein the method monitors treatment efficacy and / or guides treatment selection.

[0189] Embodiment 49: The method of any one of embodiments 1-48, wherein the 4 or more biomarkers are:

[0190] a) ANXA1, ERBB3, FGFBP1, FOLR3, GPNMB, KLK14, LGALS1, LYPD3, MIA, NECTIN4, PPY, RSPO3, S100A11, TGFA, TXLNA, and VIM;

[0191] b) CASP7, CCER2, EDIL3, ENPP7, HLA-DRA. HM0X1, ICAM2. LAMP2, MCEE. PFDN2, PRKAB1, PSRC1, SAT2, SSBP1, STC2, and TYRP1;

[0192] c) DKK1, IL4R, LRP2, OFD1, SERPINA4, and VMO1;

[0193] d) ABHD14B, ACE, BNIP2, CYTH3, FCRLB, GFRA1, KRT5, NHLRC3, REGIA, RGS8, SCPEP1, SPACA5 SPACA5B, TPBGL, and TSPAN15;

[0194] e) ATP1B3. BAMBI. CCL25, CD82. CLU, CNTN4, CPA2. EPHA4, FGF6, NT-proBNP, PMVK, SH2B3, TNFRSF13C, TNFRSF9, and ULBP2;Atorney Ref. No. 25-0029-WO

[0195] f) AGER, APOA4, C1GALT1C1, CEACAM20, CPM, CRLF1, FOLH1, GDF15, IL12B, PDCD1, SLITRK6, SUSD5. and TIMP4;

[0196] g) ARHGAP25, CA14, CCN2, CD274, CELSR2, CTRB1, FKBPL, GH1, M6PR, MATN2, MRPL58, PAGR1, PPBP, RLN1, STX4, TORI AIP1, and VCAN; or

[0197] h) ADAM23, AHSG, AXL, C4BPB, CCL27, CD33, CTSV, EIF4G3, NAGK, PBXIP1, RAB11FIP3. RNASE10, SCGB3A2, SSH3. and WFIKKN2.

[0198] EXAMPLES

[0199] The Examples that follow are illustrative of specific embodiments of the claimed invention, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the claimed invention in any way.

[0200] Example 1: Assessment of uveal melanoma using minimally invasive sampling.

[0201] Aqueous humor fluid was collected from 79 patients clinically diagnosed with UM at the time of surgery and / or biopsy and distinct aqueous protein profiles were identified and correlated to the tumor’s molecular profile (for example, using molecular prognostic testing with DecisionDx-UM, and / or DecisionDx-PRAME tests for standard of care prognostic testing). One objective of this research project is to develop a minimally invasive and widely accessible biomarker test for identification of UM tumors with high malignant potential to enable earlier interventions and treatment decisions that may improve patient outcomes. Another objective is to develop an aqueous protein-based algorithm that correlates with the DecisionDx-UM and / or DecisionDx-PRAME results in patients with uveal melanoma or indeterminate uveal melanocytic tumors.

[0202] The main goal is to be able to distinguish between Class call and PRAME status for uveal melanoma (UM) patients using protein biomarkers acquired from aqueous humor tap. Among 3,072 protein data available for analysis (aqueous humor tap samples were assayed using the Olink Explorer protein panel), first, the most significantly different proteins with False Discovery7Rate (FDR) set less than 0.01% were picked to narrow down the data set. A series of primary component analyses (PCA) were performed to identify protein groups with the highest clinical significance (Tables 2-7). A set of proteins (n=104) was then used to confirm that sample separation by GEP class.

[0203] Clinical Samples: Seventy -nine uveal melanoma aqueous samples were collected (Table 1). A total of 48 Class 1 PRAME negative, 9 Class 1 PRAME positive, 11 Class 2 PRAME negative, and 11 Class 2 PRAME positive samples were acquired. Sample inclusion was not limited into a study based on presence of BAP 1 mutation, tumor thickness, tumorAtorney Ref. No. 25-0029-WO

[0204] diameter, or AJCC staging. Limiting criteria excluded pregnant women, patients under the age of 18, patients who had previously undergone radiotherapy, patients with multiple and bilateral tumors, and patients with other eye pathology. The sample distribution was representative of atypical UM cohort: mean age 64.8+14.5 years, mean tumor diameter 11.02+3.40 mm, mean tumor thickness 4.61+3.14 mm. DecisionDx-UM identified 57 / 79 (72%) of tumors as Class 1 and 22 / 79 (28%) of tumors as Class 2. Approximately 1400 out of 3072 proteins were detected in AH samples. Proteins with statistically significant differences between Class 1 and 2 (N=386, pO.OOOl) were included in further analyses. The best fitting models were k-nearest neighbor, random forest, and neural networks with accuracies of 76%, 83%, and 83%. and kappa 0.25, 0.40, and 0.36, respectively. Within the Explorer panel, proteins grouped into the Immune Response and Oncology subpanels achieved an average accuracy of 81-85% and 77-89%, respectively.

[0205] Results: Figures 1 - 3 contain total of 79 samples with the attempted separation of Class 1 PRAME negative samples from other groups. A group of low-risk Class 1 PRAME negative samples (N=29) cluster along the negative X-axis with four Class 2 and two Class 1 PRAME positive samples joining them. The high-risk Class 2 PRAME positive group of samples is located in the bottom right quarter of the graph. The rest of Class 1 PRAME negative samples are located along the positive X-axis with mix of Class 1 PRAME positive and some of Class 2 samples. This is true for all 3 PCA analyses (Oncology only, Inflammation only, and Oncology and Inflammation combined). The same two Class 1 PRAME positive and four Class 2 samples always switched to be included with Class 1 PRAME negative sample group.

[0206] Example 2: Development and cross-validation of 16-protein signature for risk stratification of patients with UM to guide the decision to biopsy.

[0207] Patients with uveal melanocytic tumors of indeterminate malignant potential (UMTIMP) are primarily managed by observation before the decision to treat. While serial tumor biopsies are not always feasible, aqueous humor (AH) can be obtained in a doctor’s office and repeated periodically. The proteomic signature from AH that is strongly associated with aggressive tumor biology could be used as an objective measure of malignant transformation to guide the decision for a definitive tumor biopsy. An AH protein-based assay was developed for identification of high-risk UMTIMPs.

[0208] Clinical Samples: Aqueous humor samples (N=75) were prospectively collected at 3 independent sites under IRB approved protocols (Table 2). All study participants were clinically diagnosed with uveal melanoma (UM) and had the 15-GEP, PRAME and 7-geneAtorney Ref. No. 25-0029-WO

[0209] UM panel next-generation sequencing (NGS) test results available. The sample distribution was representative of atypical UM patient cohort: average age was 62.7±14.6 years, tumor diameter 11.78±3.68 mm, and tumor thickness 4.58± 2.89 mm. The 15-GEP identified 52 / 75 tumors as Class 1, and 23 / 75 tumors as Class 2. The samples were analyzed with the Olink Target 96 Oncology7II panel. Weighted Subspace and Conditional Interference Random Forest models performed in R were used to cross validate the algorithm 10-fold repeated 4 times. Largest kappa was used to select the optimal model.

[0210] Results: The training and testing identified the 16-protein Random Forest model as having the highest prediction rate. The Weighted Subspace Random Forest model had an average 74% accuracy and an average kappa of 0.347, and the Conditional Interference Random Forest model had an average 75% accuracy and an average kappa of 0.321, during cross validation. The development and cross-validation stage suggested that a 16-protein AH signature can be used to predict high-risk biology in patients with clinically diagnosed UM.

[0211] Example 3: Independent Validation of a 16-protein signature for patient risk stratification in 71 samples of UM patients to guide the decision to biopsy.

[0212] Uveal melanocytic tumors of indeterminate malignant potential (UMTIMP) are usually managed by a “watch and wait” approach before the decision to treat. Although the aggressiveness of these lesions can be assessed by tumor-biopsy based molecular testing, serial tumor biopsies may be impractical and not always feasible for routine clinical management of UMTIMPs. In contrast, aqueous humor (AH) sampling can be safely performed as an outpatient procedure and is repeatable. AH protein biomarkers strongly associated with aggressive UM could be used as a sensitive and objective biological marker of malignant transformation, facilitating earlier tumor biopsy and treatment when clinically indicated. The purpose of this study was to validate a previously developed 16-protein algorithmic test for identification of high-risk tumor biology in AH sample.

[0213] Clinical Samples: All study participants were clinically diagnosed wdth UM and had the 15-GEP, PRAME and 7-gene UM panel next-generation sequencing (NGS) test results available. AH samples (N=71) were prospectively collected at 3 independent sites under IRB approved protocols (Table 3). The samples were analyzed with the Olink Target 96 Oncology II panel. The low-risk group included Class 1 and BAP1 wild ty pe samples, and the high-risk group included Class 1 / / .4 / U-mutant samples and all Class 2. Algorithmic analyses were performed in R and demographics analysis was performed in GraphPad Prism (version 10).

[0214] Results: The sample distribution was representative of a typical UM patient cohort: average age was 62.4±15.1 years, tumor diameter 11.16±3.56 mm, and tumor thickness 4.85±Atorney Ref. No. 25-0029-WO

[0215] 2.94 mm. The 15-GEP identified 48 / 71 of tumors as Class 1, and 23 / 71 tumors as Class 2.

[0216] There was a significant difference in tumor diameter (P=0.003) and tumor thickness (P=0.001) between Class 1 and Class 2 patients. The proteins primarily belonged to Signal Transduction, Disease, and Cytokine Signaling pathways, based on Olink’s Pathway Browser (Table 10). The 16-protein test had a sensitivity of 92%, specificity 52%, NPV 92%, and PPV 51% (Table 11, Figure d).

[0217] Table 1: Comparison of clinical characteristics among Uveal Melanoma patients in Discovery Cohort:

[0218] <

[0219]

[0220] Table 2: Comparison of clinical characteristics among Uveal Melanoma patients in the Development Cohort:

[0221]

[0222] Atorney Ref. No. 25-0029-WO

[0223] <

[0224]

[0225] Table 3: Comparison of clinical characteristics among Uveal Melanoma patients in the Independent Validation Cohort:

[0226] <

[0227]

[0228] Atorney Ref. No. 25-0029-WO

[0229]

[0230] Table 4. Proteins (passing FDR<0.01%) From Oncology Panel: (N=104)

[0231]

[0232] Table 5. Proteins (passing FDR<0.01%) From Inflammation Panel: (N=230)

[0233]

[0234] Atorney Ref. No. 25-0029-WO

[0235]

[0236] Table 6. Proteins from various other panels (N=52; those proteins did not pass the FDR < 0.01%, but showed statistical significance of P<0.05 when were used to separate Class 1 from Class 2 samples):

[0237]

[0238] Table 7. Proteins from various Olink panels (N=299 proteins that show statistical significance when used to separate Class 1 from Class 2 samples):

[0239]

[0240] Atorney Ref. No. 25-0029-WO

[0241]

[0242] Table 8. Proteins from various Target-96 panels that show statistical significance when used to separate Class 1 from Class 2 samples (N=94).

[0243]

[0244] Table 9. Additional Proteins.

[0245]

[0246] Atorney Ref. No. 25-0029-WO

[0247]

[0248] Table 10. 16-protein signature for patient risk stratification.

[0249]

[0250] Table 11: Different thresholds for 16-protein model performance in Independent Validation.

[0251] > & >

[0252] >

[0253] >

[0254]

[0255] > & > Bridging Control Samples: For normalization of multiple data sets, either plate control or intensity control normalizations can be performed. Plate-control is done for a “single plate'’ project, while intensity-control is done for a “multi -plate” project. The intensity-control assumes that all samples are completely randomized. Since this study was separated into 4 plates, a “batch correction” step was included (inclusion of “bridging” samples that are the same on every plate to remove technical variations).Atorney Ref. No. 25-0029-WO

[0256] Bridging samples can be used to monitor lot-to-lot differences and to normalize intensity from plate to plate. There are a total of 8 bridging samples per plate per run and they are used to determine assay variability, i.e., the CV of control samples after normalization should be smaller than the CV of control samples before normalization.

[0257] To calculate the plate-specific adjustment factor between data sets, the NPX units of bridging controls from 2 separate plates are compared and analyzed. Usually, the values of bridging samples overlap among plates, which does not require any extra steps (6 out of 7 plates across 3 separate runs). If the values do not overlap, an adjustment factor can be calculated and added to each individual data point on that plate to bring the intensity to the same level as for other plates. When an adjustment is needed, the normal adjustment factors vary between -2 and +2. The largest adjustment factor in this study was +1.3 for one of the plates, which was still well within the boundaries of -2 to +2. The other 6 plates were within 0.3 to 0.6 units relative to each other. An adjustment was performed to normalize all plates to the same level. In the case when the adjustment factor increases between -4 and +4, assays can be visualized individually with violin plots to confirm they are still comparable between projects.

[0258] To select bridging samples, plasma, tears, saliva, and UM cell lysates of various cell lines were assayed on Target96 Oncolog ll panel. The first 2 plates were run with serial dilutions of all samples above at 1:1, 1:5, 1:10, 1:20, and 1:50. Six clinical samples in 1:1 and 1:2 dilutions were added on each plate for comparison testing. Bridging samples should include sample types that will cover the entire dynamic range of clinical samples. It was decided to include only plasma and tears as bridging controls in all subsequent assays since those were the only samples that expressed the greatest number of proteins on the panel (77 / 92 proteins are covered; 51 proteins from plasma and 56 proteins from tears) and covered the dynamic range of clinical samples (Figure 5).

[0259] The “External controls” that are included by Olink with every assay cannot be selected as bridging samples as they are not representative of the study and therefore may not cover the dynamic range of proteins that would be expressed within clinical samples.

[0260] Example 4: Protein signatures for risk stratification for patients with uveal melanocytic tumors of indeterminate malignant potential (UMTIMPs).

[0261] Uveal Melanoma Tumors of Indeterminate Malignant Potential (UMTIMPs) are vastly outnumbered in prevalence by uveal nevi, which have a population prevalence of up to 10% and overlap substantially in size with small uveal melanomas. There is an unmet need for an ocular biomarker that can be obtained from a minimally invasive office procedure thatAtorney Ref. No. 25-0029-WO

[0262] can be repeated periodically to identify patients with UNTIMPs who will benefit from earlier biopsy to assess for malignancy and subsequent treatment to prevent metastasis versus patients with low risk of metastasis or benign lesions that may be safely managed by continued monitoring. Aqueous fluid from the anterior chamber is frequently obtained safely and effectively in the office or outpatient settings as well as surgical settings for a variety of ophthalmic conditions and can be used for differentiating benign uveal nevi from high-risk uveal lesions.

[0263] Protein signatures were developed and provide a highly accurate, cost-effective, and widely accessible protein-based test to evaluate the risk that a melanocytic lesion possesses high metastatic potential in patients with indeterminate melanocytic lesions and uveal melanomas.

[0264] The protein signatures test can form the basis for a simple and safe office- / operating room-based procedure that would reduce the need for unnecessary biopsy but will detect patients who will benefit from expedited biopsy. The test will also expand test offerings to doctors, specifically - ophthalmologists, who do not perform biopsy procedures on a regular basis, but who are comfortable with acquinng aqueous humor taps from their patients.

[0265] These protein signatures can be used to determine whether detection of these proteins correlates with Class 1 / PRAME-negative (low-risk) vs all Class 2 and Class 1 PRAME-positive (high-risk) GEP result, and risk of malignancy of UMTIMPs.

[0266] Aqueous humor fluid was collected from 79 patients clinically diagnosed with UM at the time of surgery and / or biopsy and distinct aqueous protein profiles were identified and correlated to the tumor’s molecular profile (for example, using molecular prognostic testing with DecisionDx-UM, and / or DecisionDx-PRAME tests for standard of care prognostic testing).

[0267] The cohort is identical to that used in Example 1 : however, the stratification into high-risk and low-risk differs, as shown in Table 12 below.

[0268] Table 12.

[0269]

[0270] Atorney Ref. No. 25-0029-WO

[0271]

[0272] Table 13. Additional Proteins

[0273]

[0274] Using Random Forest modeling, several top-performing models were selected. Accuracy criteria were predefined to select models with a NPV > 95% and the highest possible sensitivity. A high NPV threshold was prioritized to minimize the likelihood of missing any true high-risk samples. Table 14 below summarizes the accuracy metrics for each protein set.Attorney Ref. No. 25-0029-WO

[0275] Table 14. Accuracy metrics for exemplary protein sets

[0276]

[0277] All references cited in this application are expressly incorporated by reference herein.

Claims

Atorney Ref. No. 25-0029-WOWHAT IS CLAIMED IS:Claim 1 : A method for diagnosing and treating a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising:(a) obtaining a diagnosis identifying a risk of metastasis in a sample from the patient, wherein the diagnosis was obtained by:(1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14;(2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis;(3) providing an indication as to whether the uveal melanoma has a low risk to a high risk of metastasis based on the probability score generated in step (2); and(4) identifying that the uveal melanoma has a high risk of metastasis based on the probability score and diagnosing the uveal melanoma as having a high risk of metastasis; and(b) administering to the patient an aggressive treatment when the determination is made in the affirmative that the patient has uveal melanoma with a high risk of metastasis. Claim 2: The method of claim 1 , wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.Claim 3: The method of either claim 1 or claim 2, wherein the sample is a minimally invasive sample.Claim 4: The method of any one of claims 1-3, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.Claim 5: The method of any one of claims 1-4, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry', quantitative Western blot, ELISA, or combinations thereof. Claim 6: The method of any one of claims 1-5, wherein the probability score is between 0 and 1, and wherein a value of 1 indicates a higher probability of metastasis than a value ofAtorney Ref. No. 25-0029-WOClaim 7: The method of any one of claims 1-5, wherein the probability score is a bimodal (low-risk or high-risk) analysis, wherein a patient having a value of between 0 and 0.499 is designated as low-risk and a patient having a value of between 0.500 and 1.00 is designated as high-risk.Claim 8: The method of any one of claims 1-5, wherein the probability score is a tri-modal (low-risk, moderate-risk or high-risk) analysis, wherein patients are designated as low-risk, moderate-risk, or high-risk.Claim 9: The method of any one of claims 1-8, wherein the biomarker panel further comprises at least one control biomarker.Claim 10: The method of any one of claims 1-9 further comprising identifying that the uveal melanoma is classified as having a high-risk of metastasis based on the probability score in combination with one or more clinical or pathologic risk factors.Claim 11: The method of claim 10, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.Claim 12: The method of claim 11, wherein the one or more one risk factors is PRAME status and the probability score in combination with PRAME status produces a bimodal classification of risk of metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).Claim 13: A method of treating a patient with uveal melanoma, the method comprising administering an aggressive cancer treatment regimen to the patient,wherein the patient has uveal melanoma with a moderate-risk or a high-risk as generated by comparing the expression levels of 4 or more biomarkers selected from Tables 4-10 and 13-14 from the uveal melanoma with the expression levels of the same 4 or more biomarkers from a predictive training set.Claim 14: The method of claim 13, wherein the sample is from a patient with an indeterminate uveal melanocytic lesion or a suspicious ocular lesion.Claim 15: The method of either claim 13 or claim 14, wherein the sample is a minimally invasive sample.Claim 16: The method of any one of claims 13-15, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.Claim 17: The method of any one of claims 13-16, wherein the uveal melanoma is determined to have a low-risk, a moderate-risk, or a high-risk, wherein a patient having a low-risk uveal melanoma has about a 0-10% risk for metastasis, a patient having a moderate-Atorney Ref. No. 25-0029-WOrisk uveal melanoma has about a 10-49% risk for metastasis, and a patient having a high-risk uveal melanoma has about a 50-100% risk for metastasis.Claim 18: The method of any one of claims 13-17, wherein the 4 or more biomarkers further comprises at least one control biomarker.Claim 19: The method of any one of claims 13-18 further comprising determining that the uveal melanoma has a low-risk, a moderate-risk, or a high-risk based on the expression levels of the 4 or more biomarkers in combination with one or more clinical or pathologic risk factors.Claim 20: The method of claim 19, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.Claim 21 : The method of claim 20, wherein the one or more one risk factors is PRAME status and the probability' score in combination with PRAME status produces a bimodal classification of risk of metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).Claim 22: A method for predicting risk of metastasis in a patient with uveal melanoma, the method comprising:(a) obtaining a sample from the patient;(b) determining the expression level of 4 or more biomarkers in a biomarker panel; wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14;(c) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and(d) providing an indication as to whether the uveal melanoma has a low-risk to a high-risk of metastasis, based on the probability score generated in step (c).Claim 23: The method of claim 22, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.Claim 24: The method of either claim 22 or claim 23, wherein the sample is a minimally’ invasive sample.Claim 25: The method of any one of claims 22-24, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.Atorney Ref. No. 25-0029-WOClaim 26: The method of any one of claims 22-25, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic Lab-on-a-chip readout, mass spectrometry', quantitative Western blot, ELISA, or combinations thereof. Claim 27: The method of any one of claims 22-26, wherein the probability score of metastasis is between 0 and 1, and wherein a value of 1 indicates a higher probability of metastasis than a value of 0.Claim 28: The method of any one of claims 22-27, wherein the probability' score is a bimodal (low-risk or high-risk) analysis, wherein a patient having a value of between 0 and 0.499 is designated as low-risk and a patient having a value of between 0.500 and 1.00 is designated as high-risk.Claim 29: The method of any one of claims 22-27, wherein the probability7score is a tri-modal (low-risk, moderate-risk or high-risk) analysis, wherein patients are designated as low-risk, moderate-risk, or high-risk.Claim 30: The method of any one of claims 22-29, further comprising identifying the uveal melanoma has a high-risk of metastasis based on the probability score, and administering to the patient an aggressive tumor treatment.Claim 31: The method of any one of claims 22-30, wherein the biomarker panel further comprises at least one control biomarker.Claim 32: The method of any one of claims 22-31 further comprising identifying that the uveal melanoma has a high risk of metastasis based on the probability' score in combination with one or more clinical or pathologic risk factors.Claim 33: The method of claim 32, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.Claim 34: The method of claim 33, wherein the one or more one risk factors is PRAME status and the probability' score in combination with PRAME status produces a bimodal classification of risk of metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).Claim 35. A method comprising determining the expression level of 4 or more biomarkers in a sample of a patient with uveal melanoma or suspected of having uveal melanoma by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation SequencingAtorney Ref. No. 25-0029-WO(NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof, wherein the 4 or more markers are selected from Tables 4-10 and 13-14.Claim 36: The method of claim 35, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.Claim 37: The method of either claim 35 or claim 36, wherein the sample is a minimally invasive sample.Claim 38: The method of any one of claims 35-37, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.Claim 39: A method of determining one or more treatment options for a patient with uveal melanoma or suspected of having uveal melanoma, the method comprising:(a) identifying a risk of metastasis in a sample from the patient, wherein the risk of metastasis was identified by:(1) determining the expression level of 4 or more biomarkers in a biomarker panel, wherein the 4 or more biomarkers are selected from Tables 4-10 and 13-14;(2) comparing the expression levels of the 4 or more biomarkers in the biomarker panel from the sample to the expression levels of the same 4 or more biomarkers in the biomarker panel from a predictive training set to generate a probability score of the risk of metastasis; and(b) determining that the patient receive a low intensity treatment, a moderate intensity treatment, or a high intensity treatment when the determination is made that the patient has a uveal melanoma tumor with a low-risk (Class 1 PRAME (-)), a moderate-risk (Class 1 PRAME (+)), or a high-risk (Class 2) of metastasis, respectively, based on the probability score.Claim 40: The method of claim 39, wherein the low intensity treatment comprises one or more of:(a) imaging to evaluate signs or symptoms as clinically indicated; and / or (b) surveillance imaging every 12 months for 5 years, or as clinically indicated. Claim 41 : The method of claim 39, wherein the moderate intensity treatment comprises one or more of:(a) imaging to evaluate signs or symptoms; and / or(b) surveillance imaging every 6-12 months for 10 years, then as clinically indicated.Atorney Ref. No. 25-0029-WOClaim 42: The method of claim 39, wherein the high intensity treatment comprises one or more of:(a) imaging to evaluate signs or symptoms; and / or(b) surveillance imaging every 3-6 months for 5 years, then every' 6-12 months for years 6-10, then as clinically indicated.Claim 43: The method of claim 39, further comprising performing a resection of the uveal melanoma tumor when the determination is made in the affirmative that the patient has a uveal melanoma tumor with a moderate-risk (Class 1 PRAME (+)) or a high-risk (Class 2) of metastasis.Claim 44: The method of claim 39, wherein the expression level of each biomarker in the biomarker panel is determined by immunoassay, polymerase chain reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), quantitative real-time PCR (qPCR), Next Generation Sequencing (NGS), RNA-sequencing, microfluidic lab-on-a-chip readout, mass spectrometry, quantitative Western blot, ELISA, or combinations thereof.Claim 45: The method of claim 39, wherein the sample is from a patient with a uveal melanocytic lesion of indeterminate malignant potential or a suspicious ocular lesion.Claim 46: The method of claim 39, wherein the sample is a minimally invasive sample. Claim 47: The method of claim 39, wherein the sample is an aqueous humor, vitreous humor, tears, tissue biopsy, or cell biopsy sample.Claim 48: The method of claim 39 further comprising identifying that the uveal melanoma tumor is classified as having a low-risk (Class 1 PRAME (-)), a moderate-risk (Class 1 PRAME (+)), or a high-risk (Class 2) based on the probability score in combination with one or more clinical or pathologic risk factors.Claim 49: The method of claim 48, wherein the one or more one risk factors is selected from tumor mutation status, GEP class result, GEP discriminant score, PRAME status, tumor diameter, and tumor thickness.Claim 50: The method of claim 49, wherein the one or more one risk factors is PRAME status and the probability score in combination with PRAME status produces a bimodal classification of risk of metastasis (low-risk (Class 1 PRAME (-)) or a high-risk (Class 1 PRAME (+) or Class 2)).Claim 51 : The method of claim 39, wherein the biomarker panel further comprises at least one control biomarker.Claim 52: The method of any one of claims 39-51, wherein the method monitors treatment efficacy and / or guides treatment selection.Attorney Ref. No. 25-0029-WOClaim 53: The method of any one of claims 1-52, wherein the 4 or more biomarkers in the biomarker panel are:a) ANXA1, ERBB3, FGFBP1, FOLR3, GPNMB, KLK14, LGALS1, LYPD3, MIA, NECTIN4, PPY, RSPO3, S100A11, TGFA, TXLNA, and VIM;b) CASP7, CCER2, EDIL3, ENPP7, HLA-DRA, HM0X1, ICAM2, LAMP2, MCEE. PFDN2, PRKAB1, PSRC1, SAT2, SSBP1, STC2, and TYRP1;c) DKK1, IL4R. LRP2, OFD1, SERPINA4, and VMOEd) ABHD14B, ACE, BNIP2, CYTH3, FCRLB, GFRA1, KRT5, NHLRC3, REGIA, RGS8, SCPEP1, SPACA5 SPACA5B, TPBGL, and TSPAN15;e) ATP1B3, BAMBI, CCL25, CD82, CLU, CNTN4, CPA2, EPHA4, FGF6. NT- proBNP, PMVK. SH2B3, TNFRSF13C. TNFRSF9. and ULBP2;f) AGER, APOA4, C1GALT1C1, CEACAM20, CPM, CRLF1, FOLH1, GDF15, IL12B, PDCD1, SLITRK6, SUSD5, and TIMP4;g) ARHGAP25, CAI 4, CCN2, CD274, CELSR2, CTRB1, FKBPL, GH1, M6PR, MATN2, MRPL58, PAGR1. PPBP, RLN1, STX4, TOR1AIP1, and VCAN; or h) ADAM23, AHSG, AXL. C4BPB, CCL27. CD33. CTSV, EIF4G3, NAGK. PBXIP1, RAB11FIP3, RNASE10, SCGB3A2, SSH3, and WFIKKN2.

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