Kits and methods useful for screening, diagnosing, and treating prostate cancer

Abstract

Provided herein are kits and methods useful for cancer screening, diagnosis, research and therapy. In particular, provided herein are methods of screening, diagnosing, and / or treating prostate cancer based on expression levels of cancer markers.

Description

[0001] GRGR-43680.601

[0002] KITS AND METHODS USEFUL FOR SCREENING, DIAGNOSING, AND TREATING PROSTATE CANCER

[0003] FIELD OF THE DISCLOSURE

[0004] Provided herein are kits and methods useful for cancer screening, diagnosis, research and therapy. In particular, provided herein are methods of screening, diagnosing, and / or treating prostate cancer based on expression levels of cancer markers.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Prostate cancer is one of the most common malignancies in men with an incidence of 299,000 cases and approximately 35,250 deaths yearly in the United States (www.cancer.org / cancer / types / prostate-cancer / about / key-statistics.html). Prostate cancer is primarily a malignancy of epithelial cells (classified as a carcinoma) but includes other rare subtypes. Approximately 90-95% of prostate cancers are adenocarcinomas arising from the peripheral zone of the prostate gland. The overall incidence of malignant prostate tumors is increasing and currently is the second most common form of cancer in men with the second highest death rate, behind only lung cancer (Ahmed, HU, et al. Diagnostic accuracy of multiparametric MRI and TRUS biopsy in prostate cancer (PROMIS): a paired validating confirmatory study. Lancet (2017)). Prostate tumors are increasingly diagnosed primarily as a result of screening using blood-based prostate specific antigen (PSA) testing. Despite PSA tests having relatively high sensitivity even for early-stage cancers, they have low specificity, and need follow-up, confirmatory testing, most commonly by invasive biopsies. Historically, more than half of subjects with high (positive) PSA tests that are referred for biopsy are found to have no cancer. Furthermore, PSA cannot distinguish between aggressive forms of prostate cancer that are associated with high mortality and indolent forms, which comprise the majority of cases. Imaging procedures, such as magnetic resonance imaging (MRI) have been used following screening, prior to biopsy confirmation. However, in recent large multicenter studies, although MRI was shown to lead to improved detection of clinically significant cancer, the overall sensitivity and negative predictive value (NPV) was insufficient to safely rule-out confirmatory biopsy (Ahmed, HU, et al. Diagnostic accuracy of multi-parametric MRI and TRUS biopsy in prostate cancer (PROMIS): a paired validating confirmatory study. Lancet (2017)). Non-invasive GRGR-43680.601 blood-based (Parekh, et al., A Multi-institutional Prospective Trial in the USA Confirms that the 4Kscore Accurately Identifies Men with High-grade Prostate Cancer. Eur Urol (2014); Stovsky, et al. Clinical Validation of IsoPSA, a Single Parameter, Structure Based Assay for Improved Detection of High Grade Prostate Cancer. J Urol (2019)) or urine-based (Margolis, et al., Predicting high-grade prostate cancer at initial biopsy: clinical performance of the ExoDx (EPI) Prostate Intelliscore test in three independent prospective studies. Pros Cancer Pros Dis (2021); McKiernan, et al., A Novel Urine Exosome Gene Expression Assay to Predict High-grade Prostate Cancer at Initial Biopsy. JAMA (2016); Tomlins, et al., Urine TMPRSS2:ERG Plus PCA3 for Individualized Prostate Cancer Risk Assessment. Eur Urol (2016)) molecular tests have also been developed for use following screening. However, similarly these have only modest accuracy. Therefore, there is a remaining need for improved diagnostic approaches to distinguish benign and malignant disease following a positive screening test, as well as a need for improved approaches to distinguish indolent and aggressive disease.

[0007] What is needed are additional screening and diagnostic approaches to identify and categorize subjects with or at risk for prostate cancer.

[0008] SUMMARY OF THE DISCLOSURE

[0009] Provided herein are kits and methods useful for cancer screening, diagnosis, research and therapy. In particular, provided herein are methods of screening, diagnosing, and / or treating prostate cancer based on expression levels of cancer markers.

[0010] For example, in some embodiments, provided herein is method of assaying gene expression, comprising: assaying the level of expression of two or more or all (e.g., 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, . . ., 25, . . ., 30, . . ., 40, . . . or 50) genes selected from, for example, Solute Carrier Family 16 Member 5 (SLC16A5), Integrin Subunit Alpha 3 (ITGA3), ETS Transcription Factor ERG (ERG), Mitogen- Activated Protein Kinase 13 (MAPK13), Pleckstrin Homology Domain Containing A2 (PLEKHA2), Latent Transforming Growth Factor Beta Binding Protein 3 (LTBP3), Fatty Acid Binding Protein 5 (FABP5), Calmodulin Like 4 (CALML4), TBC1 Domain Family Member 2 (TBC1D2), Protein Phosphatase, Mg2+ / Mn2+ Dependent IM (PPM1M), NGFLA Binding Protein 2 (NAB2), ENSG00000259642, Retinoic Acid Receptor Gamma (RARG), Prostate Cancer Associated 3 (PCA3), Olfactory Receptor Family 51 Subfamily E Member 2 (OR51E2), Complement Clr (C1R), Coiled-Coil Domain GRGR-43680.601

[0011] Containing 69 (CCDC69), Transglutaminase 3 (TGM3), Integrin Subunit Beta 2 (ITGB2), Signal Transducing Adaptor Family Member 1 (STAP1), Interleukin 17 Receptor E (IL17RE) ATPase Family AAA Domain Containing 3C (ATAD3C), Inhibitor Of DNA Binding 4 (ID4), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3F (APOBEC3F), EGF Containing Fibulin Extracellular Matrix Protein 2 (EFEMP2), Moesin (MSN), Growth Differentiation Factor 15 (GDF15), NIPA Like Domain Containing 3 (NIPAL3), Interleukin 4 Receptor (ZL4R), Homeobox C6 ( H0XC6), Apoptosis Inducing Factor Mitochondria Associated 2 (AIFM2), Trefoil Factor 3 (TFF3), ENSG00000234964, Zinc Finger Protein 431 (ZNF431), Docking Protein 4 (D0K4), Golgi Membrane Protein 1 (G0LM1), C3 And PZP Like Alpha-2 - Macroglobulin Domain Containing 8 (CPAMD8), UDP-N-Acetylglucosamine Pyrophosphorylase 1 Like 1 (UAP1L1), Annexin A2 (ANXA2), Cellular Communication Network Factor 3 (CCN3), Matrix Metallopeptidase 14 (MMP14), MYC Proto-Oncogene, BHLH Transcription Factor (MYC), Adaptor Related Protein Complex 5 Subunit Beta 1 (AP5B1), Heat Shock Protein Family A (Hsp70) Member 6 (HSPA6), Regulator Of G Protein Signaling 2 (RGS2), EBF Family Member 4 (EBF4), Heme Oxygenase 1 (HM0X1), Heme Oxygenase 1 (LUZP2), Periaxin (PRX), or Von Willebrand Factor A Domain Containing 5A (VWA5A) in a sample from a subject. In some embodiments, the sample is a semen sample.

[0012] Further provided is a method of diagnosing prostate cancer, comprising: a) assaying the level of expression of two or more or all (e.g., 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, . . ., 25, . . ., 30, . . ., 40, . . . or 50) genes selected from, for example, SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, or VWA5A in a sample from a subject; and b) performing a confirmatory diagnostic assay. In some embodiments, the confirmatory diagnostic assay comprises one or more of: an imaging technique (e g., ultrasound, CT, MRI, PET, etc.), a biopsy, and a different biomarker assay. In some embodiments, the sample is a semen sample. In some embodiments, the method further comprises conducting an additional screening assay (e.g., a digital rectal examination, a PSA test, etc.). GRGR-43680.601

[0013] Further provided is a method of treating prostate cancer, comprising: a) assaying the level of expression of two or more or all (e.g., 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, . . ., 25, . . ., 30, . . ., 40, . . . or 50) genes selected from, for example, SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, or VWA5A in a sample from a subject; b) optionally, performing a confirmatory diagnostic assay and c) administering a prostate cancer treatment to a subject identified as having prostate cancer based on the levels of expression of the genes. In some embodiments, the sample is a semen sample. In some embodiments, the cancer treatment is one or more of: surgery (e g., radical prostatectomy, orchiectomy, etc ), radiation therapy (e.g., external beam, brachytherapy, targeted radiotherapy, etc.), cryoablation or cryotherapy, heat ablation, high-intensity focused ultrasound (HIFU), hormone therapy (e.g., anti-androgens, luteinizing horm one-releasing hormone (LHRH) or gonadotropin-releasing hormone (GnRH) agonists and antagonists), chemotherapy, or immunotherapy.

[0014] Also provided is a method of screening for or diagnosing prostate cancer in a subject, comprising: a) assaying the level of expression of two or more or all (e.g., 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, . . ., 25, . . ., 30, . . ., 40, . . . or 50) genes selected from, for example, SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, GOLM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, or VWA5A in a sample from a subject; and b) identifying the subject as having prostate cancer when the subject is identified as having altered levels of expression of the genes relative to a subject without prostate cancer. In some embodiments, the sample is a semen sample. In some embodiments, the method further comprises conducting an additional screening assay (e.g., a digital rectal examination, a PSA test, etc.). GRGR-43680.601

[0015] In some embodiments, the two or more genes comprise SLC16A5 and ITGA3; SLC16A5 and ERG; SLC16A5 and MAPK13; SLC16A5 and PLEKHA2; SLC16A5 and LTBP3; SLC16A5 and FABP5; SLC16A5 and CALML4, SLC16A5 and TBC1D2; SLC16A5 and PPM1M; ITGA3 and ERG; ITGA3 and MAPK13; ITGA3 and PLEKHA2; ITGA3 and LTBP3; ITGA3 and FABP5; ITGA3 and CALML4; ITGA3 and TBC1D2; ITGA3 and PPM1M; ERG and MAPK13; ERG and PLEKHA2; ERG and LTBP3; ERG and FABP5; ERG and CALML4; ERG and TBC1D2; ERG and PPM1M; MAPK13 and PLEKHA2; MAPK13 and LTBP3; MAPK13 and FABP5; MAPK13 and CALML4; MAPK13 and TBC1D2; MAPK13 and PPM1M; PLEKHA2 and LTBP3; PLEKHA2 and FABP5; PLEKHA2 and CALML4; PLEKHA2 and TBC1D2; PLEKHA2 and PPM1M; LTBP3 and FABP5; LTBP3 and FABP5, LTBP3 and CALML4; LTBP3 and TBC1D2; LTBP3 and PPM1M; FABP5 and CALML4; FABP5 and CALML4; FABP5 and TBC1D2; FABP5 and PPM1M; CALML4 and TBC1D2; CALML4 and PPM1M; or TBC1D2 and PPM1M.

[0016] In some embodiments, the two or more genes is three or more genes (e.g., SLC16A5, ITGA3, and ERG; SLC16A5, ITGA3, and MAPK13; SLC16A5, ITGA3, and PLEKHA2; SLC16A5, ITGA3, and LTBP3; SLC16A5, ITGA3, and FABP5; SLC16A5, ITGA3, and CALML4; SLC16A5, ITGA3, and TBC1D2; SLC16A5, ITGA3, and PPM1M; SLC16A5, ERG, and MAPK13; SLC16A5, ERG, and PLEKHA2; SLC16A5, ERG, and LTBP3; SLC16A5, ERG, and FABP5; SLC16A5, ERG, and CALML4; SLC16A5, ERG, and TBC1D2; SLC16A5, ERG, and PPM1M; SLC16A5, MAPK13, and PLEKHA2; SLC16A5, MAPK13, and LTBP3, SLC16A5, MAPK13, and FABP5, SLC16A5, MAPK13, and CALML4, SLC16A5, MAPK13, and TBC1D2; SLC16A5, MAPK13, and PPM1M; SLC16A5, PLEKHA2, and LTBP3; SLC16A5, PLEKHA2, and FABP5; SLC16A5, PLEKHA2, and CALML4; SLC16A5, PLEKHA2, and TBC1D2; SLC16A5, PLEKHA2, and PPM1M; SLC16A5, LTBP3, and FABP5; SLC16A5, LTBP3, andCALML4; SLC16A5, LTBP3, and TBC1D2; SLC16A5, LTBP3, and PPM1M; SLC16A5, FABP5, and CALML4; SLC16A5, FABP5, and TBC1D2; SLC16A5, FABP5, and PPMIM; SLC16A5, CALML4, and TBC1D2; SLC16A5, CALML4, and PPMIM; SLC16A5, TBC1D2, and PPM1M; ITGA3, ERG, and MAPK13; ITGA3, ERG, and PLEKHA2; ITGA3, ERG, and LTBP3; ITGA3, ERG, and FABP5; ITGA3, ERG, and CALML4; ITGA3, ERG, and TBC1D2; ITGA3, ERG, and PPM1M; ITGA3, MAPK13, and PLEKHA2; ITGA3, MAPK13, and LTBP3; ITGA3, MAPK13, and FABP5, ITGA3, MAPK13, and CALML4; GRGR-43680.601

[0017] ITGA3, MAPK13, and TBC1D2; ITGA3, MAPK13, and PPM IM; ITGA3, PLEKHA2, and LTBP3; ITGA3, PLEKHA2, and FABP5; ITGA3, PLEKHA2, and CALML4; ITGA3, PLEKHA2, and TBC1D2; ITGA3, PLEKHA2, and PPM1M; ITGA3, LTBP3, and FABP5; ITGA3, LTBP3, and CALML4; ITGA3, LTBP3, and TBC1D2; ITGA3, LTBP3, and PPM1M; ITGA3, FABP5, and CALML4; ITGA3, FABP5, and TBC1D2; ITGA3, FABP5, and PPMIM; ITGA3, CALML4, and TBC1D2; ITGA3, CALML4, and PPM1M; ITGA3, TBC1D2, and PPM IM; ERG, MAPK13, and PLEKHA2; ERG, MAPK13, and LTBP3; ERG, MAPK13, and FABP5; ERG, MAPK13, and CALML4; ERG, MAPK13, and TBC1D2; ERG, MAPK13, and PPM1M; ERG, PLEKHA2, and LTBP3; ERG, PLEKHA2, and FABP5; ERG, PLEKHA2, and CALML4; ERG, PLEKHA2, and TBC1D2; ERG, PLEKHA2, and PPM1M; ERG, LTBP3, and FABP5; ERG, LTBP3, and CALML4; ERG, LTBP3, and TBC1D2; ERG, LTBP3, and PPM1M; ERG, FABP5, and CALML4; ERG, FABP5, and TBC1D2; ERG, FABP5, and PPM1M; ERG, CALML4, and TBC1D2; ERG, CALML4, and PPM1M; ERG, TBC1D2, and PPM1M;

[0018] MAPK13, PLEKHA2, LTBP3; MAPK13, PLEKHA2, and CALML4; MAPK13, PLEKHA2, and TBC1D2; MAPK13, PLEKHA2, and PPM1M; MAPK13, LTBP3, and FABP5; MAPK13, LTBP3, and CALML4; MAPK13, LTBP3, and TBC1D2; MAPK13, LTBP3, and PPM1M; MAPK13, FABP5, and CALML4; MAPK13, FABP5, and TBC1D2; MAPK13, FABP5, and PPM1M; MAPK13, CALML4, and TBC1D2; MAPK13, CALML4, and PPM1M; MAPK13, TBC1D2, and PPM1M; PLEKHA2, LTBP3, and FABP5; PLEKHA2, LTBP3, and CALML4; PLEKHA2, LTBP3, and TBC1D2; PLEKHA2, LTBP3, and PPM1M; PLEKHA2, FABP5, and CALML4; PLEKHA2, FABP5, and TBC1D2; PLEKHA2, FABP5, and PPM1M; PLEKHA2, CALML4, and TBC1D2; PLEKHA2, CALML4, and PPM IM; PLEKHA2, TBC1D2, and PPM1M; LTBP3, FABP5, and CALML4; LTBP3, FABP5, and TBC1D2; LTBP3, FABP5, and PPM1M; LTBP3, CALML4, and TBC1D2; LTBP3, CALML4, and PPMIM; LTBP3, TBC1D2, and PPM1M; FABP5, CALML4, and TBC1D2; FABP5, CALML4, and PPM1M; or CALML4, TBC1D2, or PPMIM).

[0019] In certain embodiments, the two or more genes is four or more genes (e.g., SLC16A5, ITGA3, ERG, and MAPK13, SLC16A5, ITGA3, ERG, and PLEKHA2; SLC16A5, ITGA3, ERG, and LTBP3; SLC16A5, ITGA3, ERG, and FABP5; SLC16A5, ITGA3, ERG, and CALML4; SLC16A5, ITGA3, ERG, and TBC1D2; SLC16A5, ITGA3, ERG, and PPM1M; SLC16A5, ERG, MAPK13, and PLEKHA2; SLC16A5, ERG, MAPK13, and LTBP3; GRGR-43680.601

[0020] SLC16A5, ERG, MAPK13, and FABP5; SLC16A5, ERG, MAPK13, and CALML4; SLC16A5, ERG, MAPK13, and TBC1D2; SLC16A5, ERG, MAPK13, and PPM1M; SLC16A5, MAPK13, PLEKHA2, and LTBP3; SLC16A5, MAPK13, PLEKHA2, and FABP5; SLC16A5, MAPK13, PLEKHA2, and CALCL4; SLC16A5, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, and PPMIM; SLC16A5, PLEKHA2, LTBP3, and FABP5; SLC16A5, PLEKHA2, LTBP3, and CALML4; SLC16A5, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, and PPM1M; SLC16A5, LTBP3, FABP5, and CALML4; SLC16A5, LTBP3, FABP5, and TBC1D2; SLC16A5, LTBP3, FABP5, and PPM1M; SLC16A5, FABP5, CALML4, and TBC1D2; SLC16A5, FABP5, CALML4, and PPM1M; SLC16A5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM IM; ITGA3, MAPK13, PLEKHA2, and LTBP3; ITGA3, MAPK13, PLEKHA2, and FABP5; ITGA3, MAPK13, PLEKHA2, and CALML4; ITGA3, MAPK13, PLEKHA2, and TBC1D2; ITGA3, MAPK13, PLEKHA2, and PPM1M; ITGA3, PLEKHA2, LTBP3, and FABP5; ITGA3, PLEKHA2, LTBP3, and CALML4; ITGA3, PLEKHA2, LTBP3, and TBC1D2; ITGA3, PLEKHA2, LTBP3, and PPMIM; ITGA3, LTBP3, FABP5, and CALML4; ITGA3, LTBP3, FABP5, and TBC1D2; ITGA3, LTBP3, FABP5, and PPM1M; ITGA3, FABP5, CALML4, and TBC1D2; ITGA3, FABP5, CALML4, and PPM1M; ITGA3, CALML4, TBC1D2, and PPM IM; ERG, MAPK13, PLEKHA2, and LTBP3; ERG, MAPK13, PLEKHA2, and CALML4; ERG, MAPK13, PLEKHA2, and TBC1D2; ERG, MAPK13, PLEKHA2, and SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M; ERG, PLEKHA2, LTBP3, and FABP5; ERG, PLEKHA2, LTBP3, and CALML4; ERG, PLEKHA2, LTBP3, and TBC1D2; ERG, PLEKHA2, LTBP3, and PPM1M; ERG, LTBP3, FABP5, and CALML4; ERG, LTBP3, FABP5, and TBC1D2; ERG, LTBP3, FABP5, and PPM1M; ERG, FABP5, CALML4, and TBC1D2; ERG, FABP5, CALML4, and PPM1M; ERG, CALML4, TBC1D2, and PPM1M; MAPK13, PLEKHA2, LTBP3, and FABP5; MAPK13, PLEKHA2, LTBP3, and CALML4; MAPK13, PLEKHA2, LTBP3, and TBC1D2; MAPK13, PLEKHA2, LTBP3, and PPM1M; MAPK13, LTBP3, FABP5, and CALML4; GRGR-43680.601

[0021] MAPK13, LTBP3, FABP5, and TBC1D2; MAPK13, LTBP3, FABP5, and PPM1M; MAPK13, FABP5, CALML4, and TBC1D2; MAPK13, FABP5, CALML4, and PPM1M; MAPK13, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, and CALML4; PLEKHA2, LTBP3, FABP5, and TBC1D2; PLEKHA2, LTBP3, FABP5, and PPM1M; PLEKHA2, FABP5, CALML4, and TBC1D2; PLEKHA2, FABP5, CALML4, and PPMIM; PLEKHA2, CALML4, TBC1D2, and PPM1M; or FABP5, CALML4, TBC1D2, PPM1M.

[0022] In some embodiments, the two or more genes is five or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, and PLEKHA2; SLC16A5, ITGA3, ERG, MAPK13, and LTBP3; SLC16A5, ITGA3, ERG, MAPK13, and FABP5; SLC16A5, ITGA3, ERG, MAPK13, and CALML4; SLC16A5, ITGA3, ERG, MAPK13, and TBC1D2; SLC16A5, ITGA3, ERG, MAPK13, and PPM1M; SLC16A5, ERG, MAPK13, PLEKHA2, and LTBP3; SLC16A5, ERG, MAPK13, PLEKHA2, and FABP5; SLC16A5, ERG, MAPK13, PLEKHA2, and CALML4; SLC16A5, ERG, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, ERG, MAPK13, PLEKHA2, and PPM1M; SLC16A5, MAPK13, PLEKHA2, LTBP3, and FABP5; SLC16A5, MAPK13, PLEKHA2, LTBP3, and CALML4; SLC16A5, MAPK13, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, LTBP3, and PPM IM; SLC16A5, PLEKHA2, LTBP3, FABP5, and CALML4; SLC16A5, PLEKHA2, LTBP3, FABP5, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, FABP5, and PPM1M; SLC16A5, LTBP3, FABP5, CALML4, and TBC1D2; SLC16A5, LTBP3, FABP5, CALML4, and PPM1M; SLC16A5, FABP5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, PLEKHA2, and LTBP3; ITGA3, ERG, MAPK13, PLEKHA2, and FABP5; ITGA3, ERG, MAPK13, PLEKHA2, and CALML4; ITGA3, ERG, MAPK13, PLEKHA2, and TBC1D2; ITGA3, ERG, MAPK13, PLEKHA2, and PPM IM; ITGA3, MAPK13, PLEKHA2, LTBP3, and FABP5; ITGA3, MAPK13, PLEKHA2, LTBP3, and CALML4; ITGA3, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ITGA3, MAPK13, PLEKHA2, LTBP3, and PPM1M; ITGA3, PLEKHA2, LTBP3, FABP5, and CALML4; ITGA3, PLEKHA2, LTBP3, FABP5, and TBC1D2; ITGA3, PLEKHA2, LTBP3, FABP5, and PPM1M; ITGA3, LTBP3, FABP5, CALML4, and TBC1D2; ITGA3, LTBP3, FABP5, CALML4, and PPM1M; ITGA3, FABP5, CALML4, TBC1D2, and PPM1M; ERG, MAPK13, PLEKHA2, LTBP3, and FABP5; ERG, MAPK13, PLEKHA2, LTBP3, and CALML4; ERG, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ERG, MAPK13, PLEKHA2, LTBP3, and PPM1M; ERG, PLEKHA2, LTBP3, FABP5, and CALML4; ERG, PLEKHA2, GRGR-43680.601

[0023] LTBP3, FABP5, and TBC1D2; ERG, PLEKHA2, LTBP3, FABP5, and PPM1M; ERG, LTBP3, FABP5, CALML4, and TBC1D2; ERG, LTBP3, FABP5, CALML4, and PPM1M; MAPK13, PLEKHA2, LTBP3, FABP5, and CALML4; MAPK13, PLEKHA2, LTBP3, FABP5, and TBC1D2; MAPK13, PLEKHA2, LTBP3, FABP5, and PPM1M; MAPK13, LTBP3, FABP5, CALML4, and TBC1D2; MAPK13, LTBP3, FABP5, CALML4, and PPM1M; MAPK13, FABP5, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, CALML4, and TBC1D2; PLEKHA2, LTBP3, FABP5, CALML4, PPM1M; PLEKHA2, FABP5, CALML4, TBC1D2, and PPM1M; or LTBP3, FABP5, CALML4, TBC1D2, and PPM1M).

[0024] In some embodiments, the two or more genes is 10 or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, and PPM1M). In some embodiments, the two or more genes is 25 or more genes. In some embodiments, the two or more genes comprises 2 to 50 genes. In some embodiments, the two or more genes comprises 2 to 100 genes. In some embodiments, the level of expression of one or more additional genes is determined.

[0025] In some embodiments, the two or more genes is 50 or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, GOLM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HMOX1, LUZP2, PRX, and VWA5A).

[0026] In some embodiments, the method further comprises administering a prostate cancer treatment to said subject (e.g., one or more of surgery (e.g., radical prostatectomy, orchiectomy, etc.), radiation therapy (e.g., external beam, brachytherapy, targeted radiotherapy, etc.), cryoablation or cryotherapy, heat ablation, high-intensity focused ultrasound (HIFU), hormone therapy (e.g., anti-androgens, luteinizing hormone-releasing hormone (LHRH) or gonadotropinreleasing hormone (GnRH) agonists and antagonists), chemotherapy, or immunotherapy).

[0027] The present disclosure is not limited to particular sample types. Examples include but are not limited to, urine, prostate secretions, prostate cells, blood, and semen.

[0028] The present disclosure is not limited by the vasectomy status of the subject. Both subjects that have and have not undergone vasectomies can provide samples (e.g., urine, prostate secretions, prostate cells, blood or semen) for use in the methods described herein. GRGR-43680.601

[0029] In some embodiments, the subject has not had a prior prostate biopsy or has had a prior negative prostate biopsy result. In some embodiments the prostate biopsy is positive, resulting in a diagnosis of cancer, and the cancer is characterized as low-risk (e.g., as a candidate for active surveillance). In some embodiments the prostate biopsy is positive, resulting in a diagnosis of cancer, and the cancer is characterized as high-risk (e.g., as a candidate for active treatment). In some embodiments, the method further comprises performing a prostate biopsy on the subject.

[0030] In some embodiments, the method further comprises identifying and analyzing at least one additional variable in combination with the gene expression analysis. In some embodiments, the one or more additional variables is the subject’s age, race, family history of prostate cancer, digital rectal examination (DRE) result, prostate biopsy result, prostate specific antigen (PSA) expression value (e.g., based on a serum sample), multi-perimetric MRI (mpMRI) result, prostate volume (as measured by either DRE or MRI), PSA density (PSAD), or any combination thereof.

[0031] In some embodiments, the level or amount of expression is the amount of mRNA or protein expressed by the genes. In some embodiments, the level of expression is increased or decreased relative to the level in a subject that does not have prostate cancer. In some embodiments, detecting the level or amount of expression of said genes comprises detecting an amount of mRNA expression of the genes (e.g., using a nucleic acid amplification assay and / or a nucleic acid sequencing assay). In some embodiments, the nucleic acid amplification assay comprises performing a reverse transcription polymerase chain reaction.

[0032] Also provided is a device, system, or kit comprising one or more of: a) reagents for detecting the level of expression of at least two (e.g., 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, . . ., 25, . . ., 30, . . ., 40, . . . or 50) genes selected from, for example, SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, or VWA5A; b) software comprising instructions that cause a computing device having a processor to carryout out one or more of: i) collecting expression data (e.g., from a detection instrument); ii) receiving patientspecific information (e.g., from a patient, a caregiver, an electronic medical record, etc.); iii) calculating a risk score or profile based on the expression data results and / or one or more GRGR-43680.601 additional patient-specific variables; iv) displaying expression information, additional information, and / or a risk score or profile; v) listing or recommending a potential treatment course of action; and vi) transmitting or communicating expression information, additional information, a risk score or profile, and / or a treatment listing or recommendation to a second computing device (e.g., at a remove location). c) instruction for using the device, system, or kit; d) control reagents (e.g., positive controls, negative controls, standards); e) detection assay components (e.g., a reverse transcriptase, a polymerase, primers, probes, dNTPs, buffers, adapters, ligases, detectable labels (e.g., fluorophores), antibodies, etc.); f) reagent containers, dispensers, multi-well plates, flow cells, etc.; and g) packaging.

[0033] In some embodiments, any of the methods described herein are employed before and / or after a treatment event. For example, in some embodiments, a first expression analysis is conducted prior to an intervention and a second expression analysis is conducted following the treatment event (i.e., test-treat-test). Any order of combination of testing and treating steps are contemplated (e.g., test / treat; treat / test; test / treat / test; test / treat / test / treat; treat / test / treat; test / treat / test / treat / test; treat / test / test / treat; etc.). Such methods find use to evaluate the efficacy of an intervention, to identify when a change in intervention is warranted, to move from watch- and-wait to treatment or vice versa, and the like.

[0034] Additional embodiments are described herein.

[0035] DESCRIPTION OF THE FIGURES

[0036] FIG 1 shows ROC-AUC of validation set samples.

[0037] FIG 2 shows score distribution of classifier for cancer (Class 1) and no-cancer (Class 0) samples.

[0038] DEFINITIONS

[0039] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

[0040] As used herein, the terms "detect", "detecting" or "detection" may describe either the general act of discovering or discerning or the specific observation of a composition. Detecting a GRGR-43680.601 composition may comprise determining the presence or absence of a composition. Detecting may comprise quantifying a composition. For example, detecting comprises determining the expression level of a composition. The composition may comprise a nucleic acid molecule. For example, the composition may comprise at least a portion of the cancer markers disclosed herein. Alternatively, or additionally, the composition may be a detectably labeled composition.

[0041] As used herein, the term "subject" refers to any organisms that are screened using the diagnostic or therapeutic methods described herein. Such organisms preferably include, but are not limited to, mammals, and most preferably includes humans. In some embodiments, the subject is a mammal having a prostate. In some embodiments, the subject is a human having a prostate.

[0042] The term "diagnosed, " as used herein, refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like.

[0043] As used herein, the language "characterizing cancer in a subject" refers to the identification of one or more properties of a cancer sample in a subject, including but not limited to, the presence of benign, pre-cancerous or cancerous tissue, the stage of the cancer, and the subject's prognosis. Cancers may be characterized by the identification of the level of expression of one or more cancer marker genes, including but not limited to, the cancer markers disclosed herein.

[0044] As used herein, the language "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria useful to determine the stage of a cancer include, but are not limited to, the size of the tumor and the extent of metastases (e.g., localized or distant). In some embodiments, prostate cancer is classified by Gleason scores and ISUP grade groups (See e.g., Leenders et al., Am J Surg Pathol. 2020 Aug; 44(8): e87-e99; herein incorporated by reference in its entirety). A Gleason score less than or equal to 6 is considered low / very low risk (Grade Group 1); score of 7 is considered intermediate risk (Grade Group 2 (Gleason Score 7 (3 + 4)) or Grade Group 3 (Gleason Score 7 (4 + 3)); score of 8 or more is considered high / very high risk (Grade Group 4 (Gleason Score 8); Grade Group 5 (Gleason Score 9-10)).

[0045] As used herein, the language "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The nucleic acid molecule may comprise one or more nucleotides. The language may include nucleotide polymers in which the GRGR-43680.601 nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. The term further encompasses sequences that may include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl- methyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine,

[0046] 1 -methyl adenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethyl- guanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino- methyl-2 -thiouracil, beta-D-mannosylqueosine, 5'-methoxy carbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-

[0047] 2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), the sequence also includes an RNA sequence i.e., A, U, G, C) in which "U" replaces "T."

[0048] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragments are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end, such that the “gene” corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or GRGR-43680.601

[0049] "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0050] As used herein, the term "oligonucleotide," refers to a short length of single- stranded polynucleotide chain. Oligonucleotides are typically less than 200 nucleotide residues long (e.g., between 15 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24- residue oligonucleotide is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

[0051] The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to: dyes; radiolabels such as32P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (e.g., a positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. In some embodiments, nucleic acids are detected directly without a label (e.g., directly reading a sequence).

[0052] As used herein, the term "sample" includes a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids (e.g., blood, urine, semen), solids, tissues, and gases. Biological samples can include urine, urine supernatant, and urine cell pellet, semen, prostate secretions, as well as blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure. GRGR-43680.601

[0053] As used herein, the term "altered, " for example in the context of "altered levels of expression of one or more of the genes, " refers to a level of gene expression that is different e.g., increased or decreased) than the level of expression in, e.g., a subject without prostate cancer.

[0054] As used herein, the term "about" means ± 10% variation from nominal value unless otherwise indicated or inferred. When the term "about" is used before a number, the present disclosure also includes the specific number itself, unless specifically stated otherwise.

[0055] DETAILED DESCRIPTION OF THE DISCLOSURE

[0056] Provided herein are kits and methods useful for cancer screening, diagnosis, research and therapy. In particular, provided herein are methods of screening, diagnosing, and / or treating prostate cancer based on expression levels of cancer markers.

[0057] Described herein are methods and kits incorporating one or more of a set of markers useful for screening, diagnosing or treating prostate cancer.

[0058] The methods described herein are useful to identify subjects with prostate cancer for further actions such as, for example, biopsy, imaging, watch-and-wait, and / or treatment.

[0059] I. Methods of assaying marker expression

[0060] As described herein, embodiments of the present disclosure provide methods for screening, diagnosis or treatment that utilize detection of an expression amount or level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) genes selected from, for example, SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, D0K4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, or VWA5A. Illustrative, non-limiting methods are described herein. GRGR-43680.601

[0061] Genes for Detecting

[0062] In some embodiments, the level or amount of expression of one or more genes is determined. In some embodiments, the level or amount of expression is the level or amount of mRNA or protein expressed by the genes.

[0063] In some embodiments, the genes are selected from SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, H0XC6, AIFM2, TFF3, ENSG00000234964, ZNF431, D0K4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A.

[0064] In some embodiments, the level of expression of two or more genes is determined. In some embodiments, the two or more genes comprise SLC16A5 and ITGA3; SLC16A5 and ERG; SLC16A5 and MAPK13; SLC16A5 and PLEKHA2; SLC16A5 and LTBP3; SLC16A5 and FABP5; SLC16A5 and CALML4, SLC16A5 and TBC1D2; SLC16A5 and PPM1M; ITGA3 and ERG; ITGA3 and MAPK13; ITGA3 and PLEKHA2; ITGA3 and LTBP3; ITGA3 and FABP5; ITGA3 and CALML4; ITGA3 and TBC1D2; ITGA3 and PPM1M; ERG and MAPK13; ERG and PLEKHA2; ERG and LTBP3; ERG and FABP5; ERG and CALML4; ERG and TBC1D2; ERG and PPM1M; MAPK13 and PLEKHA2; MAPK13 and LTBP3; MAPK13 and FABP5; MAPK13 and CALML4; MAPK13 and TBC1D2; MAPK13 and PPM1M; PLEKHA2 and LTBP3; PLEKHA2 and FABP5; PLEKHA2 and CALML4; PLEKHA2 and TBC1D2; PLEKHA2 and PPM1M; LTBP3 and FABP5; LTBP3 and FABP5, LTBP3 and CALML4; LTBP3 and TBC1D2; LTBP3 and PPM1M; FABP5 and CALML4; FABP5 and CALML4; FABP5 and TBC1D2; FABP5 and PPM1M; CALML4 and TBC1D2; CALML4 and PPM1M; or TBC1D2 and PPM1M.

[0065] In some embodiments, the two or more genes is three or more genes (e.g., SLC16A5, ITGA3, and ERG; SLC16A5, ITGA3, and MAPK13; SLC16A5, ITGA3, and PLEKHA2; SLC16A5, ITGA3, and LTBP3; SLC16A5, ITGA3, and FABP5; SLC16A5, ITGA3, and CALML4; SLC16A5, ITGA3, and TBC1D2; SLC16A5, ITGA3, and PPM1M; SLC16A5, ERG, and MAPK13; SLC16A5, ERG, and PLEKHA2; SLC16A5, ERG, and LTBP3; SLC16A5, ERG, and FABP5; SLC16A5, ERG, and CALML4; SLC16A5, ERG, and TBC1D2; SLC16A5, ERG, and PPM1M; SLC16A5, MAPK13, and PLEKHA2; SLC16A5, MAPK13, and LTBP3, GRGR-43680.601

[0066] SLC16A5, MAPK13, and FABP5, SLC16A5, MAPK13, and CALML4, SLC16A5, MAPK13, and TBC1D2; SLC16A5, MAPK13, and PPMIM; SLC16A5, PLEKHA2, and LTBP3;

[0067] SLC16A5, PLEKHA2, and FABP5; SLC16A5, PLEKHA2, and CALML4; SLC16A5, PLEKHA2, and TBC1D2; SLC16A5, PLEKHA2, and PPM1M; SLC16A5, LTBP3, and FABP5; SLC16A5, LTBP3, andCALML4; SLC16A5, LTBP3, and TBC1D2; SLC16A5, LTBP3, and PPM1M; SLC16A5, FABP5, and CALML4; SLC16A5, FABP5, and TBC1D2; SLC16A5, FABP5, and PPMIM; SLC16A5, CALML4, and TBC1D2; SLC16A5, CALML4, and PPMIM; SLC16A5, TBC1D2, and PPM1M; ITGA3, ERG, and MAPK13; ITGA3, ERG, and PLEKHA2; ITGA3, ERG, and LTBP3; ITGA3, ERG, and FABP5; ITGA3, ERG, and CALML4; ITGA3, ERG, and TBC1D2; ITGA3, ERG, and PPM1M; ITGA3, MAPK13, and PLEKHA2; ITGA3, MAPK13, and LTBP3; ITGA3, MAPK13, and FABP5, ITGA3, MAPK13, and CALML4;

[0068] ITGA3, MAPK13, and TBC1D2; ITGA3, MAPK13, and PPM IM; ITGA3, PLEKHA2, and LTBP3; ITGA3, PLEKHA2, and FABP5; ITGA3, PLEKHA2, and CALML4; ITGA3, PLEKHA2, and TBC1D2; ITGA3, PLEKHA2, and PPM1M; ITGA3, LTBP3, and FABP5; ITGA3, LTBP3, and CALML4; ITGA3, LTBP3, and TBC1D2; ITGA3, LTBP3, and PPM1M; ITGA3, FABP5, and CALML4; ITGA3, FABP5, and TBC1D2; ITGA3, FABP5, and PPMIM; ITGA3, CALML4, and TBC1D2; ITGA3, CALML4, and PPM1M; ITGA3, TBC1D2, and PPM IM; ERG, MAPK13, and PLEKHA2; ERG, MAPK13, and LTBP3; ERG, MAPK13, and FABP5; ERG, MAPK13, and CALML4; ERG, MAPK13, and TBC1D2; ERG, MAPK13, and PPM1M; ERG, PLEKHA2, and LTBP3; ERG, PLEKHA2, and FABP5; ERG, PLEKHA2, and CALML4; ERG, PLEKHA2, and TBC1D2; ERG, PLEKHA2, and PPM1M; ERG, LTBP3, and FABP5; ERG, LTBP3, and CALML4; ERG, LTBP3, and TBC1D2; ERG, LTBP3, and PPM1M; ERG, FABP5, and CALML4; ERG, FABP5, and TBC1D2; ERG, FABP5, and PPM1M; ERG, CALML4, and TBC1D2; ERG, CALML4, and PPM1M; ERG, TBC1D2, and PPM1M;

[0069] MAPK13, PLEKHA2, LTBP3; MAPK13, PLEKHA2, and CALML4; MAPK13, PLEKHA2, and TBC1D2; MAPK13, PLEKHA2, and PPM1M; MAPK13, LTBP3, and FABP5; MAPK13, LTBP3, and CALML4; MAPK13, LTBP3, and TBC1D2; MAPK13, LTBP3, and PPM1M; MAPK13, FABP5, and CALML4; MAPK13, FABP5, and TBC1D2; MAPK13, FABP5, and PPM1M; MAPK13, CALML4, and TBC1D2; MAPK13, CALML4, and PPM1M; MAPK13, TBC1D2, and PPM1M; PLEKHA2, LTBP3, and FABP5; PLEKHA2, LTBP3, and CALML4; PLEKHA2, LTBP3, and TBC1D2; PLEKHA2, LTBP3, and PPM1M; PLEKHA2, FABP5, and GRGR-43680.601

[0070] CALML4; PLEKHA2, FABP5, and TBC1D2; PLEKHA2, FABP5, and PPM1M; PLEKHA2, CALML4, and TBC1D2; PLEKHA2, CALML4, and PPM1M; PLEKHA2, TBC1D2, and PPM1M; LTBP3, FABP5, and CALML4; LTBP3, FABP5, and TBC1D2; LTBP3, FABP5, and PPM1M; LTBP3, CALML4, and TBC1D2; LTBP3, CALML4, and PPM1M; LTBP3, TBC1D2, and PPM1M; FABP5, CALML4, and TBC1D2; FABP5, CALML4, and PPM1M; or CALML4, TBC1D2, or PPMIM).

[0071] In certain embodiments, the two or more genes is four or more genes (e.g., SLC16A5, ITGA3, ERG, and MAPK13, SLC16A5, ITGA3, ERG, and PLEKHA2; SLC16A5, ITGA3, ERG, and LTBP3; SLC16A5, ITGA3, ERG, and FABP5; SLC16A5, ITGA3, ERG, and CALML4; SLC16A5, ITGA3, ERG, and TBC1D2; SLC16A5, ITGA3, ERG, and PPM1M; SLC16A5, ERG, MAPK13, and PLEKHA2; SLC16A5, ERG, MAPK13, and LTBP3; SLC16A5, ERG, MAPK13, and FABP5; SLC16A5, ERG, MAPK13, and CALML4; SLC16A5, ERG, MAPK13, and TBC1D2; SLC16A5, ERG, MAPK13, and PPMIM; SLC16A5, MAPK13, PLEKHA2, and LTBP3; SLC16A5, MAPK13, PLEKHA2, and FABP5; SLC16A5, MAPK13, PLEKHA2, and CALCL4; SLC16A5, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, and PPMIM; SLC16A5, PLEKHA2, LTBP3, and FABP5; SLC16A5, PLEKHA2, LTBP3, and CALML4; SLC16A5, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, and PPM1M; SLC16A5, LTBP3, FABP5, and CALML4; SLC16A5, LTBP3, FABP5, and TBC1D2; SLC16A5, LTBP3, FABP5, and PPM1M; SLC16A5, FABP5, CALML4, and TBC1D2; SLC16A5, FABP5, CALML4, and PPM1M; SLC16A5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM IM; ITGA3, MAPK13, PLEKHA2, and LTBP3; ITGA3, MAPK13, PLEKHA2, and FABP5; ITGA3, MAPK13, PLEKHA2, and CALML4; ITGA3, MAPK13, PLEKHA2, and TBC1D2; ITGA3, MAPK13, PLEKHA2, and PPM1M; ITGA3, PLEKHA2, LTBP3, and FABP5; ITGA3, PLEKHA2, LTBP3, and CALML4; ITGA3, PLEKHA2, LTBP3, and TBC1D2; ITGA3, PLEKHA2, LTBP3, and PPMIM; ITGA3, LTBP3, FABP5, and CALML4; ITGA3, LTBP3, FABP5, and TBC1D2; ITGA3, LTBP3, FABP5, and PPM1M; GRGR-43680.601

[0072] ITGA3, FABP5, CALML4, and TBC1D2; ITGA3, FABP5, CALML4, and PPM1M; ITGA3, CALML4, TBC1D2, and PPM IM; ERG, MAPK13, PLEKHA2, and LTBP3; ERG, MAPK13, PLEKHA2, and CALML4; ERG, MAPK13, PLEKHA2, and TBC1D2; ERG, MAPK13, PLEKHA2, and SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M; ERG, PLEKHA2, LTBP3, and FABP5; ERG, PLEKHA2, LTBP3, and CALML4; ERG, PLEKHA2, LTBP3, and TBC1D2; ERG, PLEKHA2, LTBP3, and PPM1M; ERG, LTBP3, FABP5, and CALML4; ERG, LTBP3, FABP5, and TBC1D2; ERG, LTBP3, FABP5, and PPM1M; ERG, FABP5, CALML4, and TBC1D2; ERG, FABP5, CALML4, and PPM1M; ERG, CALML4, TBC1D2, and PPM1M; MAPK13, PLEKHA2, LTBP3, and FABP5; MAPK13, PLEKHA2, LTBP3, and CALML4; MAPK13, PLEKHA2, LTBP3, and TBC1D2; MAPK13, PLEKHA2, LTBP3, and PPM1M; MAPK13, LTBP3, FABP5, and CALML4; MAPK13, LTBP3, FABP5, and TBC1D2; MAPK13, LTBP3, FABP5, and PPM1M; MAPK13, FABP5, CALML4, and TBC1D2; MAPK13, FABP5, CALML4, and PPM1M; MAPK13, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, and CALML4; PLEKHA2, LTBP3, FABP5, and TBC1D2; PLEKHA2, LTBP3, FABP5, and PPM1M; PLEKHA2, FABP5, CALML4, and TBC1D2; PLEKHA2, FABP5, CALML4, and PPMIM; PLEKHA2, CALML4, TBC1D2, and PPM1M; or FABP5, CALML4, TBC1D2, PPM1M.

[0073] In some embodiments, the two or more genes is five or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, and PLEKHA2; SLC16A5, ITGA3, ERG, MAPK13, and LTBP3; SLC16A5, ITGA3, ERG, MAPK13, and FABP5; SLC16A5, ITGA3, ERG, MAPK13, and CALML4; SLC16A5, ITGA3, ERG, MAPK13, and TBC1D2; SLC16A5, ITGA3, ERG, MAPK13, and PPM1M; SLC16A5, ERG, MAPK13, PLEKHA2, and LTBP3; SLC16A5, ERG, MAPK13, PLEKHA2, and FABP5; SLC16A5, ERG, MAPK13, PLEKHA2, and CALML4; SLC16A5, ERG, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, ERG, MAPK13, PLEKHA2, and PPM1M; SLC16A5, MAPK13, PLEKHA2, LTBP3, and FABP5; SLC16A5, MAPK13, PLEKHA2, LTBP3, and CALML4; SLC16A5, MAPK13, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, LTBP3, and PPM IM; SLC16A5, PLEKHA2, LTBP3, FABP5, and CALML4; SLC16A5, PLEKHA2, LTBP3, FABP5, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, FABP5, and PPM1M; SLC16A5, LTBP3, FABP5, CALML4, and TBC1D2; SLC16A5, LTBP3, FABP5, CALML4, and PPM1M; SLC16A5, FABP5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, PLEKHA2, and LTBP3; ITGA3, ERG, GRGR-43680.601

[0074] MAPK13, PLEKHA2, and FABP5; ITGA3, ERG, MAPK13, PLEKHA2, and CALML4; ITGA3, ERG, MAPK13, PLEKHA2, and TBC1D2; ITGA3, ERG, MAPK13, PLEKHA2, and PPM IM; ITGA3, MAPK13, PLEKHA2, LTBP3, and FABP5; ITGA3, MAPK13, PLEKHA2, LTBP3, and CALML4; ITGA3, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ITGA3, MAPK13, PLEKHA2, LTBP3, and PPM1M; ITGA3, PLEKHA2, LTBP3, FABP5, and CALML4; ITGA3, PLEKHA2, LTBP3, FABP5, and TBC1D2; ITGA3, PLEKHA2, LTBP3, FABP5, and PPM1M; ITGA3, LTBP3, FABP5, CALML4, and TBC1D2; ITGA3, LTBP3, FABP5, CALML4, and PPM1M; ITGA3, FABP5, CALML4, TBC1D2, and PPM1M; ERG, MAPK13, PLEKHA2, LTBP3, and FABP5; ERG, MAPK13, PLEKHA2, LTBP3, and CALML4; ERG, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ERG, MAPK13, PLEKHA2, LTBP3, and PPM1M; ERG, PLEKHA2, LTBP3, FABP5, and CALML4; ERG, PLEKHA2, LTBP3, FABP5, and TBC1D2; ERG, PLEKHA2, LTBP3, FABP5, and PPM1M; ERG, LTBP3, FABP5, CALML4, and TBC1D2; ERG, LTBP3, FABP5, CALML4, and PPM1M; MAPK13, PLEKHA2, LTBP3, FABP5, and CALML4; MAPK13, PLEKHA2, LTBP3, FABP5, and TBC1D2; MAPK13, PLEKHA2, LTBP3, FABP5, and PPM1M; MAPK13, LTBP3, FABP5, CALML4, and TBC1D2; MAPK13, LTBP3, FABP5, CALML4, and PPMIM; MAPK13, FABP5, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, CALML4, and TBC1D2; PLEKHA2, LTBP3, FABP5, CALML4, PPM1M; PLEKHA2, FABP5, CALML4, TBC1D2, and PPM1M; or LTBP3, FABP5, CALML4, TBC1D2, and PPM1M).

[0075] In some embodiments, the two or more genes is 10 or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, and PPM1M). In some embodiments, the two or more genes is 25 or more genes.

[0076] In some embodiments, the two or more genes comprises 2 to 50 genes or any range therewith (e.g., 2-49, 2-48, 2-47, . . . ; 3-50, 3-49, 3-48, . . . , 4-50, 4-49, 4-48, . . ., 5-50, 5-49, 5- 48, > . . . . . . , 49-50). In some embodiments, the two or more genes comprises 2 to 100 genes. In some embodiments, the level of expression of one or more additional genes is determined.

[0077] In some embodiments, the two or more genes is 50 or more genes (e.g., SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, GRGR-43680.601

[0078] ENSG00000234964, ZNF431, D0K4, G0LM1 , CPAMD8, UAP1L1 , ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A).

[0079] In some embodiments, fusions of the genes described herein with a second gene are detected (e.g., fusion of gene described herein with another gene described herein or a gene non described herein).

[0080] In some embodiments, the level of expression of the genes is altered (e.g., increased or decreased) relative to the level of expression of the gene in a subject (e.g., average value for a plurality of subjects) that does not have prostate cancer (e.g., as determined by the Log2 Fold Change of the gene shown in Table 3), the level of expression from a prior time point from the same subject, or the an established threshold level of expression.

[0081] Methods for Detecting Expression o f Genes

[0082] The level or amount of expression of one or more genes of the present disclosure can be detected using any of a variety of nucleic acid techniques, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and nucleic acid amplification.

[0083] In some embodiments, nucleic acid amplification is employed to assess gene expression. For example, reverse transcription polymerase chain reaction (RT-PCR) finds use for evaluating gene expression by quantifying RNA levels in a sample. The process begins with the conversion of RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This cDNA then serves as a template for amplification during the PCR process. RT-PCR is highly sensitive, allowing for the detection and quantification of low-abundance mRNA transcripts, making it a powerful tool for studying gene expression patterns. There are two main types of RT-PCR: endpoint RT-PCR and quantitative RT-PCR (qRT-PCR). While end-point RT-PCR provides qualitative insights, such as whether a gene is expressed, qRT-PCR allows for the precise quantification of gene expression levels in real-time by measuring the accumulation of PCR products during each cycle. The use of fluorescent dyes or probes in qRT-PCR enables the monitoring of the amplification process, allowing for the accurate determination of relative or absolute gene expression levels.

[0084] In some embodiments, gene expression is measured using hybridization technologies. For example, gene expression analysis using hybridization-based approaches, such as microarrays, involves the measurement of RNA levels by detecting the hybridization of nucleic GRGR-43680.601 acids to complementary probes. In microarray technology, thousands of short, single-stranded DNA probes representing specific genes are immobilized on a solid surface, typically a glass slide. RNA extracted from a sample is first reverse-transcribed into cDNA, which is then labeled with fluorescent dyes. When this labeled cDNA is applied to the microarray, it hybridizes with complementary DNA probes on the array. The intensity of the fluorescence at each probe spot correlates with the abundance of the corresponding RNA in the sample, allowing for quantification of gene expression levels across thousands of genes simultaneously. This approach is particularly useful for comparing gene expression profiles between different conditions, such as healthy versus diseased tissues, or for identifying genes that are up- or down-regulated in response to a treatment.

[0085] In some embodiments, gene expression is measured by nucleic acid sequencing. In some embodiments, the nucleic acid sequencing comprises next-generation sequencing. Modem nextgeneration sequencing (NGS) technologies have revolutionized nucleic acid analysis by enabling the rapid and high-throughput sequencing of DNA and RNA with unparalleled accuracy and depth. NGS platforms can sequence millions to billions of nucleic acid fragments simultaneously. For example, Illumina’s sequencing by synthesis (SBS) technology is widely used for its accuracy and scalability. It employs reversible dye terminators and a massively parallel sequencing approach. Another example is Oxford Nanopore’s long-read sequencing technology, which allows for the direct sequencing of long DNA or RNA fragments in real-time, making it especially useful for resolving complex genomic regions, structural variants, and full- length RNA transcripts. These advancements have significantly lowered the cost and time required for sequencing. NGS can be employed in transcriptomics through RNA sequencing (RNA-seq), which allows for the comprehensive analysis of gene expression, alternative splicing, and non-coding RNA discovery.

[0086] Protein analysis for assessing gene expression involves measuring the levels of proteins in a sample, which provides a direct insight into the functional outcomes of gene expression. Unlike RNA-based methods, which measure transcripts, protein analysis captures the final product of gene expression, revealing the actual biological activity. Techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry find use for these purposes. Western blotting allows for the detection and quantification of specific proteins by separating them based on size via gel electrophoresis, followed by transfer to a membrane and GRGR-43680.601 probing with specific antibodies. ELISA, on the other hand, is a highly sensitive technique that quantifies proteins in a sample using antibody-antigen interactions, often used in clinical diagnostics and research to measure protein concentrations. Mass spectrometry provides a more comprehensive approach, enabling the identification and quantification of thousands of proteins simultaneously, offering a global view of protein expression profiles, post-translational modifications, and protein interactions.

[0087] In some embodiments, the level or amount of expression of any one of the genes described herein is normalized to a level or an amount of expression of a reference gene. In some embodiments, the amount of expression of mRNA is normalized to the level or amount of expression of the genes in a subject that does not have prostate cancer.

[0088] Compositions for use in the methods described herein, such as reagent compositions, include, but are not limited to, antibodies, probes, amplification oligonucleotides, and the like.

[0089] The compositions and kits can comprise 1 or more, 2 or more, 3 or more, or 4 or more antibodies, probes, pairs of probes, pairs of amplification oligonucleotide, or sequencing primers.

[0090] The probes or primers can hybridize to 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, or 21 or more target molecules. The target molecules may be RNA, DNA, cDNA, mRNA, a portion or fragment thereof or a combination thereof. In some instances, at least a portion of the target molecules are cancer markers. The probes may hybridize to 1 or more, or 2 or more cancer markers disclosed herein.

[0091] Typically, the probes or primers comprise a target specific sequence. The target specific sequence may be complementary to at least a portion of the target molecule. The target specific sequence may be at least about 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 100% complementary to at least a portion of the target molecule.

[0092] The target specific sequence can be at least about 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more nucleotides in length. In some instances, the target specific sequence is between about 8 to about 20 nucleotides, 10 to about 18 nucleotides, or 12 to about 16 nucleotides in length. GRGR-43680.601

[0093] The compositions and kits can comprise a plurality of probes or primers, wherein the two or more probes of the plurality of probes comprise identical target specific sequences. The compositions and kits may comprise a plurality of probes, wherein the two or more probes of the plurality of probes comprise different target specific sequences.

[0094] The primers or probes can further comprise a unique sequence. The unique sequence is noncomplementary to the cancer marker. The unique sequence may comprise a label, barcode, or unique identifier. The unique sequence may comprise a random sequence, nonrandom sequence, or a combination thereof. The unique sequence may be at least about 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more nucleotides in length. In some instances, the unique sequence is between about 8 to about 20 nucleotides, 10 to about 18 nucleotides, or 12 to about 16 nucleotides in length.

[0095] The probes can further comprise a universal sequence. The universal sequence may comprise a primer binding site. The universal sequence may enable detection of the target sequence. The universal sequence may enable amplification of the target sequence. The universal sequence may enable transcription or reverse transcription of the target sequence. The universal sequence may enable sequencing of the target sequence.

[0096] In some embodiments, target-specific capture probes are provided to selectively capture target molecules of interest to enrich a sample for the molecules of interest. For example, in some embodiments, target-specific capture probes are bound to beads (e.g., magnetic beads) and incubated with a sample to bind to target nucleic acids, if present in a sample. The bound beads are then separated from the remainder of the sample (e.g., by washing), enriching the reaction for the target nucleic acid molecules of interest. The target may then be further analyzed. Capture can occur prior to conducting a molecule detection assay or may be included as a step within a detection assay protocol.

[0097] The probe or primer compositions of the present disclosure can be provided on a solid support. The solid support can comprise one or more beads, plates, solid surfaces, wells, chips, or a combination thereof. The beads can be magnetic, antibody coated, protein A crosslinked, protein G crosslinked, streptavidin coated, oligonucleotide conjugated, silica coated, or a combination thereof. Examples of beads include, but are not limited to, Ampure beads, AMPure GRGR-43680.601

[0098] XP beads, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads (e.g.,, anti-immunoglobulin microbead), protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo-dT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and BcMag™ Carboxy-Terminated Magnetic Beads.

[0099] The compositions and kits can comprise primers and primer pairs capable of amplifying target molecules, or fragments or subsequences or complements thereof. The nucleotide sequences of the target molecules may be provided in computer-readable media for in silica applications and as a basis for the design of appropriate primers for amplification of one or more target molecules.

[0100] Primers based on the nucleotide sequences of target molecules can be designed for use in amplification of the target molecules. For use in amplification reactions such as PCR, a pair of primers can be used. The exact composition of the primer sequences is not critical to the disclosure, but for most applications the primers may hybridize to specific sequences of the target molecules or the universal sequence of the probe under stringent conditions, particularly under conditions of high stringency, as known in the art. The pairs of primers are usually chosen so as to generate an amplification product of at least about 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more nucleotides. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. These primers may be used in standard quantitative or qualitative PCR-based assays to assess transcript expression levels of target molecules. Alternatively, these primers may be used in combination with probes, such as molecular beacons in amplifications using real-time PCR.

[0101] The nucleotide sequence of the entire length of the primer does not need to be derived from the target sequence. Thus, for example, the primer may comprise nucleotide sequences at the 5' and / or 3' termini that are not derived from the target molecule. In some embodiments, primers or probes hybridize to, span, or otherwise detect a fusion junction of a gene fusion (e.g., TMPRSS2:ERG, etc.). Nucleotide sequences which are not derived from the nucleotide sequence of the target molecule may provide additional functionality to the primer. For example, GRGR-43680.601 they may provide a restriction enzyme recognition sequence or a "tag" that facilitates detection, isolation, purification or immobilization onto a solid support. Alternatively, the additional nucleotides may provide a self-complementary sequence that allows the primer to adopt a hairpin configuration. Such configurations may be necessary for certain primers, for example, molecular beacon and Scorpion primers, which can be used in solution hybridization techniques.

[0102] The probes or primers can incorporate moieties useful in detection, isolation, purification, or immobilization, if desired. Such moieties are well-known in the art (see, for example, Ausubel el al., (1997 & updates) Current Protocols in Molecular Biology, Wiley & Sons, New York) and are chosen such that the ability of the probe to hybridize with its target molecule is not affected.

[0103] Examples of suitable moieties are detectable labels, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, and fluorescent microparticles, as well as antigens, antibodies, haptens, avidin / streptavidin, biotin, haptens, enzyme cofactors / substrates, enzymes, and the like.

[0104] A label can optionally be attached to or incorporated into a probe or primer to allow detection and / or quantitation of a target polynucleotide representing the target molecule of interest. The target polynucleotide may be the expressed target molecule RNA itself, a cDNA copy thereof, or an amplification product derived therefrom, and may be the positive or negative strand, so long as it can be specifically detected in the assay being used. Similarly, an antibody may be labeled.

[0105] In certain multiplex formats, labels used for detecting different target molecules may be distinguishable. The label can be attached directly (e.g., via covalent linkage) or indirectly, e.g., via a bridging molecule or series of molecules (e.g., a molecule or complex that can bind to an assay component, or via members of a binding pair that can be incorporated into assay components, e.g., biotin-avidin or streptavidin). Many labels are commercially available in activated forms which can readily be used for such conjugation (for example through amine acylation), or labels may be attached through known or determinable conjugation schemes, many of which are known in the art.

[0106] Labels useful in the disclosure described herein include any substance which can be detected when bound to or incorporated into the target molecule. Any effective detection method can be used, including optical, spectroscopic, electrical, piezoelectrical, magnetic, Raman scattering, surface plasmon resonance, colorimetric, calorimetric, etc. A label is typically GRGR-43680.601 selected from a chromophore, a lumiphore, a fluorophore, one member of a quenching system, a chromogen, a hapten, an antigen, a magnetic particle, a material exhibiting nonlinear optics, a semiconductor nanocrystal, a metal nanoparticle, an enzyme, an antibody or binding portion or equivalent thereof, an aptamer, and one member of a binding pair, and combinations thereof. Quenching schemes may be used, wherein a quencher and a fluorophore as members of a quenching pair may be used on a probe, such that a change in optical parameters occurs upon binding to the target introduce or quench the signal from the fluorophore. One example of such a system is a molecular beacon. Suitable quencher / fluorophore systems are known in the art. The label may be bound through a variety of intermediate linkages. For example, a target polynucleotide may comprise a biotin-binding species, and an optically detectable label may be conjugated to biotin and then bound to the labeled target polynucleotide. Similarly, a polynucleotide sensor may comprise an immunological species such as an antibody or fragment, and a secondary antibody containing an optically detectable label may be added.

[0107] Chromophores useful in the methods described herein include any substance which can absorb energy and emit light. For multiplexed assays, a plurality of different signaling chromophores can be used with detectably different emission spectra. The chromophore can be a lumophore or a fluorophore. Typical fluorophores include fluorescent dyes, semiconductor nanocrystals, lanthanide chelates, polynucleotide-specific dyes and green fluorescent protein.

[0108] Coding schemes may optionally be used, comprising encoded particles and / or encoded tags associated with different polynucleotides of the disclosure. A variety of different coding schemes are known in the art, including fluorophores, including SCNCs, deposited metals, and RF tags.

[0109] Subjects and Samples

[0110] The methods and kits described herein are suitable for detecting a level or an amount of expression of one or more of the genes described herein in a sample from a subject. In some embodiments, a subject from whom a sample is obtained can be selected by the skilled practitioner. In some embodiments, selection of the subject is based upon consideration or analysis of one or more factors. Such factors for consideration include, but are not limited to, family history of a specific disease, genetic predisposition for the disease, increased risk for the disease, physical symptoms which indicate the disease, or environmental reasons. Environmental GRGR-43680.601 reasons can include, but are not limited to, lifestyle or exposure to agents which cause or contribute to the specific disease. In some embodiments, selection of a subject is based on the subject’s previous history with the disease, positive diagnosis prior to therapy or after therapy, treatment for the disease, or remission or recovery from the disease. In some embodiments, the subject is a member of the general population with no enhanced risk factors.

[0111] In some embodiments, the sample or portion of the sample comprising or suspected of comprising cancer tissue or cells can be any source of biological material, including cells, tissue, secretions, or fluid, including bodily fluids. Non-limiting examples of the source of the sample include an aspirate, a needle biopsy, a cytology pellet, a bulk tissue preparation or a section thereof obtained for example by surgery or autopsy, lymph fluid, blood, plasma, serum, tumors, and organs. Alternatively, or additionally, the source of the sample can be semen, urine, bile, excrement, sweat, tears, spinal fluid, and stool.

[0112] While the present disclosure is illustrated with semen samples, the present disclosure should not be construed as being limited to the use of semen. Any suitable sample that allows for detection of expression of the recited markers may be used.

[0113] The present disclosure is not limited to the vasectomy status of the subject. Both subjects that have and have not undergone vasectomies can provide samples for use in the methods described herein.

[0114] In some embodiments, a sample is combined with a buffer, e.g., for processing. In some embodiments, an amount of expression of one or more genes described herein is determined from a composition, e.g., a solution or suspension, comprising the sample and a buffer. In some embodiments, the composition further comprises a preservative for adequate stability of the sample. In some embodiments, the buffer to sample ratio is 2:5. In some embodiments, the buffer to sample ratio is 1:6, 2:5, 3:5, 4:5, 1 :1, or 2:1.

[0115] The samples may be archival samples, having a known and documented medical outcome, or may be samples from current patients whose ultimate medical outcome is not yet known.

[0116] Determining Likelihood of Having or Developing Prostate Cancer

[0117] In some embodiments, the level or amount of expression of one or more genes described herein determines the likelihood of detecting prostate cancer in a subject. For example, as GRGR-43680.601 described in Example 1, the level of expression of the genes described herein is increased or decreased relative to the level of expression in a subject that does not have prostate cancer.

[0118] In some embodiments, a computer-based analysis program is used to translate the raw data generated by a detection assay (e.g., the presence, absence, or amount of a given marker or markers) into data of predictive value for a clinician, subject or subject’s healthcare provider. The clinician, subject or subject’s healthcare provider can access the raw data using any suitable means. Thus, in some embodiments, the present disclosure provides the further benefit that the clinician, subject or subject’s healthcare provider, who might not be trained in genetics or molecular biology, need not understand the raw data. The data can be presented directly to the clinician subject or subject’s healthcare provider in its most useful form. This enables the clinician or healthcare provider to immediately utilize the information in order to optimize the care of the subject.

[0119] The information can be received, processed or transmitted to or from one or more laboratories conducting the assays, information providers, medical personnel, or subjects using any suitable method. For example, in some embodiments of the present disclosure, a sample (e.g., a semen, biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject can visit a medical center to have the sample obtained and sent to the profiling center, or the subject itself can collect the sample (e.g., a semen or urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information can be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample can be processed and a profile can be produced (i.e., expression data), useful for the diagnostic or prognostic information desired for the subject.

[0120] The profile data is then prepared in a format suitable for interpretation by one or more medical personnel (e.g., a treating clinician, physician assistant, nurse, or pharmacist). For example, rather than providing raw expression data, the prepared format may represent a GRGR-43680.601 diagnosis or risk assessment (e.g., levels of the cancer markers described herein) for the subject, along with recommendations for particular treatment options. The data may be displayed to the medical personnel by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the medical personnel e.g., at the point of care) or displayed to the medical personnel on a computer monitor.

[0121] In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to convert the raw data to information useful for medical personnel or subject. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the medical personnel, the subject, or researchers.

[0122] In some embodiments, the subject or the subject’s healthcare provider is able to directly access the data using the electronic communication system. The subject may choose further intervention or counseling based on the results.

[0123] In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.

[0124] Kits and Devices

[0125] In some embodiments, the disclosure provides kits for analyzing a sample comprising reagents and or other components necessary, sufficient, or useful to conduct any of the methods described herein. For example, in some embodiments, kits comprise one or more of a) software comprising instructions that cause a computing device having a processor to carryout out one or more of: i) collecting expression data (e.g., from a detection instrument); ii) receiving patientspecific information (e.g., from a patient, a caregiver, an electronic medical record, etc.); iii) calculating a risk score or profile based on the expression data results and / or one or more additional patient-specific variables; iv) displaying expression information, additional information, and / or a risk score or profile; v) listing or recommending a potential treatment GRGR-43680.601 course of action; and vi) transmitting or communicating expression information, additional information, a risk score or profile, and / or a treatment listing or recommendation to a second computing device (e.g., at a remove location); b) instruction for using the device, system, or kit; c) control reagents (e.g., positive controls, negative controls, standards); d) detection assay components (e.g., a reverse transcriptase, a polymerase, primers, probes, dNTPs, buffers, adapters, ligases, detectable labels (e.g., fluorophores), antibodies, etc.); e) reagent containers, dispensers, multi-well plates, flow cells, etc.; or f) packaging.

[0126] Devices useful for performing methods of the disclosure are also provided. The devices can comprise components for characterizing the expression level of a target molecule of the disclosure, for example components for performing one or more methods of nucleic acid extraction, amplification, sequencing, and / or detection. Such components may include one or more of an amplification chamber (for example, a thermal cycler), a plate reader, a spectrophotometer, capillary electrophoresis apparatus, a chip reader, a sequencer, a flow cell, and or robotic sample handling components. These components ultimately can obtain data that reflects the expression level of the target molecules used in the assay being employed.

[0127] The devices can include an excitation and / or a detection means. Any instrument that provides a wavelength that can excite a species of interest and is shorter than the emission wavelength(s) to be detected can be used for excitation. Commercially available devices can provide suitable excitation wavelengths as well as suitable detection component.

[0128] Illustrative excitation sources include a broadband UV light source such as a deuterium lamp with an appropriate filter, the output of a white light source such as a xenon lamp or a deuterium lamp after passing through a monochromator to extract out the desired wavelength(s), a continuous wave (cw) gas laser, a solid-state diode laser, or any of the pulsed lasers. Emitted light can be detected through any suitable device or technique; many suitable approaches are known in the art. For example, a fluorimeter or spectrophotometer may be used to detect whether the test sample emits light of a wavelength characteristic of a label used in an assay.

[0129] The devices can comprise a components for identifying a given sample, and of linking the results obtained to that sample. Such means can include manual labels, barcodes, and other indicators which can be linked to a sample vessel, and / or may optionally be included in the sample itself, for example where an encoded particle is added to the sample. The results may be linked to the sample, for example in a computer memory that contains a sample designation and GRGR-43680.601 a record of expression levels obtained from the sample. Linkage of the results to the sample can also include a linkage to a particular sample receptacle in the device, which is also linked to the sample identity.

[0130] The devices can also comprise components for correlating the expression levels of the target molecules being studied with a prognosis of disease outcome. Such components may comprise one or more of a variety of correlative techniques, including lookup tables, algorithms, multivariate models, and linear or nonlinear combinations of expression models or algorithms. The expression levels may be converted to one or more likelihood scores, reflecting a likelihood that the subject providing the sample may exhibit a particular disease outcome. The models and / or algorithms can be provided in machine readable format and can optionally further designate a treatment modality for a subject or class of subjects.

[0131] The devices can also comprise output means for outputting the disease status, prognosis and / or a treatment modality. Such output means can take any form which transmits the results to a subject and / or a healthcare provider, researcher, patient, or other user, and may include a display, a printed format, or both. The device may use a computer system comprising a processor running software for performing one or more of the steps provided.

[0132] II. Screening, diagnosis or treatment

[0133] The methods, compositions, and kits disclosed herein are useful for the screening, diagnosis, predication, monitoring and / or treatment of cancer (e.g., prostate cancer) in a subject as well as for cancer research and for evaluating and monitoring the efficacy of interventions. In some embodiments, the methods described herein identify a subject as having prostate cancer or as suspected of having prostate cancer (e.g., in a screening assay). In such embodiments, the subject sometimes receives further diagnostic intervention (e.g., prostate biopsy).

[0134] In some embodiments, at least one additional variable is analyzed in combination with the gene expression analysis. In some embodiments, the one or more additional variables is the subject’s age, race, family history of prostate cancer, digital rectal examination (DRE) result, prostate biopsy result, prostate specific antigen (PSA) expression value (e.g., based on a serum sample), multi-perimetric MRI (mpMRI) result, prostate volume (as measured by either DRE or MRI), PSA density (PSAD), or any combination thereof. In some embodiments, PSAD is calculated as the PSA score (ng / ml) divided by the prostate volume (ml). GRGR-43680.601

[0135] In some embodiments, the methods comprise determining, recommending or administering a therapeutic regimen. In some embodiments, the therapeutic regimen is an anticancer therapy. In some embodiments, the methods comprise modifying a therapeutic regimen. Modifying a therapeutic regimen can comprise increasing a therapeutic dosage, decreasing a therapeutic dosage, or terminating a therapeutic regimen.

[0136] Some subjects are administered prostate cancer therapy or other intervention (e.g., one or more of surgery, radiation therapy, hormonal therapy, targeted therapy, chemotherapy, immunotherapy, radiopharmaceuticals, or bone-modifying drugs).

[0137] In some embodiments, the prostate cancer therapy comprises administering a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes, topoisomerase inhibitors, and cytotoxic antibiotics. Cisplatin, carboplatin, and oxaliplatin are examples of alkylating agents. Other alkylating agents include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide. Alkylating agents may impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Alternatively, alkylating agents may chemically modify a cell's DNA.

[0138] Biological therapy (sometimes called immunotherapy, biotherapy, or biological response modifier (BRM) therapy) uses the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. Biological therapies include interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents.

[0139] In some embodiments, the biological therapy is immune checkpoint therapy. Immune checkpoint inhibitors target CTLA-4, PD-1, or PD-L1. Examples include but are not limited to, ipilimumab, nivolumab, cemiplimab, avelumab, durvalumab, tremelimumab, dostarlimab, pembrolizumab, spartalizumab, and atezolizumab.

[0140] In some embodiments, the prostate cancer therapy is FDA-approved for treating prostate cancer. In some embodiments, the prostate cancer therapy is: abiraterone acetate, apulutamide, bicalutamide, cabazitaxel, casodex, darolutamide, degarelix, docetaxel, eligard, enzalutamide, erleada, firmagon, flutamide, goserelin acetate, jevtana, leuprolide acetate, Lupron depot, lutetium lu 177 vipivotide tetraxetan, Lynparza, mitoxantrone hydrochloride, nilandron, GRGR-43680.601 nilutamide, nubeqa, Olaparib, orgovyx, pluvicto, provenge, radium 223 dichloride, relugolix, rubraca, rucaparib camsylate, sipuleucel-t, taxotere, xofigo, xtandi, yonsa, zoladex, xytiga, or any combination thereof.

[0141] In some embodiments, any of the methods described herein are employed before and / or after a treatment event. For example, in some embodiments, a first expression analysis is conducted prior to an intervention and a second expression analysis is conducted following the treatment event (i.e., test-treat-test). Any order of combination of testing and treating steps are contemplated (e.g., test / treat; treat / test; test / treat / test; test / treat / test / treat; treat / test / treat; test / treat / test / treat / test; treat / test / test / treat; etc.). Such methods find use to evaluate the efficacy of an intervention, to identify when a change in intervention is warranted, to move from watch- and-wait to treatment or vice versa, and the like.

[0142] For example, in some embodiments, subjects are given an option to avoid a biopsy or treatment and opt for watchful waiting or minimal treatments. For example, in some embodiments the subject has had a prior positive biopsy and diagnosis of prostate cancer. In some embodiments, prostate cancer has been characterized as low-risk (DOI: 10.6004 / jnccn.2024.0019; www.cancer.org / cancer / types / prostate-cancer / detection-diagnosis- staging / risk-groups.html). In some embodiments, low-risk prostate cancer cases are frequently managed by active surveillance involving the recommendation for serial biopsies on an annual or biannual basis. In some embodiments a sample is obtained prior to serial biopsies and the level of expression of cancer markers described herein is determined at the time of initial diagnosis and at regular time points after. In such embodiments, a change in the level of expression of one or more markers may result in either continued active surveillance or the recommendation of active treatment.

[0143] In some embodiments the diagnosed prostate cancer is characterized as high-risk, requiring treatment. In these cases a sample is before and at a time period following the treatment to evaluate effectiveness of the treatment. GRGR-43680.601

[0144] EXPERIMENTAL

[0145] The following Examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.

[0146] Example 1

[0147] Methods

[0148] Study Population

[0149] The study population consisted of men >50 years of age scheduled for prostate biopsy and meeting all other eligibility criteria. Subjects were enrolled across 22 sites. Study protocols were approved by institutional review boards and all subjects provided written, informed consent to participate in the study.

[0150] Sample Collection and Eligibility

[0151] Prior to sample collection, subjects abstained from ejaculation for at least 48 hours. Subjects produced semen samples by masturbation into a specimen cup at the clinic or at the subject’s home, noting the time of collection. To allow liquefaction, samples remained at ambient temperature for a minimum of 1 hour and a maximum of 3 hours at which point clinic site staff transferred samples to 15 mL conical tubes and froze samples at -20°C or -80°C. After freezing, clinics shipped samples on dry ice to a testing laboratory for storage at -80°C. The testing laboratory thawed samples on ice, homogenized samples via pipetting up and down, created 350 pL single-use aliquots, and froze the aliquots for further processing and RNA extraction. If remnant material did not create a full aliquot, the remnant volume was recorded and the material was frozen. To be eligible for inclusion in the study, a minimum sample volume of 250 pL of semen was required. Additionally, clinical and demographic information from each subject was recorded. Importantly this included results of the biopsy that all subjects underwent. Diagnosis of cancer, including the Gleason score, was performed at each site. All clinical data was retained in an electronic database, and was subsequently reviewed for accuracy and consistency of data capture prior to the point where samples were selected for analysis. GRGR-43680.601

[0152] Sample Processing and RNA Extraction

[0153] Prior to processing samples for RNA extraction, randomized study IDs were assigned, blinding laboratory personnel to cancer status and subject information. At the time of RNA extraction, single-use semen aliquots were thawed on ice and spun at 2,000xg for 10 minutes to pellet sperm cells and debris. 250 pL of seminal plasma supernatant was isolated for RNA extraction, and samples with less than 250 pL seminal plasma were brought to volume using PBS (Sigma-Aldrich, Saint Louis, MO). RNA was extracted using the Qiagen AllPrep DNA / RNA / miRNA Universal Kit (QIAGEN, Aarhus, Denmark) with the modifications to the manufacturer’s protocol for purification from cells described in brief below. For lysis, 250 pL of Buffer RLT was added to seminal plasma and vortexed for 10 seconds and subjected to DNA binding. Following DNA binding, 36 pL of Proteinase K and 143 pL of 100% ethanol were added to the flow through and incubated 10 minutes at ambient temperature. A volume of 286 pL of 100% ethanol was added to samples prior to completing RNA purification per the manufacturer’s instructions. RNA was quantified using the Qubit™ RNA High Sensitivity (HS) assay kit (Invitrogen, Waltham, MA) and read on a Promega Quantus Fluorometer (Promega, Madison, WI). RNA was frozen at -80°C prior to preparation for RNA-sequencing (RNA-seq).

[0154] Sequencing Library Prep, RNA-seq, and Data Processing

[0155] All samples providing a minimum of 100 ng of RNA as determined via the Qubit assay were eligible for RNA sequencing (RNA-seq). Samples were submitted to the University of Wisconsin Biotechnology Center Gene Expression Center for sequencing library prep and RNA- seq using qualified kits and procedures. Prior to RNA-seq library prep, samples were assayed on Agilent Bioanalyzer with a Nano or Picochip (Agilent, Santa Clara, CA). The volume of RNA to yield 100 ng, as determined by the Bioanalyzer, was utilized as template for RNA-seq library prep. Sequencing libraries were prepared using the RNA Prep with Enrichment (L) Tagmentation kit, DNA / RNA UD Indexes Set D, Illumina RNA Fast Hyb Enrichment Beads, and Enrichment Exome Panel per the manufacturer’s instructions (Illumina, San Diego, CA) in singleplex. In process and final library concentration checks were determined using the Quant- iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen, Waltham, MA). RNA seq was performed by the University of Wisconsin DNA Sequencing Core using either a NovSeq 6000 or NovaSeq X Plus (Illumina, San Diego, CA) targeting 50 million paired-end reads per sample. Raw fastq files GRGR-43680.601 were processed using Skewer (Jiang et al., BMC Bioinformatics, 15: 182, 2014) to trim adapter sequences, and STAR (Spliced Transcripts Alignment to a Reference) (Dobin et al., Bioinformatics, 29(1): 15-21, 2013) to align and map reads to the reference genome. Copies of all raw data fdes (fastq) in additional to aligned read files (BAM) were obtained. Alignment QC data was used to identify technical outlier samples. Plots of precent duplicate reads versus reads per kilobase (RPK) were reviewed, and samples that showed atypical trends of increased duplication rate or decreased RPK were flagged as outliers and removed from further data analysis. RSEM (RNASeq by Expectation Maximization, (Li and Dewey, BMC Bioinformatics, 12:323, 2011) was used to create a normalized gene expression matrix reporting transcripts per million mapped reads (TPM), and batch effects were identified and corrected using ComBat (Zhang et al., NAR Genom. Bioinform., 2(3), 1-10, 2020). Genes with very low expression levels, defined as <2 cpm (gene expression) in <20 samples, were removed to reduce the effects of noise and improve reproducibility of predictive models.

[0156] Machine Learning Feature Selection and Model Generation

[0157] A finalized sample set was based on evaluable samples that met quality metrics for minimum RNA yield, RNA quality, sequencing library QC, and sequencing QC. Samples were analyzed in a total of 4 RNA-seq batches, with each batch containing a combination of cancer and no-cancer samples. Samples in 3 of the batches were assigned to a training set, and the 4thbatch was reserved as an independent validation set. The clinical and demographic variables in the training and validation sets were compared to ensure that both were representative of the intended-use population (e.g., men undergoing screening and / or diagnosis for prostate cancer) and the two sample sets were not significantly different from each other. All feature selection and model-fitting was done within the training set. The model was then locked for subsequent analysis of the validation set samples.

[0158] Feature Selection

[0159] To minimize model over-fitting and maximize classifier generalizability, a preliminary feature selection step was implemented to reduce the dimensionality of the RNA-seq data set and focus model development on the most informative features. Top-ranked features were selected using a t-test comparing cancer versus benign samples within the training cohort. GRGR-43680.601

[0160] Model Training

[0161] Classification models were developed within the training set samples. Several types of models (for example, logistic regression, random forest, elastic net, and XGBoost) were evaluated and optimized. Models were evaluated by overall Receiver Operator Curve (ROC) area under the curve (AUC). Training was based on performing 100-fold training exercises using an 80:20 split of the training set samples in each case. For each of the 100-fold iterations 80% of the samples were used for training and the remaining 20% were used as a hold-out test set. Samples were randomly selected for each of k- fold runs, insuring that all samples were equally represented in training and testing. The overall AUC was reported as a mean of the 100-fold routines. For model selection, the above models were trained on the training set and their performance compared using 100-fold cross-validation. It was found that XGBoost had the highest median AUC among all models, and therefore, it was chosen as the final model architecture. Finally, an XGBoost model was trained on the entire training dataset (100% of the training set) and the model was locked.

[0162] Model Testing

[0163] The locked XGBoost model was tested on the independent validation set which comprised of samples that had been sequenced in a separate RNA-seq run (independent batch) with clinical specimens that were well-matched to samples in the training set. Overall performance of the final classifier was reported based on model prediction performance (AUC) on the independent validation set with samples had not been used to develop the classifier.

[0164] Results

[0165] Final Sample Sets

[0166] The final sample set was made up of 279 total samples, including 128 subjects with nocancer, and 151 subjects diagnosed with cancer. Demographic and clinical variables for samples in the training and validation sets are shown in Table 1. All variables were well-matched between the two sample sets, with no significant differences observed for any of the variables. The training set consisted of 199 samples, whereas the validation was comprised of 80 samples. GRGR-43680.601

[0167] Gene sets & Classifier Development

[0168] In total 217 genes (Table 2) were found to be significantly differentially expressed between cancer and no-cancer samples with an FDR corrected q-value of <0.05. The top 50 genes (Table 3) were selected for classifier development.

[0169] An XGBoost model was found to have the best performance in the training set, and was subsequently locked for evaluation of the independent validation set. The classifier was based on the 50 input genes, however, the weighting of the genes by the XGBoost classifier led to a distinct ranking of gene importance, compared to the q-value ranking shown in Table 3. The classifier feature importance ranking is shown in Table 4.

[0170] Performance of the final classifier in the validation set

[0171] The overall AUC was found to be 0.83 (Figure 1), and with a score threshold of 0.10, to differentiate cancer and benign samples, the sensitivity was found to be 90% with a specificity of 56%>. The classifier score distribution demonstrated a clear separation of cancer and no-cancer samples (Figure 2). GRGR-43680.601 aCalculated values exclude samples for which prostate volume data was unavailable.b% of cohort (i.e. training cohort or validation cohort) GRGR-43680.601

[0172] Table 2 - Differentially expressed gene comparing no-cancer and cancer GRGR-43680.601 GRGR-43680.601 GRGR-43680.601 GRGR-43680.601 GRGR-43680.601 GRGR-43680.601 GRGR-43680.601

[0173] Table 3 - Top-ranked differentially expressed genes between cancer and no-cancer samples, with FDR q-value<0.05 GRGR-43680.601 GRGR-43680.601

[0174] GRGR-43680.601

[0175] Table 4 - Gene weights of the final classifier GRGR-43680.601

[0176] GRGR-43680.601

[0177] All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

GRGR-43680.601CLAIMSWe claim:

1. A method of assaying gene expression, comprising: assaying the level of expression of two or more genes selected from the group consisting of Solute Carrier Family 16 Member 5 (SLC16A5), Integrin Subunit Alpha 3 (ITGA3), ETS Transcription Factor ERG (ERG), Mitogen-Activated Protein Kinase 13 (MAPK13), Pleckstrin Homology Domain Containing A2 (PLEKHA2), Latent Transforming Growth Factor Beta Binding Protein 3 (LTBP3), Fatty Acid Binding Protein 5 (FABP5), Calmodulin Like 4 (CALML4), TBC1 Domain Family Member 2 (TBC1D2), Protein Phosphatase, Mg2+ / Mn2+ Dependent IM (PPM1M), NGFLA Binding Protein 2 (NAB2), ENSG00000259642, Retinoic Acid Receptor Gamma (RARG), Prostate Cancer Associated 3 (PCA3), Olfactory Receptor Family 51 Subfamily E Member 2 (OR51E2), Complement Clr (C1R), Coiled-Coil Domain Containing 69 (CCDC69), Transglutaminase 3 (TGM3), Integrin Subunit Beta 2 (ITGB2), Signal Transducing Adaptor Family Member 1 (STAP1), Interleukin 17 Receptor E (IL17RE) ATPase Family AAA Domain Containing 3C (ATAD3C), Inhibitor Of DNA Binding 4 (ID4), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3F (APOBEC3F), EGF Containing Fibulin Extracellular Matrix Protein 2 (EFEMP2), Moesin (MSN), Growth Differentiation Factor 15 (GDF15), NIPA Like Domain Containing 3 (NIPAL3), Interleukin 4 Receptor (IL4R), Homeobox C6 ( H0XC6), Apoptosis Inducing Factor Mitochondria Associated 2 (AIFM2), Trefoil Factor 3 (TFF3), ENSG00000234964, Zinc Finger Protein 431 (ZNF431), Docking Protein 4 (D0K4), Golgi Membrane Protein 1 (G0LM1), C3 And PZP Like Alpha-2 - Macroglobulin Domain Containing 8 (CPAMD8), UDP-N-Acetylglucosamine Pyrophosphorylase 1 Like 1 (UAPILI), Annexin A2 (ANXA2), Cellular Communication Network Factor 3 (CCN3), Matrix Metallopeptidase 14 (MMP14), MYC Proto-Oncogene, BHLH Transcription Factor (MYC), Adaptor Related Protein Complex 5 Subunit Beta 1 (AP5B1), Heat Shock Protein Family A (Hsp70) Member 6 (HSPA6), Regulator Of G Protein Signaling 2 (RGS2), EBF Family Member 4 (EBF4), Heme Oxygenase 1 (HM0X1), Heme Oxygenase 1 (LUZP2), Periaxin (PRX), and Von Willebrand Factor A Domain Containing 5A (VWA5A) in a sample from a subject.GRGR-43680.6012. A method of diagnosing prostate cancer in a subject, comprising: assaying the level of expression of two or more genes selected from the group consisting of SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, H0XC6, AIFM2, TFF3, ENSG00000234964, ZNF431, D0K4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A in a sample from a subject; and b) performing a confirmatory diagnostic assay.

3. The method of claim 2, wherein said confirmatory diagnostic assay is selected from the group consisting of an imaging technique, a biopsy, and a different biomarker assay.

4. The method of claim 3, wherein said imaging technique is selected from the group consisting of ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

5. A method of treating prostate cancer, comprising: a) assaying the level of expression of two or more genes selected from the group consisting of SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, H0XC6, AIFM2, TFF3, ENSG00000234964, ZNF431, D0K4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B 1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A in a sample from a subject; and b) administering a prostate cancer treatment to a subject identified as having prostate cancer based on level of expression of said genes associated.

6. The method of claim 5, further comprising the step of performing a confirmatory diagnostic assay prior to said administering a prostate cancer treatment.GRGR-43680.6017. The method of claim 5 or 6, wherein said prostate cancer treatment is selected from the group consisting of surgery, radiation therapy, cryoablation or cryotherapy, heat ablation, high-intensity focused ultrasound (HIFU), hormone therapy, chemotherapy, and immunotherapy8. A method of screening for or diagnosing prostate cancer in a subject, comprising: assaying the level of expression of two or more genes selected from the group consisting of SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, H0XC6, AIFM2, TFF3, ENSG00000234964, ZNF431, D0K4, G0LM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A in a sample from a subject; and b) identifying said subject as having prostate cancer when said subject is identified as having altered levels of expression of said genes relative to a subject without prostate cancer.

9. The method of any one of the preceding claims, wherein said sample is semen.

10. The method of any of the preceding claims, wherein said two or more genes comprise SLC16A5 and ITGA3; SLC16A5 and ERG; SLC16A5 and MAPK13; SLC16A5 and PLEKHA2; SLC16A5 and LTBP3; SLC16A5 and FABP5; SLC16A5 and CALML4, SLC16A5 and TBC1D2; SLC16A5 and PPM1M; ITGA3 and ERG; ITGA3 and MAPK13; ITGA3 and PLEKHA2; ITGA3 and LTBP3; ITGA3 and FABP5; ITGA3 and CALML4; ITGA3 and TBC1D2; ITGA3 and PPM1M; ERG and MAPK13; ERG and PLEKHA2; ERG and LTBP3; ERG and FABP5; ERG and CALML4; ERG and TBC1D2; ERG and PPM1M; MAPK13 and PLEKHA2; MAPK13 and LTBP3; MAPK13 and FABP5; MAPK13 and CALML4; MAPK13 and TBC1D2; MAPK13 and PPM1M; PLEKHA2 and LTBP3; PLEKHA2 and FABP5; PLEKHA2 and CALML4; PLEKHA2 and TBC1D2; PLEKHA2 and PPM1M; LTBP3 and FABP5; LTBP3 and FABP5, LTBP3 and CALML4; LTBP3 and TBC1D2; LTBP3 and PPM1M;GRGR-43680.601FABP5 and CALML4; FABP5 and CALML4; FABP5 and TBC1D2; FABP5 and PPM1M; CALML4 and TBC1D2; CALML4 and PPM1M; or TBC1D2 and PPM1M.

11. The method of any one of the preceding claims, wherein said two or more genes is three or more genes.

12. The method of claim 11, wherein said three or more genes comprise SLC16A5, ITGA3, and ERG; SLC16A5, ITGA3, and MAPK13; SLC16A5, ITGA3, and PLEKHA2; SLC16A5, ITGA3, and LTBP3; SLC16A5, ITGA3, and FABP5; SLC16A5, ITGA3, and CALML4; SLC16A5, ITGA3, and TBC1D2; SLC16A5, ITGA3, and PPMIM; SLC16A5, ERG, and MAPK13; SLC16A5, ERG, and PLEKHA2; SLC16A5, ERG, and LTBP3; SLC16A5, ERG, and FABP5; SLC16A5, ERG, and CALML4; SLC16A5, ERG, and TBC1D2; SLC16A5, ERG, and PPM1M; SLC16A5, MAPK13, and PLEKHA2; SLC16A5, MAPK13, and LTBP3, SLC16A5, MAPK13, and FABP5, SLC16A5, MAPK13, and CALML4, SLC16A5, MAPK13, and TBC1D2; SLC16A5, MAPK13, and PPM1M; SLC16A5, PLEKHA2, and LTBP3; SLC16A5, PLEKHA2, and FABP5; SLC16A5, PLEKHA2, and CALML4; SLC16A5, PLEKHA2, and TBC1D2; SLC16A5, PLEKHA2, and PPM1M; SLC16A5, LTBP3, and FABP5; SLC16A5, LTBP3, andCALML4; SLC16A5, LTBP3, and TBC1D2; SLC16A5, LTBP3, and PPM1M; SLC16A5, FABP5, and CALML4; SLC16A5, FABP5, and TBC1D2; SLC16A5, FABP5, and PPMIM; SLC16A5, CALML4, and TBC1D2; SLC16A5, CALML4, and PPMIM; SLC16A5, TBC1D2, and PPM1M; ITGA3, ERG, and MAPK13; ITGA3, ERG, and PLEKHA2; ITGA3, ERG, and LTBP3; ITGA3, ERG, and FABP5; ITGA3, ERG, and CALML4; ITGA3, ERG, and TBC1D2; ITGA3, ERG, and PPM1M; ITGA3, MAPK13, and PLEKHA2; ITGA3, MAPK13, and LTBP3; ITGA3, MAPK13, and FABP5, ITGA3, MAPK13, and CALML4; ITGA3, MAPK13, and TBC1D2; ITGA3, MAPK13, and PPM IM; ITGA3, PLEKHA2, and LTBP3; ITGA3, PLEKHA2, and FABP5; ITGA3, PLEKHA2, and CALML4; ITGA3, PLEKHA2, and TBC1D2; ITGA3, PLEKHA2, and PPM1M; ITGA3, LTBP3, and FABP5; ITGA3, LTBP3, and CALML4; ITGA3, LTBP3, and TBC1D2; ITGA3, LTBP3, and PPM1M; ITGA3, FABP5, and CALML4; ITGA3, FABP5, and TBC1D2; ITGA3, FABP5, and PPM1M; ITGA3, CALML4, and TBC1D2; ITGA3, CALML4, and PPM1M; ITGA3, TBC1D2, and PPM IM; ERG, MAPK13, and PLEKHA2; ERG, MAPK13, and LTBP3; ERG, MAPK13, andGRGR-43680.601FABP5; ERG, MAPK13, and CALML4; ERG, MAPK13, and TBC1D2; ERG, MAPK13, and PPM1M; ERG, PLEKHA2, and LTBP3; ERG, PLEKHA2, and FABP5; ERG, PLEKHA2, and CALML4; ERG, PLEKHA2, and TBC1D2; ERG, PLEKHA2, and PPM1M; ERG, LTBP3, and FABP5; ERG, LTBP3, and CALML4; ERG, LTBP3, and TBC1D2; ERG, LTBP3, and PPM1M; ERG, FABP5, and CALML4; ERG, FABP5, and TBC1D2; ERG, FABP5, and PPM1M; ERG, CALML4, and TBC1D2; ERG, CALML4, and PPM1M; ERG, TBC1D2, and PPM1M;MAPK13, PLEKHA2, LTBP3; MAPK13, PLEKHA2, and CALML4; MAPK13, PLEKHA2, and TBC1D2; MAPK13, PLEKHA2, and PPM1M; MAPK13, LTBP3, and FABP5; MAPK13, LTBP3, and CALML4; MAPK13, LTBP3, and TBC1D2; MAPK13, LTBP3, and PPM1M; MAPK13, FABP5, and CALML4; MAPK13, FABP5, and TBC1D2; MAPK13, FABP5, and PPM1M; MAPK13, CALML4, and TBC1D2; MAPK13, CALML4, and PPM1M; MAPK13, TBC1D2, and PPM1M; PLEKHA2, LTBP3, and FABP5; PLEKHA2, LTBP3, and CALML4; PLEKHA2, LTBP3, and TBC1D2; PLEKHA2, LTBP3, and PPM1M; PLEKHA2, FABP5, and CALML4; PLEKHA2, FABP5, and TBC1D2; PLEKHA2, FABP5, and PPM1M; PLEKHA2, CALML4, and TBC1D2; PLEKHA2, CALML4, and PPM1M; PLEKHA2, TBC1D2, and PPM1M; LTBP3, FABP5, and CALML4; LTBP3, FABP5, and TBC1D2; LTBP3, FABP5, and PPM1M; LTBP3, CALML4, and TBC1D2; LTBP3, CALML4, and PPM1M; LTBP3, TBC1D2, and PPM1M; FABP5, CALML4, and TBC1D2; FABP5, CALML4, and PPM1M; or CALML4, TBC1D2, and PPM1M.

13. The method of any one of the preceding claims, wherein said two or more genes is four or more genes.

14. The method of claim 13, wherein said four or more genes comprise SLC16A5, ITGA3, ERG, and MAPK13, SLC16A5, ITGA3, ERG, and PLEKHA2; SLC16A5, ITGA3, ERG, and LTBP3; SLC16A5, ITGA3, ERG, and FABP5; SLC16A5, ITGA3, ERG, and CALML4; SLC16A5, ITGA3, ERG, and TBC1D2; SLC16A5, ITGA3, ERG, and PPM1M; SLC16A5, ERG, MAPK13, and PLEKHA2; SLC16A5, ERG, MAPK13, and LTBP3; SLC16A5, ERG, MAPK13, and FABP5; SLC16A5, ERG, MAPK13, and CALML4; SLC16A5, ERG, MAPK13, and TBC1D2; SLC16A5, ERG, MAPK13, and PPMIM; SLC16A5, MAPK13, PLEKHA2, and LTBP3; SLC16A5, MAPK13, PLEKHA2, and FABP5; SLC16A5, MAPK13,GRGR-43680.601PLEKHA2, and CALCL4; SLC16A5, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, and PPM1M; SLC16A5, PLEKHA2, LTBP3, and FABP5; SLC16A5, PLEKHA2, LTBP3, and CALML4; SLC16A5, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, and PPM1M; SLC16A5, LTBP3, FABP5, and CALML4; SLC16A5, LTBP3, FABP5, and TBC1D2; SLC16A5, LTBP3, FABP5, and PPM1M; SLC16A5, FABP5, CALML4, and TBC1D2; SLC16A5, FABP5, CALML4, and PPM1M; SLC16A5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM1M; ITGA3, ERG, MAPK13, and PLEKHA2; ITGA3, ERG, MAPK13, and LTBP3; ITGA3, ERG, MAPK13, and FABP5; ITGA3, ERG, MAPK13, and CALML4; ITGA3, ERG, MAPK13, and TBC1D2; ITGA3, ERG, MAPK13, and PPM IM; ITGA3, MAPK13, PLEKHA2, and LTBP3; ITGA3, MAPK13, PLEKHA2, and FABP5; ITGA3, MAPK13, PLEKHA2, and CALML4; ITGA3, MAPK13, PLEKHA2, and TBC1D2; ITGA3, MAPK13, PLEKHA2, and PPM1M; ITGA3, PLEKHA2, LTBP3, and FABP5; ITGA3, PLEKHA2, LTBP3, and CALML4; ITGA3, PLEKHA2, LTBP3, and TBC1D2; ITGA3, PLEKHA2, LTBP3, and PPM1M; ITGA3, LTBP3, FABP5, and CALML4; ITGA3, LTBP3, FABP5, and TBC1D2; ITGA3, LTBP3, FABP5, and PPM1M; ITGA3, FABP5, CALML4, and TBC1D2; ITGA3, FABP5, CALML4, and PPM1M; ITGA3, CALML4, TBC1D2, and PPM IM; ERG, MAPK13, PLEKHA2, and LTBP3; ERG, MAPK13, PLEKHA2, and CALML4; ERG, MAPK13, PLEKHA2, and TBC1D2; ERG, MAPK13, PLEKHA2, and SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M; ERG, PLEKHA2, LTBP3, and FABP5; ERG, PLEKHA2, LTBP3, and CALML4; ERG, PLEKHA2, LTBP3, and TBC1D2; ERG, PLEKHA2, LTBP3, and PPM1M; ERG, LTBP3, FABP5, and CALML4; ERG, LTBP3, FABP5, and TBC1D2; ERG, LTBP3, FABP5, and PPM1M; ERG, FABP5, CALML4, and TBC1D2; ERG, FABP5, CALML4, and PPM1M; ERG, CALML4, TBC1D2, and PPM1M; MAPK13, PLEKHA2, LTBP3, and FABP5; MAPK13, PLEKHA2, LTBP3, and CALML4; MAPK13, PLEKHA2, LTBP3, and TBC1D2; MAPK13, PLEKHA2, LTBP3, and PPM1M; MAPK13, LTBP3, FABP5, and CALML4;MAPK13, LTBP3, FABP5, and TBC1D2; MAPK13, LTBP3, FABP5, and PPM1M; MAPK13, FABP5, CALML4, and TBC1D2; MAPK13, FABP5, CALML4, and PPM1M; MAPK13, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, and CALML4; PLEKHA2,GRGR-43680.601LTBP3, FABP5, and TBC1D2; PLEKHA2, LTBP3, FABP5, and PPM1M; PLEKHA2, FABP5, CALML4, and TBC1D2; PLEKHA2, FABP5, CALML4, and PPMIM; PLEKHA2, CALML4, TBC1D2, and PPM1M; or FABP5, CALML4, TBC1D2, PPM1M.

15. The method of any one of the preceding claims, wherein said two or more genes is five or more genes.

16. The method of claim 15, wherein said five or more genes comprise SLC16A5, ITGA3, ERG, MAPK13, and PLEKHA2; SLC16A5, ITGA3, ERG, MAPK13, and LTBP3; SLC16A5, ITGA3, ERG, MAPK13, and FABP5; SLC16A5, ITGA3, ERG, MAPK13, and CALML4; SLC16A5, ITGA3, ERG, MAPK13, and TBC1D2; SLC16A5, ITGA3, ERG, MAPK13, and PPM1M; SLC16A5, ERG, MAPK13, PLEKHA2, and LTBP3; SLC16A5, ERG, MAPK13, PLEKHA2, and FABP5; SLC16A5, ERG, MAPK13, PLEKHA2, and CALML4; SLC16A5, ERG, MAPK13, PLEKHA2, and TBC1D2; SLC16A5, ERG, MAPK13, PLEKHA2, and PPM1M; SLC16A5, MAPK13, PLEKHA2, LTBP3, and FABP5; SLC16A5, MAPK13, PLEKHA2, LTBP3, and CALML4; SLC16A5, MAPK13, PLEKHA2, LTBP3, and TBC1D2; SLC16A5, MAPK13, PLEKHA2, LTBP3, and PPM1M; SLC16A5, PLEKHA2, LTBP3, FABP5, and CALML4; SLC16A5, PLEKHA2, LTBP3, FABP5, and TBC1D2; SLC16A5, PLEKHA2, LTBP3, FABP5, and PPM1M; SLC16A5, LTBP3, FABP5, CALML4, and TBC1D2; SLC16A5, LTBP3, FABP5, CALML4, and PPMIM; SLC16A5, FABP5, CALML4, TBC1D2, and PPM1M; ITGA3, ERG, MAPK13, PLEKHA2, and LTBP3; ITGA3, ERG, MAPK13, PLEKHA2, and FABP5; ITGA3, ERG, MAPK13, PLEKHA2, and CALML4; ITGA3, ERG, MAPK13, PLEKHA2, and TBC1D2; ITGA3, ERG, MAPK13, PLEKHA2, and PPM IM; ITGA3, MAPK13, PLEKHA2, LTBP3, and FABP5; ITGA3, MAPK13, PLEKHA2, LTBP3, and CALML4; ITGA3, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ITGA3, MAPK13, PLEKHA2, LTBP3, and PPM1M; ITGA3, PLEKHA2, LTBP3, FABP5, and CALML4; ITGA3, PLEKHA2, LTBP3, FABP5, and TBC1D2; ITGA3, PLEKHA2, LTBP3, FABP5, and PPM1M; ITGA3, LTBP3, FABP5, CALML4, and TBC1D2; ITGA3, LTBP3, FABP5, CALML4, and PPM1M; ITGA3, FABP5, CALML4, TBC1D2, and PPM1M; ERG, MAPK13, PLEKHA2, LTBP3, and FABP5; ERG, MAPK13, PLEKHA2, LTBP3, and CALML4; ERG, MAPK13, PLEKHA2, LTBP3, and TBC1D2; ERG, MAPK13, PLEKHA2,GRGR-43680.601LTBP3, and PPM1M; ERG, PLEKHA2, LTBP3, FABP5, and CALML4; ERG, PLEKHA2, LTBP3, FABP5, and TBC1D2; ERG, PLEKHA2, LTBP3, FABP5, and PPM1M; ERG, LTBP3, FABP5, CALML4, and TBC1D2; ERG, LTBP3, FABP5, CALML4, and PPM1M; MAPK13, PLEKHA2, LTBP3, FABP5, and CALML4; MAPK13, PLEKHA2, LTBP3, FABP5, and TBC1D2; MAPK13, PLEKHA2, LTBP3, FABP5, and PPM1M; MAPK13, LTBP3, FABP5, CALML4, and TBC1D2; MAPK13, LTBP3, FABP5, CALML4, and PPM1M; MAPK13, FABP5, CALML4, TBC1D2, and PPM1M; PLEKHA2, LTBP3, FABP5, CALML4, and TBC1D2; PLEKHA2, LTBP3, FABP5, CALML4, PPM1M; PLEKHA2, FABP5, CALML4, TBC1D2, and PPM1M; or LTBP3, FABP5, CALML4, TBC1D2, and PPM1M.

17. The method of any one of the preceding claims, wherein said two or more genes is 10 or more genes.

18. The method of claim 17, wherein said 10 or more genes are SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, and PPM1M.

19. The method of any one of the preceding claims, wherein said two or more genes is 25 or more genes.

20. The method of any one of the preceding claims, wherein said two or more genes is 50 or more genes.

21. The method of claim 20, wherein said 50 or more genes comprises SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, GOLM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A.

22. The method of any one of the preceding claims, wherein said two or more genes comprises 2 to 50 genes.GRGR-43680.60123. The method of any one of the preceding claims, wherein said two or more genes comprises 2 to 100 genes.

24. The method of any one of the preceding claims, further comprising administering a prostate cancer treatment to said subject.

25. The method of claim 24, wherein said prostate cancer treatment is one or more of surgery, radiation therapy, cryotherapy, cryoablation, heat ablation, high-intensity focused ultrasound (HIFU), hormonal therapy, chemotherapy, and immunotherapy.

26. The method of claim 25, wherein said surgery is selected from the group consisting of radical prostatectomy and orchiectomy.

27. The method of claim 25, wherein said radiation therapy is selected from the group consisting of external beam, brachytherapy, and targeted radiotherapy.

28. The method of claim 25, wherein said hormonal therapy is selected from the group consisting of anti-androgens, luteinizing hormone-releasing hormone (LHRH) or gonadotropin-releasing hormone (GnRH) agonists and antagonists.

29. The method of any one of the preceding claims, wherein said sample is selected from the group consisting of urine, prostate secretions, prostate cells, blood, and semen.

30. The method of any one of the preceding claims, further comprising conducting an additional screening assay.

31. The method of claim 21, wherein said additional screening assay is selected from the group consisting of a digital rectal examination, and a PSA test.GRGR-43680.60132. The method of any one of the preceding claims, wherein the subject has not had a prior prostate biopsy.

33. The method of any one of the preceding claims, wherein the subject has had a prior negative prostate biopsy result.

34. The method of any one of the preceding claims, wherein the subject has had a prior positive prostate biopsy result.

35. The method of any one of the preceding claims, further comprising identifying and analyzing at least one additional variable in combination with said gene expression.

36. The method of claim 35, wherein said additional variable is selected from the group consisting of the subject’s age, race, family history of prostate cancer, digital rectal examination (DRE) result, prostate biopsy result, prostate specific antigen (PSA) expression value based on a serum sample, multi-perimetric MRI (mpMRI) result, prostate volume, prostate density, or any combination thereof.

37. The method of any one of the preceding claims, wherein said level or amount of expression is the amount of mRNA or protein expressed by said genes.

38. The method of claim 37, wherein said level of expression is increased or decreased relative to the level in a subject that does not have prostate cancer.

39. The method of any one of the preceding claims, wherein detecting the level or amount of expression of said genes comprises detecting an amount of mRNA expression of the genes.

40. The method of claim 39, wherein the detecting an amount of mRNA expression of the genes comprises a nucleic acid amplification assay and / or a nucleic acid sequencing assay.GRGR-43680.60141 . The method of claim 40, wherein the nucleic acid amplification assay comprises performing a reverse transcription polymerase chain reaction.

42. The method of any one of the preceding claims, wherein said method is performed before and / or after administration of a prostate cancer treatment or intervention.

43. The method of claim 42, wherein said method is repeated one or more times.

44. The method of claim 42, wherein said treatment or intervention is altered based on the results of said method.

45. A device, system, or kit comprising one or more of: a) reagents for detecting an amount of expression of at least two genes selected from the group consisting of SLC16A5, ITGA3, ERG, MAPK13, PLEKHA2, LTBP3, FABP5, CALML4, TBC1D2, PPM1M, NAB2, ENSG00000259642, RARG, PCA3, OR51E2, C1R, CCDC69, TGM3, ITGB2, STAP1, IL17RE, ATAD3C, ID4, APOBEC3F, EFEMP2, MSN, GDF15, NIPAL3, IL4R, HOXC6, AIFM2, TFF3, ENSG00000234964, ZNF431, DOK4, GOLM1, CPAMD8, UAP1L1, ANXA2, CCN3, MMP14, MYC, AP5B1, HSPA6, RGS2, EBF4, HM0X1, LUZP2, PRX, and VWA5A; b) software comprising instructions that cause a computing device having a processor to carryout out one or more of: i) collecting expression data; ii) receiving patient-specific information; iii) calculating a risk score or profile based on the expression data results and / or one or more additional patient-specific variables; iv) displaying expression information, additional information, and / or a risk score or profile; v) listing or recommending a potential treatment course of action; and vi) transmitting or communicating expression information, additional information, a risk score or profile, and / or a treatment listing or recommendation to a second computing device; c) instruction for using the device, system, or kit; d) control reagents; e) detection assay components; f) one or more of reagent containers, dispensers, multi-well plates, or flow cells; and g) packaging.GRGR-43680.60146. The device, system, or kit of claim 45, wherein said expression data is collected from a detection instrument.

47. The device, system, or kit of claim 45, wherein said patient-specific information from a is collected from a patient, a caregiver, an electronic medical record.

48. The device, system, or kit of claim 45, wherein said control reagents are selected from the group consisting of positive controls, negative controls, and standards.

49. The device, system, or kit of claim 45, wherein said detection assay components are selected from the group consisting of a reverse transcriptase, a polymerase, primers, probes, dNTPs, buffers, adapters, ligases, detectable labels, and antibodies.

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