Compositions and methods for detecting prostate cancer

Abstract

In one aspect, the present disclosure provides a technique for detecting prostate cancer, for example, for early detection of prostate cancer.In another aspect, the technique provided herein is useful for selecting and / or monitoring and / or evaluating the effectiveness of treatment administered to a subject who is determined to have or be susceptible to prostate cancer.In some embodiments, the technique provided herein is useful for developing companion diagnostics, for example, by measuring tumor burden and tumor burden changes in conjunction with therapeutic agents.In some embodiments, the technique provided herein is useful for developing companion diagnostics, for example, by identifying biomarkers in subject's body fluid samples (e.g., blood samples) that are associated with therapeutic response.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 224,380, filed July 21, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Early detection of cancer greatly increases the chance of successful treatment. However, many cancers, including prostate cancer, still lack effective screening recommendations. Typical problems for cancer screening tests include limited sensitivity and specificity. High false positive rate results can be particularly concerning, because it can create difficult management decisions for clinicians and patients who do not want to unnecessarily administer (or receive) anti-cancer therapy that potentially has undesirable side effects. Conversely, high false negative rate results will miss patients who need therapy, resulting in delayed treatment and consequently reduced chances of success, and therefore cannot meet the purpose of screening tests. Summary of the Invention

[0003] Abstract The present disclosure provides, among other things, insights and techniques for achieving effective prostate cancer screening from biological samples. In some embodiments, such biological samples are or include bodily fluid-derived samples, for example, in some embodiments, blood-derived samples. In some embodiments, the present disclosure provides, among other things, insights and techniques that are particularly useful for prostate adenocarcinoma screening. In some embodiments, the provided techniques are effective for detecting early-stage prostate cancer (e.g., prostate colon adenocarcinoma). In some embodiments, the provided techniques are effective even when applied to a population that includes or consists of asymptomatic individuals (e.g., due to sufficiently high sensitivity and / or low false positive and / or false negative rates). In some embodiments, the provided techniques are effective when applied to a population that includes or consists of individuals without a genetic risk for developing prostate cancer (e.g., prostate adenocarcinoma) (e.g., asymptomatic individuals). In some embodiments, the provided techniques are effective when applied to a population that includes or consists of symptomatic individuals (e.g., individuals suffering from one or more symptoms of prostate cancer). In some embodiments, the provided technology is effective when applied to a population that includes or consists of individuals at risk for prostate cancer (e.g., individuals with genetic and / or lifestyle-related risk factors for prostate cancer). In some embodiments, the provided technology can be or can include one or more compositions (e.g., molecular entities or complexes, systems, cells, collections, combinations, kits, etc.) and / or methods (e.g., methods of making, using, assessing, etc.), as would be apparent to one of skill in the art upon reading the disclosure provided herein.

[0004] In some embodiments, the present disclosure identifies the origin of certain prior technology challenges, including, for example, certain conventional approaches to the detection and diagnosis of prostate cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as digital rectal examination (DRE), urinalysis, CT scanning, and / or cell-free nucleic acid-based molecular tests, serum biomarkers (e.g., PSA), and / or bulk analysis of extracellular vesicles, are time-consuming, expensive, and / or lack sufficient sensitivity and / or specificity to provide a reliable, comprehensive diagnostic assessment. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that address such challenges by, among other things, detecting the co-localization of a target biomarker signature for prostate cancer in individual extracellular vesicles, which includes at least one extracellular vesicle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker, an internal biomarker, and an RNA biomarker. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems by detecting such target biomarker signatures for prostate cancer using, among other things, a target entity detection approach developed by applicant and described in U.S. Patent Application No. 16 / 805,637 (published as US2020 / 0299780; issued as US11,085,089) and International Application No. PCT / US2020 / 020529 (published as WO2020180741), both filed February 28, 2020, and entitled "Systems, Compositions, and Methods for Target Entity Detection," which is based on the interaction and / or co-localization of at least two or more target entities (e.g., target biomarker signatures) in individual extracellular vesicles.

[0005] In some embodiments, extracellular vesicles for detection as described herein can be isolated from a subject's bodily fluids by size-exclusion-based methods. As will be understood by those skilled in the art, in some embodiments, size-exclusion-based methods can provide samples containing nanoparticles of a desired size range, including extracellular vesicles. Thus, in some embodiments, the techniques provided herein involve the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for prostate cancer on individual nanoparticles of a desired size range (e.g., in some embodiments, about 30 nm to about 1000 nm), including extracellular vesicles. Those skilled in the art who read this disclosure will understand that various embodiments described herein in the context of "extracellular vesicles" may also be applicable in the context of "nanoparticles" as described herein.

[0006] In some embodiments, the present disclosure provides, among other things, insight that screening asymptomatic individuals, e.g., periodic screening before the onset of symptoms or otherwise in their absence, may be beneficial and even important for the effective management (e.g., successful treatment) of prostate cancer (e.g., in some embodiments, prostate adenocarcinoma). In some embodiments, the present disclosure provides prostate cancer screening systems that can be implemented to detect prostate cancer (e.g., in some embodiments, prostate adenocarcinoma), including early stage cancer, in some embodiments, early stage cancer in asymptomatic individuals. In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals. The present disclosure provides compositions (e.g., reagents, kits, components, etc.), including, for example, strategies involving periodic testing of one or more individuals (e.g., symptomatic or asymptomatic individuals), as well as methods of providing and / or using them. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them.

[0007] In some embodiments, the provided techniques achieve detection (e.g., early detection, e.g., in asymptomatic individuals and / or populations) of one or more features of prostate cancer (e.g., incidence, progression, response to therapy, recurrence, etc.) with sensitivity and / or specificity (e.g., resulting false positive and / or false negative rates) appropriate to enable useful application of the provided techniques to single and / or regular (e.g., periodic) assessments. In some embodiments, the provided techniques are useful in conjunction with regular screening tests, including, but not limited to, physical exams, general practitioner visits, cholesterol / lipid blood tests, diabetes (type 2) screening, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV / Pap smears, prostate cancer screening, and / or vaccinations. In some embodiments, the provided techniques are useful in conjunction with treatment regimens, and in some embodiments, the provided techniques may improve one or more characteristics (e.g., success rate by accepted parameters) of such treatment regimens.

[0008] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to prostate cancer (e.g., in some embodiments, prostate adenocarcinoma). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to prostate cancer (e.g., in some embodiments, prostate adenocarcinoma). In some embodiments, the provided methods or assays include (a) detecting extracellular vesicles expressing a target biomarker signature of prostate cancer (e.g., in some embodiments, prostate adenocarcinoma) in a body fluid-derived sample (e.g., without limitation, a blood-derived sample, a urine-derived sample, etc.) from a subject in need thereof, wherein the target biomarker signature includes at least one extracellular vesicle-associated surface biomarker, and at least one extracellular vesicle-associated surface biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein). (b) comparing sample information indicating the level of target biomarker signature-expressing extracellular vesicles in the body fluid-derived sample (for example, but not limited to, a blood-derived sample, a urine-derived sample, etc.) with reference information including a reference threshold level; and (c) classifying the subject as having or susceptible to prostate cancer (e.g., in some embodiments, prostate adenocarcinoma) if the body fluid-derived sample (for example, but not limited to, a blood-derived sample, a urine-derived sample, etc.) shows an elevated level of target biomarker signature-expressing extracellular vesicles compared to a classification cutoff that references the reference threshold level.

[0009] In some embodiments, one or more surface biomarkers that may be included in the target biomarker signature are (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MU C1, NAAA, NDUFA2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM 3, SLC35F2, SLC39A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TME M9, TMPRSS2, TRPM4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV , KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, and combinations thereof; and / or (ii) a carbohydrate-dependent marker selected from the following: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), and combinations thereof.

[0010] In some embodiments, the one or more surface biomarkers that may be included in the target biomarker signature are (i) polypeptides encoded by the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, FOLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis It is selected from the X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, and combinations thereof.

[0011] In some embodiments, one or more intravesicular biomarkers that may be included in the target biomarker signature are selected from the following human genes: ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANTXR1, ANTXR2, AP1M2, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ATP8B1, ATP9A, BAIAP2L1, BCAM, BHLHA15, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD74, CDC42EP1, CDC42EP5, CGN, CGNL1, CHMP4C, CLGN, CMTM4, COLCA 1, CORO2A, CPE, CPNE4, CPQ, CRYM, CTSF, CX3CL1, CXADR, CXCR4, CYB561, DPYSL3, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, EMP2, EPHX1, EPN3, ERBB3, ERG, ES RP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLNC, FNBP1L, FOS, FOXA1, GADD45G, GAT A2, GGT1, GJB1, GLYATL1, GNG4, GNMT, GRHL2, GUCY1A3, HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQ GAP2, IRF6, ITGA6, KIAA1522, KIF5C, KLK2, KRT14, KRT15, ​​KRT17, KRT18, KRT19, KRT5, KRT7, KRT8, LCP1, LIFR, LIMCH1, LMOD1, LRP11, LRP3, LSR, MAL2, M AOA, MAP7, MARC1, MARCKSL1, MB, MESP1, MLPH, MME, MPZL2, MT1G, MYBPC1, MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX, NKX3-1, NPDC1, NUPR1, OBSL1, PALLD,PARVA, PCDH1, PCP4, PDIA5, PEBP4, PGM5, PKP1, PKP3, PPL, PPM1H, PPP1R1B, PPP3CA, PRAC1, PRR15L, PSAT1, PTPRF, PYCR1, RAB3B, RAMP1, RAP1GAP, RASEF, RBM47, RCAN3, RDH11, REEP6, REPS2, RG S10, RHPN2, RNF144B, RORC, RPS4Y1, S100A16, SELENBP1, SERINC5, SH3BGRL2, SH3BP4, SHC2, SLC 1A5, SLC22A3, SLC2A12, SLC40A1, SLC4A4, SLC7A8, SLC9A3R2, SMOC2, SORD, SPDEF, SPINT1, SPIN In some embodiments, the intravesicular biomarkers described herein may include at least one post-translational modification.

[0012] In some embodiments, one or more intravesicular RNAs (e.g., mRNAs) that may be included in the target biomarker signature include the following human genes: ABCC4, ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANO7, ANPEP, ANTXR1, ANTXR2, AP1M2, APCDD1, AQP3, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ASTN2, ATP8B1, ATP9A, BAIAP2L 1, BCAM, BHLHA15, BIK, BMPR1B, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD7 4, CDC42EP1, CDC42EP5, CDH1, CGN, CGNL1, CHMP4C, CHRM1, CHRNA2, CLDN3, CLDN4, CLDN7, CLDN8, CLGN, CMTM4, COLCA1, COLEC12, CORO2A, CPE, CPNE4, CPQ , CRB3, CREB3L1, CREB3L4, CRYM, CTSF, CWH43, CX3CL1, CXADR, CXCR4, CYB561, DPP4, DPYSL3, DSG2, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, ELOVL7, EMB, E MP2, ENPP5, EPCAM, EPHX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM189A2, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FA T1, FBLIM1, FBP1, FLNC, FNBP1L, FOLH1, FOS, FOXA1, FXYD3, GADD45G, GALNT3, GALNT7, GATA2, GCNT1, GGT1, GJB1, GLYATL1, GNG4, GNMT, GOLM1, GPR160, G REB1, GRHL2, GUCY1A3, HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HPN, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, ITGA6, KCNN4,KIAA1324、KIAA1522、KIF5C、KLK2、KRT14、KRT15、KRT17、KRT18、KRT19、KRT 5、KRT7、KRT8、KRTCAP3、LCP1、LIFR、LIMCH1、LMOD1、LPAR3、LRP11、LRP3、LR RC26、LSR、MAL2、MAOA、MAP7、MARC1、MARCKSL1、MB、MBOAT2、MESP1、MLPH、MM E、MPZL2、MT1G、MYBPC1、MYL9、MYLK、NCAPD3、NEDD4L、NEFH、NFIX、NKX3-1、NP DC1、NUPR1、OBSL1、OR51E1、OR51E2、PALLD、PARM1、PARVA、PCDH1、PCP4、PDI A5、PDLIM5、PDZK1IP1、PDZRN3、PEBP4、PGM5、PIGR、PKP1、PKP3、PLA2G2A、PM EPA1、PODXL2、PPL、PPM1H、PPP1R1B、PPP3CA、PRAC1、PROM2、PRR15L、PRSS8、 PSAT1、PSCA、PTPRF、PTPRN2、PYCR1、RAB25、RAB27B、RAB3B、RAMP1、RAP1GAP、 RASEF、RBM47、RCAN3、RDH11、REEP6、REPS2、RGS10、RHPN2、RNF144B、RORC、R PS4Y1、S100A16、SCNN1A、SDC1、SELENBP1、SERINC2、SERINC5、SEZ6L2、SH3B GRL2, SH3BP4, SHC2, SLC15A2, SLC1A5, SLC22A3, SLC2A10, SLC2A12, SLC30A4, SLC35F2, SLC40A1, SLC44A4, SLC45A3, SLC4A4, SLC7A8, SLC9A3R2, SMIM22 、SMOC2、SORD、SPDEF、SPINT1、SPINT2、SPTBN2、ST14、STAP2、STEAP1、STEAP 2、STEAP4、STK39、SULT2B1、SYNE4、SYT7、SYTL1、TACSTD2、TAGLN、TBC1D16、T BX3、TC2N、TCEA3、TEAD3、TESC、THBS4、TM4SF1、TMC4、TMC5、TMED3、TMEFF2、 TMEM125、TMEM150C、TMEM30B、TMEM45B、TMEM54、TMEM98、TMPRSS2、TMSB15A、The RNA transcript (e.g., mRNA transcript) is selected from the group consisting of TP53I11, TPD52, TPM1, TRPM4, TRPM8, TRPV6, TSPAN1, TSPAN13, TSPAN6, TSPAN8, TUSC3, UPK3A, VAMP8, VSIG2, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, and combinations thereof.

[0013] In some embodiments, the methods or assays described herein may be performed for one more additional target biomarker signature (e.g., including at least one, at least two, at least three, or more additional target biomarker signatures). In some such embodiments, the classification cutoff may refer to additional reference threshold levels corresponding to each additional target biomarker signature.

[0014] In some embodiments, the extracellular vesicle-associated surface biomarkers for use in the target biomarker signatures for prostate cancer used and / or described herein may be or include tumor-specific and / or tissue-specific biomarkers (e.g., prostate tissue-specific biomarkers). In some embodiments, such extracellular vesicle-associated surface biomarkers may be or include non-specific markers, for example, present in one or more non-target tumors and / or one or more non-target tissues.In some embodiments, such extracellular vesicle-associated surface biomarkers are selected from the group consisting of (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PMEP A1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39A6, S LC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSP AN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP 1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, or any combination thereof; and / or (ii) one or more of the following carbohydrate-dependent markers: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), or a combination thereof.

[0015] In some embodiments, the extracellular vesicle-associated surface biomarker may be or may include (i) a polypeptide encoded by the human gene MUC1; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), or a combination thereof.

[0016] In some embodiments, a target biomarker signature for prostate cancer (e.g., prostate adenocarcinoma) may include extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one (e.g., including 1, 2, 3, or more) additional target surface biomarkers, which in some embodiments are selected from the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4 , GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39A6, SLC39A7 , SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSPAN1, UNC13B , VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, or a combination thereof;and / or may be or include one or more of the following carbohydrate markers: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), or a combination thereof;

[0017] In some embodiments, a target biomarker signature for prostate cancer (e.g., prostate adenocarcinoma) may include extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one (e.g., including 1, 2, 3, or more) additional surface biomarkers, including (i) the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, F and / or (ii) a polypeptide encoded by OLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, and combinations thereof; and / or (ii) a carbohydrate-dependent marker selected from the following: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, and combinations thereof.

[0018] In some embodiments, a target biomarker signature for prostate cancer (e.g., prostate adenocarcinoma) may include extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one target intravesicle RNA biomarker, which in some embodiments is selected from the following human genes: ABCC4, ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANO7, ANPEP, ANTXR1, ANTXR2, AP1M2, APCDD1, AQP3, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ASTN2, ATP8B1, ATP9A, BAIAP2L1, BCAM, BHLHA15, BIK, BMPR1B, BOK, C15orf48, C19orf48, C1S, C 1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD74, CDC42EP1, CDC42EP5, CDH1, CGN, CGNL1, CHMP4C, CHRM1, CHRNA2 , CLDN3, CLDN4, CLDN7, CLDN8, CLGN, CMTM4, COLCA1, COLEC12, CORO2A, CPE, CPNE4, CPQ, CRB3, CREB3L1, CREB3L4, CRYM, CTSF, CWH43, CX3CL1, CXADR, CX CR4, CYB561, DPP4, DPYSL3, DSG2, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, ELOVL7, EMB, EMP2, ENPP5, EPCAM, EPHX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FA AH, FAM129A, FAM129B, FAM189A2, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLNC, FNBP1L, FOLH1, FOS, FOXA1, FXYD3, GADD4 5G, GALNT3, GALNT7, GATA2, GCNT1, GGT1, GJB1, GLYATL1, GNG4, GNMT, GOLM1, GPR160, GREB1, GRHL2, GUCY1A3, HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2,HOMER2, HOXB13, HPN, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, ITG A6、KCNN4、KIAA1324、KIAA1522、KIF5C、KLK2、KRT14、KRT15、KRT17、KRT18、 KRT19, KRT5, KRT7, KRT8, KRTCAP3, LCP1, LIFR, LIMCH1, LMOD1, LPAR3, LRP1 1、LRP3、LRRC26、LSR、MAL2、MAOA、MAP7、MARC1、MARCKSL1、MB、MBOAT2、MESP1 MLPH, MME, MPZL2, MT1G, MYBPC1, MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX. NKX3-1, NPDC1, NUPR1, OBSL1, OR51E1, OR51E2, PALLD, PARM1, PARVA, PCDH1, PCP4、PDIA5、PDLIM5、PDZK1IP1、PDZRN3、PEBP4、PGM5、PIGR、PKP1、PKP3、PL A2G2A, PMEPA1, PODXL2, PPL, PPM1H, PPP1R1B, PPP3CA, PRAC1, PROM2, PRR15L PRSS8, PSAT1, PSCA, PTPRF, PTPRN2, PYCR1, RAB25, RAB27B, RAB3B, RAMP1 RAP1GAP, RASEF, RBM47, RCAN3, RDH11, REEP6, REPS2, RGS10, RHPN2, RNF144 B、RORC、RPS4Y1、S100A16、SCNN1A、SDC1、SELENBP1、SERINC2、SERINC5、SEZ 6L2、SH3BGRL2、SH3BP4、SHC2、SLC15A2、SLC1A5、SLC22A3、SLC2A10、SLC2A12 、SLC30A4、SLC35F2、SLC40A1、SLC44A4、SLC45A3、SLC4A4、SLC7A8、SLC9A3R 2、SMIM22、SMOC2、SORD、SPDEF、SPINT1、SPINT2、SPTBN2、ST14、STAP2、STEAP 1、STEAP2、STEAP4、STK39、SULT2B1、SYNE4、SYT7、SYTL1、TACSTD2、TAGLN、T BC1D16、TBX3、TC2N、TCEA3、TEAD3、TESC、THBS4、TM4SF1、TMC4、TMC5、TMED3、The gene may be or may comprise at least one RNA transcript (e.g., an mRNA transcript) encoded by TMEFF2, TMEM125, TMEM150C, TMEM30B, TMEM45B, TMEM54, TMEM98, TMPRSS2, TMSB15A, TP53I11, TPD52, TPM1, TRPM4, TRPM8, TRPV6, TSPAN1, TSPAN13, TSPAN6, TSPAN8, TUSC3, UPK3A, VAMP8, VSIG2, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, or a combination thereof.

[0019] In some embodiments, a target biomarker signature for prostate cancer may include extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one additional target intravesicular biomarker, which in some embodiments includes the following human genes: ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANTXR1, ANTXR2, AP1M2, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, AS AP3, ATP8B1, ATP9A, BAIAP2L1, BCAM, BHLHA15, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, C BLC, CD24, CD74, CDC42EP1, CDC42EP5, CGN, CGNL1, CHMP4C, CLGN, CMTM4, COLCA1, CORO2A, CPE, CPNE4, CPQ, CRYM, CTSF, CX3CL1, CXADR, CXCR4, CYB561, D PYSL3, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, EMP2, EPHX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM210B, FAM3B, FAM3D, FAM83H , FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLNC, FNBP1L, FOS, FOXA1, GADD45G, GATA2, GGT1, GJB1, GLYATL1, GNG4, GNMT, GRHL2, GUCY1A3, HGD, HID1, HIST1H 2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, ITGA6, KIAA1522, KIF5C, KLK2, KRT14, KRT15, ​​KRT17, KRT18, KRT19, KRT5, KRT7, KRT8, LCP1, LIFR, LIMCH1, LMOD1, LRP11, LRP3, LSR, MAL2, MAOA, MAP7, MARC1, MARCKSL1, MB, MESP1, MLPH, MME, MPZL2, MT1G, MYBPC1,MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX, NKX3-1, NPDC1, NUPR1, OBSL1, PALLD, PARVA, PCDH1, PCP4, PDIA5, PE BP4, PGM5, PKP1, PKP3, PPL, PPM1H, PPP1R1B, PPP3CA, PRAC1, PRR15L, PSAT1, PTPRF, PYCR1, RAB3B, RAMP1, RAP 1GAP, RASEF, RBM47, RCAN3, RDH11, REEP6, REPS2, RGS10, RHPN2, RNF144B, RORC, RPS4Y1, S100A16, SELENBP1, SERINC5, SH3BGRL2, SH3BP4, SHC2, SLC1A5, SLC22A3, SLC2A12, SLC40A1, SLC4A4, SLC7A8, SLC9A3R2, SMOC2, S The polypeptide may be or may comprise at least one polypeptide encoded by ORD, SPDEF, SPINT1, SPINT2, SPTBN2, ST14, STAP2, STK39, SULT2B1, SYNE4, SYT7, SYTL1, TAGLN, TBC1D16, TBX3, TC2N, TCEA3, TEAD3, TESC, THBS4, TM4SF1, TMED3, TMEM150C, TMEM54, TMEM98, TMSB15A, TP53I11, TPD52, TPM1, TSPAN13, TSPAN6, TSPAN8, VAMP8, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, ACSL3, CADH7, KCNN2, OPRK1, PDE9A, PLPP1, SPON2, TMEM121B, or a combination thereof. In some embodiments, the intravesicular biomarkers described herein may include at least one post-translational modification.

[0020] In some embodiments, the reference threshold level for use in the provided methods or assays described herein is determined by the level of target biomarker signature-expressing extracellular vesicles observed in comparable samples from a population of non-prostate cancer subjects.

[0021] In some embodiments, extracellular vesicle-associated surface biomarkers included in the target biomarker signature may be detected using affinity agents (for example, but not limited to, antibody-based agents). In some embodiments, extracellular vesicle-associated surface biomarkers may be detected using a capture assay comprising an antibody-based agent. For example, in some embodiments, a capture assay for detecting the presence of extracellular vesicle-associated surface biomarkers in extracellular vesicles may include contacting a body fluid-derived sample (for example, but not limited to, a blood-derived sample, a urine-derived sample, etc.) containing extracellular vesicles with a capture agent that targets such extracellular vesicle-associated surface biomarkers. In some embodiments, such a capture agent may include a binding moiety (e.g., as described herein) that targets the extracellular vesicle-associated surface biomarker, which may be conjugated to a solid substrate, if desired. Without limitation, an exemplary capture agent for an extracellular vesicle-associated surface biomarker may be or include a solid substrate (e.g., magnetic beads) and a binding moiety (e.g., an antibody agent) that targets the extracellular vesicle-associated surface biomarker.

[0022] In some embodiments, target biomarkers included in a target biomarker signature may be detected using suitable methods known in the art, which may vary depending on the type of analyte being detected (e.g., surface analytes vs. intravesicular analytes; and / or polypeptides and / or glycoforms vs. carbohydrates vs. RNA). For example, those skilled in the art reading this disclosure will recognize that in some embodiments, surface biomarkers and / or intravesicular biomarkers may be detected using affinity agents (e.g., antibody-based agents), while in some embodiments, intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) may be detected using nucleic acid-based agents, e.g., using quantitative reverse transcription PCR.

[0023] For example, in some embodiments in which the target biomarker is or includes a surface biomarker and / or an intravesicular marker, such target biomarker may be detected, for example, by a proximity ligation assay following a capture assay (e.g., as described herein) for capturing extracellular vesicles displaying extracellular vesicle-associated surface biomarkers (e.g., as used and / or described herein). In some embodiments, such a proximity ligation assay may include contacting a body fluid-derived sample (e.g., but not limited to, a blood-derived sample, a urine-derived sample, etc.) containing extracellular vesicles with a set of detection probes each targeting a target biomarker, the set including at least two distinct detection probes, such that a combination including the extracellular vesicles and the set of detection probes is created, the two detection probes each including: (i) a binding moiety targeting the surface biomarker and / or the intravesicular biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain including a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain. The single-stranded overhang portion of the detection probe is characterized in that when the detection probes are bound to the same extracellular vesicle, they can hybridize with each other.The combination of such a set of extracellular vesicles and a set of detection probes is then maintained under conditions that allow the set of detection probes to bind to their respective targets on the extracellular vesicles, so that their oligonucleotide domains are sufficiently close to anneal and form a double-stranded complex.This double-stranded complex can be detected by contacting the double-stranded complex with nucleic acid ligase to form a ligated template; and detecting the ligated template.In some embodiments, the ligated template can be detected using quantitative PCR.The presence of such a ligated template indicates the presence of extracellular vesicles that are positive for the target biomarker signature of prostate cancer (e.g., prostate adenocarcinoma).Such proximity ligation assays may perform better, e.g., with greater specificity and / or sensitivity, than other existing proximity ligation assays, although those of skill in the art reading this disclosure will recognize that other forms of proximity ligation assays known in the art may alternatively be used.

[0024] In some embodiments where the target biomarker is or includes an intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) marker, such target biomarker may be detected including a nucleic acid detection assay. In some embodiments, an exemplary nucleic acid detection assay may be or include reverse transcription PCR.

[0025] In some embodiments in which the target biomarker is or comprises an intravesicular biomarker and / or an intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarker, such target biomarker may be detected by treating the sample (e.g., fixation and / or permeabilization) prior to a detection assay (e.g., a proximity ligation assay described herein) to expose such biomarker within the extracellular vesicles for subsequent detection.

[0026] The present disclosure recognizes, among other things, that detection of a single prostate cancer-associated serum protein (e.g., prostate-specific antigen (PSA)) or multiple prostate cancer-associated biomarkers based on a bulk sample (e.g., a bulk sample of extracellular vesicles), rather than at single extracellular vesicle resolution, typically does not provide sufficient specificity and / or sensitivity in determining whether the subject from whom the sample was obtained is likely to suffer from or be predisposed to prostate cancer. The present disclosure provides technologies, including systems, compositions, and / or methods, that solve such problems, including, among other things, by specifically requiring that individual extracellular vesicles for detection be characterized by the presence of a target biomarker signature comprising a combination of at least one or more extracellular vesicle-associated surface biomarkers and at least one or more target biomarkers. In certain embodiments, the present disclosure teaches techniques that require such individual extracellular vesicles characterized by the presence (e.g., by expression) of such target biomarker signatures for prostate cancer (e.g., prostate adenocarcinoma), whereas extracellular vesicles that do not contain the target biomarker signature do not produce a detectable signal (e.g., a level above a reference level, e.g., at least 10% or more above, where in some embodiments the reference level may be the level observed in a negative control sample, such as a sample in which individual extracellular vesicles containing such target biomarker signature are absent).

[0027] As will be understood by those skilled in the art, in some embodiments, a sample containing extracellular vesicles may also contain nanoparticles having a size range of interest, including extracellular vesicles. Thus, in some embodiments, the techniques provided herein are applicable to the detection of nanoparticles having a size range of interest, including extracellular vesicles, in the context of extracellular vesicles. Thus, in some embodiments, the present disclosure provides techniques for, among other things, the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for prostate cancer in individual nanoparticles having a size range of interest, including extracellular vesicles (e.g., in some embodiments, about 30 nm to about 1000 nm).

[0028] In some embodiments, the present disclosure provides a method for detecting a prostate cancer cell line comprising: (a) providing or obtaining a sample comprising nanoparticles isolated from a subject's bodily fluid-derived sample (e.g., but not limited to, a blood-derived sample, a urine-derived sample, etc.), the nanoparticles having a size within a range of about 30 nm to about 1000 nm; (b) detecting, on the surface of the nanoparticles, the co-localization of at least two surface biomarkers whose combined expression level has been determined to be associated with prostate cancer, wherein the surface biomarkers are (i) a human gene selected from the group consisting of: AB CC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJ C30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVE LD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROO M2, SHROOM3, SLC35F2, SLC39A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM 9, polypeptides encoded by TMPRSS2, TRPM4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4;and / or (ii) a carbohydrate-dependent marker selected from the following: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), and combinations thereof; (c) comparing the detected co-localization level with the determined level; and (d) classifying the subject as having or susceptible to prostate cancer if the detected co-localization level is at or above the determined level.

[0029] In some embodiments, the first surface biomarker and the second surface biomarker are (i) polypeptides encoded by the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, FOLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis Each is independently selected from the group consisting of an X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), a T antigen, a Tn antigen, and combinations thereof.

[0030] Thus, in some embodiments, the techniques provided herein may be useful for detecting the occurrence or recurrence of prostate cancer in a subject and / or an entire population of subjects. In some embodiments, a target biomarker signature may be selected for the detection of prostate cancer. In some embodiments, a target biomarker signature may be selected for the detection of a specific category of prostate cancer, for example, but not limited to, prostate adenocarcinoma. In some embodiments, a target biomarker signature may be selected for the detection of early stage (e.g., stage I and / or stage II) prostate cancer, for example, but not limited to, prostate adenocarcinoma. In some embodiments, a target biomarker signature may be selected for the detection of late stage (e.g., stage III and / or stage IV) prostate cancer, for example, but not limited to, prostate adenocarcinoma. In some embodiments, a target biomarker signature may be selected for the detection of low-grade (e.g., Gleason score of 6) prostate cancer, for example, but not limited to, prostate adenocarcinoma. In some embodiments, a target biomarker signature may be selected for the detection of intermediate-grade (e.g., Gleason score of 7) prostate cancer, including, but not limited to, prostate adenocarcinoma. In some embodiments, a target biomarker signature may be selected for the detection of high-grade (e.g., Gleason score of 8-10) prostate cancer, including, but not limited to, prostate adenocarcinoma. In some embodiments, the techniques provided herein can be used periodically (e.g., annually) to screen human subjects or entire populations of human subjects for early-stage prostate cancer or recurrence of prostate cancer.

[0031] In some embodiments, the subject suitable for the technology provided herein for detecting the occurrence or recurrence of prostate adenocarcinoma may be asymptomatic human subject and / or the entire asymptomatic population.Such asymptomatic subject may be the subject with a family history of prostate cancer, the subject with a life history that puts the patient at increased risk for prostate cancer, the subject who has been previously treated for prostate cancer, the subject who is at risk of prostate cancer recurrence after cancer treatment, the subject who is in remission after prostate cancer treatment, and / or the subject who has previously or periodically been screened for the presence of at least one serum biomarker of prostate cancer (e.g., PSA).In some embodiments, such asymptomatic subject may be the subject who has been determined to have normal medical diagnosis results, for example, from digital rectal examination (DRE), urinalysis, CT scanning, and / or molecular testing, and / or based on cell-free nucleic acid. In some embodiments, such an asymptomatic subject may be a subject who has been determined to have an abnormal medical diagnostic result, e.g., based on cell-free nucleic acid, from, e.g., a digital rectal exam (DRE), a urinalysis, a CT scan, and / or a molecular test, when compared to results typically observed in non-prostate cancer subjects and / or normal healthy subjects. Alternatively, in some embodiments, an asymptomatic subject may be a subject who has not previously been screened for prostate cancer, a subject who has not been diagnosed with prostate cancer, and / or a subject who has not previously received prostate cancer therapy.

[0032] In some embodiments, the subject or population of subjects may be selected based on one or more characteristics such as age, race, geographic location, genetic history, personal history and / or medical history (e.g., smoking, alcohol, drugs, carcinogens, diet, obesity, diabetes, physical activity, sun exposure, radiation exposure, chronic inflammation of the prostate, chronic urinary tract infections, and / or occupational hazards).

[0033] In some embodiments, the techniques provided herein may be useful for selecting a surgical procedure or therapy for a subject suffering from or susceptible to prostate adenocarcinoma. In some embodiments, prostate cancer surgery, therapy, and / or adjuvant therapy may be selected in light of findings based on the techniques provided herein.

[0034] In some embodiments, the techniques provided herein may be useful for monitoring and / or evaluating the effectiveness of a therapy administered to a subject (e.g., a prostate cancer subject).

[0035] In some embodiments, the present disclosure provides technologies for managing patient care, for example, for one or more individual subjects and / or entire populations of subjects. To give some examples, in some embodiments, the present disclosure provides technologies that can be utilized in screening (e.g., temporally or contingently motivated screening and / or non-temporally or contingently motivated screening, e.g., periodic screening, annually, twice a year, biennially, or some other frequency). For example, in some embodiments, the provided technologies for use in temporally motivated screening may be useful for screening one or more individual subjects or entire populations of subjects (e.g., asymptomatic subjects) who are older than a certain age (e.g., over 40, 45, 50, 55, 60, 65, 70 years of age, or older). In some embodiments, the provided technologies for use in temporally motivated screening may be useful for screening one or more individual subjects or entire populations of subjects (e.g., asymptomatic subjects) who are in the 50-70 year age range. In some embodiments, provided technologies for use in temporally motivated screening may be useful for screening one or more individual subjects or entire populations of subjects (e.g., asymptomatic subjects) between the ages of 55 and 69. In some embodiments, provided technologies for use in incidentally motivated screening may be useful for screening individual subjects who may have experienced an event or occurrence that motivated screening for prostate cancer as described herein.For example, in some embodiments, the incidental motivation for determining one or more indicators of cancer or susceptibility to cancer may be or may include, for example, a family history event thereof (e.g., a close relative, such as a blood relative, previously diagnosed with prostate cancer), the identification of one or more risk factors associated with prostate cancer (e.g., lifestyle history risk factors, including, but not limited to, smoking, alcohol, diet, obesity, occupational hazards, etc.), and / or a prior incidental finding from genetic testing (e.g., genomic sequencing), and / or an imaging diagnostic test (e.g., ultrasound, computed tomography (CT) and / or magnetic resonance imaging (MRI) scan), the occurrence of one or more signs or symptoms characteristic of prostate cancer (e.g., difficulty urinating, blood in the urine or blood-stained semen, abnormal medical results such as erectile dysfunction (ED), pain in the buttocks, back (spine), chest (ribs), weakness or numbness in the legs or feet, loss of bladder control, and / or symptoms potentially indicative of prostate cancer, etc.).

[0036] In some embodiments, the provided technology for managing patient care can inform treatment and / or payment (e.g., reimbursement for treatment) decisions and / or actions. For example, in some embodiments, the provided technology can provide a determination of whether an individual subject has one or more indicators of the development or recurrence of prostate cancer, thereby informing a physician and / or patient on when to initiate therapy, taking such findings into account. Additionally or alternatively, in some embodiments, the provided technology can inform a physician and / or patient on treatment selection, for example, based on findings of specific response biomarkers (e.g., prostate cancer response biomarkers). In some embodiments, the provided technology can provide a determination of whether an individual subject is responsive to current treatment, for example, based on findings of changes in the levels of one or more molecular targets associated with prostate cancer, thereby informing a physician and / or patient on the effectiveness of such therapy and / or decisions to maintain or modify therapy, taking such findings into account.

[0037] In some embodiments, the provided technology can inform decisions by health insurance providers regarding, for example, (1) whether to reimburse (or not) for the screening itself (e.g., reimbursement available only for periodic / routine screening, or only for time- and / or incidentally-motivated screening); and / or (2) whether to initiate, maintain, and / or modify therapy in light of findings from the provided technology. For example, in some embodiments, the present disclosure provides methods for (a) receiving results of the screenings described herein and also receiving claims for reimbursement for the screening and / or for a particular treatment regimen; (b) approving reimbursement for the screening if the screening was performed on the subject according to the appropriate schedule or response to the relevant event, and / or approving reimbursement for the treatment regimen if the received screening results represent appropriate treatment; and, as appropriate, (c) issuing the reimbursement or providing notification that the reimbursement was denied. In some embodiments, a treatment regimen is applied in consideration of the received screening result if the received screening result detects a biomarker that represents an approved biomarker for the associated treatment regimen (e.g., as may be stated on the prescribing information label and / or via an approved companion diagnostic). Alternatively, or in addition, the present disclosure contemplates a reporting system (e.g., implemented via a suitable electronic device and / or communication system) that permits or facilitates reporting and / or processing of the screening results and / or reimbursement determinations described herein.

[0038] Some aspects provided herein relate to systems and kits for use in the provided techniques. In some embodiments, the system or kit may include a detection agent (for example, as described herein) for a tumor biomarker signature of prostate cancer.

[0039] In some embodiments, such a system or kit may include: (a) a capture agent (e.g., as used and / or described herein) for extracellular vesicle-associated surface biomarkers present in extracellular vesicles associated with prostate cancer; and (b) at least one or more detection agents directed to one or more target biomarkers of a target biomarker signature for prostate cancer, which may be or include additional surface biomarkers (e.g., as used and / or described herein), intravesicular biomarkers (e.g., as used and / or described herein), and / or intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers (e.g., as used and / or described herein).

[0040] In some embodiments, the capture agent included in the system and / or kit may comprise a binding moiety (e.g., as described herein) directed to an extracellular vesicle-associated surface biomarker. In some embodiments, such a binding moiety may be conjugated to a solid substrate, which in some embodiments may be or comprise a solid substrate. In some embodiments, such a solid substrate may be or comprise a magnetic bead. In some embodiments, an exemplary capture agent included in the provided system and / or kit may be or comprise a solid substrate (e.g., magnetic bead) and an affinity reagent (e.g., but not limited to, an antibody agent) directed to an extracellular vesicle-associated surface biomarker conjugated thereto.

[0041] In some embodiments, in which the target biomarkers include surface biomarkers and / or intravesicular biomarkers, the system and / or kit may include a detection agent (e.g., as described herein) for performing a proximity ligation assay. In some embodiments, such a detection agent for performing a proximity ligation assay may include a set of detection probes each directed to a target biomarker of the target biomarker signature, the set including at least two detection probes, each of which includes: (i) a polypeptide binding moiety directed to the target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhanging portion extending from one end of the oligonucleotide domain, wherein the single-stranded overhanging portion of the detection probes allows the detection probes to hybridize with each other when the detection probes bind to the same extracellular vesicle.

[0042] In some embodiments, the provided systems and / or kits may include multiple (e.g., 2, 3, 4, 5, or more) sets of detection probes, each set including two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) detection probes. In some embodiments, at least one set of detection probes may be directed to the detection of prostate cancer. For example, in some embodiments, the provided systems and kits may include at least one set of detection probes for the detection of prostate cancer and at least one set of detection probes for the detection of a different cancer (e.g., pancreatic cancer). In some embodiments, the two or more detection probes may be directed to different categories of prostate cancer (e.g., including prostate adenocarcinoma). In some embodiments, the two or more sets may be directed to the detection of different stages of prostate cancer. In some embodiments, the two or more sets may be directed to the detection of the same stage of prostate cancer.

[0043] In some embodiments, the detection probes in the provided kits may be provided as a single mixture in a container. In some embodiments, multiple sets of detection probes may be provided as individual mixtures in separate containers. In some embodiments, each detection probe is provided individually in a separate container.

[0044] In some embodiments, where the target biomarkers include intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers, such systems and / or kits may include detection agents for performing nucleic acid detection assays. In some embodiments, such systems and / or kits may include detection agents for performing quantitative reverse transcription PCR, which may include, for example, primers directed to intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) targets.

[0045] Those skilled in the art will understand that systems or kits for detecting extracellular vesicles can also be used to detect nanoparticles having a desired size range, including extracellular vesicles. Thus, in some embodiments, the system or kit may include (i) a capture agent (e.g., as described herein) for a first surface biomarker of a prostate cancer-associated biomarker signature present on the surface of nanoparticles having a desired size range, including extracellular vesicles; and (ii) at least one or more detection agents targeting a second surface biomarker of a prostate cancer-specific biomarker signature. In some embodiments, such nanoparticles have a size within the range of about 30 nm to about 1000 nm.

[0046] In some embodiments, the present disclosure provides a kit for detecting prostate cancer, comprising: (a) a capture agent comprising a target capture moiety directed to a first surface biomarker; and (b) at least one set of detection probes, the set comprising at least two detection probes each directed to a second surface biomarker, the detection probes each comprising: (i) a target binding moiety for the second surface biomarker; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the at least two detection probes are capable of hybridizing to each other when the at least two detection probes are bound to the same nanoparticle having a size in the range of about 30 nm to about 1000 nm;At least a first surface biomarker and a second surface biomarker form a target biomarker signature determined to be associated with prostate cancer, the first and second surface biomarkers being selected from the group consisting of (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNA JC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1 , MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM 141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1 , IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, and combinations thereof; and / or (ii) a carbohydrate-dependent marker, each independently selected from the following: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), and combinations thereof.

[0047] In some embodiments, the first surface biomarker and the second surface biomarker are (i) polypeptides encoded by the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, FOLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis Each is independently selected from the group consisting of an X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), a T antigen, a Tn antigen, and combinations thereof.

[0048] In some embodiments, the provided systems and / or kits may include at least one chemical reagent, e.g., a chemical reagent for processing a sample and / or nanoparticles therein (e.g., including, in some embodiments, extracellular vesicles). In some embodiments, the provided systems and / or kits may include at least one chemical reagent for processing nanoparticles (e.g., including, in some embodiments, extracellular vesicles) in a sample, including, for example, but not limited to, a fixative, a permeabilizing agent, and / or a blocking agent. In some embodiments, the provided systems and / or kits may include a nucleic acid ligase and / or a nucleic acid polymerase. In some embodiments, the provided systems and / or kits may include one or more primers and / or probes. In some embodiments, the provided systems and / or kits may include, for example, one or more primer pairs for PCR, e.g., quantitative PCR (qPCR), reactions. In some embodiments, the provided systems and / or kits may include one or more probes, such as hydrolysis probes (e.g., TaqMan probes), which, in some embodiments, may be designed to increase the specificity of qPCR. In some embodiments, the provided systems and / or kits may include one or more multiplexing probes, as may be useful, for example, when simultaneous or parallel qPCR reactions are used (e.g., to facilitate or improve readout).

[0049] In some embodiments, the provided systems and / or kits can be used for screening (e.g., periodic screening) and / or other assessment of individuals (e.g., asymptomatic or symptomatic subjects) for detection (e.g., early detection) of prostate cancer. In some embodiments, the provided systems and / or kits can be used for screening and / or other assessment of individuals predisposed to prostate cancer (e.g., individuals with known genetic, environmental, or experiential risk, etc.). In some embodiments, the provided systems and / or kits can be used for monitoring the recurrence of prostate cancer in previously treated subjects. In some embodiments, the provided systems and / or kits can be used as a companion diagnostic in combination with a therapy for a subject suffering from prostate cancer. In some embodiments, the provided systems and / or kits can be used to monitor or evaluate the effectiveness of a therapy administered to a subject suffering from prostate cancer. In some embodiments, the provided systems and / or kits can be used to select a therapy for a subject suffering from prostate cancer. In some embodiments, the provided systems and / or kits can be used to make therapy decisions and / or select a therapy for a subject with one or more symptoms (e.g., non-specific symptoms) associated with prostate cancer.

[0050] The complex formed by carrying out the method described herein and / or using the system and / or kit described herein is also within the scope of the present disclosure.For example, in some embodiments, the complex comprises an extracellular vesicle expressing a target biomarker signature, which comprises at least one extracellular vesicle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker (e.g., as described herein), an intravesicular biomarker (e.g., as described herein), and an intravesicular RNA biomarker (e.g., as described herein), and the extracellular vesicle is immobilized on a solid substrate comprising a binding moiety that targets the extracellular vesicle-associated surface biomarker. In some embodiments, such complexes further comprise at least two detection probes directed to at least one target biomarker in the target biomarker signature present in the extracellular vesicles, each detection probe binding to an individual target biomarker and comprising: (i) a binding moiety directed to the target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, wherein the oligonucleotide domain comprises a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, and the single-stranded overhang portions of the detection probes hybridize to each other.

[0051] In some embodiments, the extracellular vesicle-associated surface biomarkers present in the complexed extracellular vesicles may include one or more of the surface biomarkers described herein. In some embodiments, such extracellular vesicle-associated biomarkers include (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PME PA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39A6 , SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1 , PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), or a combination thereof.

[0052] In some embodiments, the extracellular vesicle-associated biomarker may be or include (i) a polypeptide encoded by the human gene MUC1; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, and combinations thereof.

[0053] In some embodiments, the surface biomarkers present on the complexed extracellular vesicles are selected from the group consisting of (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, and GALNT3. , GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA 2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC 39A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRP M4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLA and / or (ii) at least one polypeptide encoded by C1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), or a combination thereof.

[0054] In some embodiments, the surface biomarkers present on the complexed extracellular vesicles are selected from the group consisting of (i) one or more of the polypeptides encoded by the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, FOLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, or a combination thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis It may be or include one or more of the X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, or a combination thereof.

[0055] In some embodiments, the intravesicular biomarkers present in the complexed extracellular vesicles are selected from the group consisting of human genes: ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANTXR1, ANTXR2, AP1M2, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ATP8B1, ATP9A, BAIAP2L1, BCAM, BHLHA15, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf 72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD74, CDC42EP1, CDC42EP5, CGN, CGNL1, CHMP4C, CLGN, CMTM4, COLCA1, CORO2A, CPE, CPNE4, CPQ, CRYM, CTSF, CX3CL1, CXADR, CXCR4, CYB561, DPYSL3, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, EMP2, EPHX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLNC, FNBP1L, FOS, FOXA1, GADD45G, GATA2, GGT1, GJB1 , GLYATL1, GNG4, GNMT, GRHL2, GUCY1A3, HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, IT GA6, KIAA1522, KIF5C, KLK2, KRT14, KRT15, ​​KRT17, KRT18, KRT19, KRT5, KRT7, KRT8, LCP1, LIFR, LIMCH1, LMOD1, LRP11, LRP3, LSR, MAL2, MAOA, MAP7, MAR C1, MARCKSL1, MB, MESP1, MLPH, MME, MPZL2, MT1G, MYBPC1, MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX, NKX3-1, NPDC1, NUPR1, OBSL1, PALLD, PARVA, PCDH1,PCP4, PDIA5, PEBP4, PGM5, PKP1, PKP3, PPL, PPM1H, PPP1R1B, PPP3CA, PRAC1, PRR15L, PSAT1, PTPR F, PYCR1, RAB3B, RAMP1, RAP1GAP, RASEF, RBM47, RCAN3, RDH11, REEP6, REPS2, RGS10, RHPN2, RNF14 4B, RORC, RPS4Y1, S100A16, SELENBP1, SERINC5, SH3BGRL2, SH3BP4, SHC2, SLC1A5, SLC22A3, SLC2 A12, SLC40A1, SLC4A4, SLC7A8, SLC9A3R2, SMOC2, SORD, SPDEF, SPINT1, SPINT2, SPTBN2, ST14, STA The vesicular biomarkers may be or may include at least one polypeptide encoded by P2, STK39, SULT2B1, SYNE4, SYT7, SYTL1, TAGLN, TBC1D16, TBX3, TC2N, TCEA3, TEAD3, TESC, THBS4, TM4SF1, TMED3, TMEM150C, TMEM54, TMEM98, TMSB15A, TP53I11, TPD52, TPM1, TSPAN13, TSPAN6, TSPAN8, VAMP8, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, ACSL3, CADH7, KCNN2, OPRK1, PDE9A, PLPP1, SPON2, TMEM121B, or a combination thereof. In some embodiments, the intravesicular biomarkers described herein may include at least one post-translational modification.

[0056] In some embodiments, the intravesicular RNA biomarkers present in the complexed extracellular vesicles are selected from the group consisting of human genes: ABCC4, ACP5, ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANO7, ANPEP, ANTXR1, ANTXR2, AP1M2, APCDD1, AQP3, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ASTN2, ATP8B1, ATP9A, BAIAP2L1, BCAM, BHLHA15, BI K, BMPR1B, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD74, CDC42EP1, CDC42EP 5, CDH1, CGN, CGNL1, CHMP4C, CHRM1, CHRNA2, CLDN3, CLDN4, CLDN7, CLDN8, CLGN, CMTM4, COLCA1, COLEC12, CORO2A, CPE, CPNE4, CPQ, CRB3, CREB3L1, CREB 3L4, CRYM, CTSF, CWH43, CX3CL1, CXADR, CXCR4, CYB561, DPP4, DPYSL3, DSG2, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, ELOVL7, EMB, EMP2, ENPP5, EPCAM, EP HX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM189A2, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLN C, FNBP1L, FOLH1, FOS, FOXA1, FXYD3, GADD45G, GALNT3, GALNT7, GATA2, GCNT1, GGT1, GJB1, GLYATL1, GNG4, GNMT, GOLM1, GPR160, GREB1, GRHL2, GUCY1A3 , HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HPN, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, ITGA6, KCNN4, KIAA1324, KIAA1522,KIF5C、KLK2、KRT14、KRT15、KRT17、KRT18、KRT19、KRT5、KRT7、KRT8、KRTCAP 3、LCP1、LIFR、LIMCH1、LMOD1、LPAR3、LRP11、LRP3、LRRC26、LSR、MAL2、MAOA MAP7, MARC1, MARCKSL1, MB, MBOAT2, MESP1, MLPH, MME, MPZL2, MT1G, MYBPC1, MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX, NKX3-1, NPDC1, NUPR1, OBSL1, OR 51E1、OR51E2、PALLD、PARM1、PARVA、PCDH1、PCP4、PDIA5、PDLIM5、PDZK1IP1 、PDZRN3、PEBP4、PGM5、PIGR、PKP1、PKP3、PLA2G2A、PMEPA1、PODXL2、PPL、PPM 1H、PPP1R1B、PPP3CA、PRAC1、PROM2、PRR15L、PRSS8、PSAT1、PSCA、PTPRF、PT PRN2、PYCR1、RAB25、RAB27B、RAB3B、RAMP1、RAP1GAP、RASEF、RBM47、RCAN3、R DH11、REEP6、REPS2、RGS10、RHPN2、RNF144B、RORC、RPS4Y1、S100A16、SCNN1 A、SDC1、SELENBP1、SERINC2、SERINC5、SEZ6L2、SH3BGRL2、SH3BP4、SHC2、SL C15A2, SLC1A5, SLC22A3, SLC2A10, SLC2A12, SLC30A4, SLC35F2, SLC40A1, SLC44A4, SLC45A3, SLC4A4, SLC7A8, SLC9A3R2, SMIM22, SMOC2, SORD, SPDEF, S PINT1、SPINT2、SPTBN2、ST14、STAP2、STEAP1、STEAP2、STEAP4、STK39、SULT 2B1、SYNE4、SYT7、SYTL1、TACSTD2、TAGLN、TBC1D16、TBX3、TC2N、TCEA3、TEAD 3、TESC、THBS4、TM4SF1、TMC4、TMC5、TMED3、TMEFF2、TMEM125、TMEM150C、TM EM30B、TMEM45B、TMEM54、TMEM98、TMPRSS2、TMSB15A、TP53I11、TPD52、TPM1、The gene may be or may include at least one RNA transcript (e.g., an mRNA transcript) encoded by TRPM4, TRPM8, TRPV6, TSPAN1, TSPAN13, TSPAN6, TSPAN8, TUSC3, UPK3A, VAMP8, VSIG2, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, or a combination thereof.

[0057] In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a CLDN3 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include an ABCC4 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a RAB3B polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a FOLH1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a PMEPA1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a TMPRSS2 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a TSPAN1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a PODXL2 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a GOLM1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a SORD polypeptide.In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a CLGN polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a GRHL2 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a CANT1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a SYT7 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a PPP3CA polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a CDH1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include an RDH11 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a CLDN4 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include an EPCAM polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a TRPM4 polypeptide.In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a FAAH polypeptide.

[0058] In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a MUC1 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a Lewis Y antigen. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include an sTn antigen. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include an sLex antigen. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a T antigen. In some embodiments, the extracellular vesicle-associated surface biomarker and / or surface biomarker included in the target biomarker signature may be or may include a Tn antigen.

[0059] Also within the scope of the present disclosure are complexes comprising nanoparticles of interest, including extracellular vesicles, and comprising a prostate cancer-specific biomarker signature, which comprise at least two surface biomarkers described herein, and the nanoparticles are immobilized on a solid substrate comprising a binding moiety that targets the first surface biomarker of the prostate cancer-specific biomarker signature.In some embodiments, such complexes are also bound to at least two detection probes that respectively target the surface biomarkers of the prostate cancer-specific biomarker signature (which can be the same or different surface biomarkers), each detection probe binds to a respective surface biomarker and comprises (i) a binding moiety that targets the surface biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, wherein the oligonucleotide domain comprises a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, and the single-stranded overhang portions of the detection probes hybridize with each other.

[0060] In some embodiments, the present disclosure provides (a) nanoparticles having a size in the range of about 30 nm to about 1000 nm, comprising at least a first surface biomarker and a second surface biomarker, the combination of which has been determined to be a target biomarker signature for prostate cancer, wherein the first surface biomarker and the second surface biomarker are selected from the group consisting of (i) the following human genes: ABCC4, ABHD17C, ADI1, AGTRAP, AP1M2, APOO, ARFGE, and EGFR. F3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, G ALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUF A2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39 A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSP Polypeptides encoded by AN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4, and combinations thereof;and / or (ii) the following carbohydrate-dependent markers: Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis (b) a solid substrate comprising a target capture moiety directed to a first surface biomarker, the target capture moiety binding to the first surface biomarker on the nanoparticle such that the nanoparticle is immobilized on the solid substrate; and (c) at least a first detection probe and a second detection probe coupled to the nanoparticle, each detection probe comprising: (i) a target binding moiety directed to a second surface biomarker; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, the single-stranded overhang portions of the first and second detection probes hybridizing to one another.

[0061] In some embodiments, the first surface biomarker and the second surface biomarker are (i) polypeptides encoded by the following human genes: ABCC4, AP1M2, ARFGEF3, CANT1, CD38, CDH1, CLDN3, CLDN4, CLGN, ENPP5, FOLH1, GOLM1, GRHL2, MAP7, MARCKSL1, MUC1, PMEPA1, PODXL2, PPP3CA, PSCA, RAB3B, RAB3D, RDH11, SLC39A6, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYT7, TMPRSS2, TRPM4, TSPAN1, UNC13B, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis Y antigen (also known as CD174), sialyl Tn (sTn) antigen, sialyl Lewis Each is independently selected from the group consisting of an X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), a T antigen, a Tn antigen, and combinations thereof.

[0062] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1 is a schematic diagram illustrating an exemplary workflow for profiling individual extracellular vesicles (EVs). The diagram shows the purification of EVs from plasma using size exclusion chromatography (SEC) and immunoaffinity capture of EVs displaying specific EV-associated surface markers (Panel A); and the detection of co-localized target markers (e.g., intravesicular or surface biomarkers) on the captured EVs using a target entity detection assay according to some embodiments described herein (Panel B).

[0064] [Figure 2]2 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein. In some embodiments, the target entity detection assay uses a combination of detection probes, which are specific for the detection of cancer. In some embodiments, a duplex system includes a first detection probe for target biomarker 1 and a second detection probe for target biomarker 2, added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each include a target binding moiety (e.g., an affinity agent, such as an antibody agent for the target biomarker) coupled to an oligonucleotide domain, which includes a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. When the separate target binding moieties (e.g., affinity agents, such as an antibody agent for target biomarker 1 and target biomarker 2, respectively) of the first and second detection probes are localized in close proximity to the same biological entity (e.g., extracellular vesicles) such that the corresponding single-stranded overhangs hybridize with each other, thus resulting in ligation of the oligonucleotide domains. A detection signal is generated when the separate target binding moieties (e.g., affinity agents, such as an antibody agent for target biomarker 1 and target biomarker 2, respectively) of the first and second detection probes are localized in the same biological entity (e.g., extracellular vesicles) such that the corresponding single-stranded overhangs hybridize with each other, thus resulting in ligation of the oligonucleotide domains. For example, a control entity (e.g., a biological entity derived from a healthy subject sample) does not express one or both of target biomarker 1 and target biomarker 2, and as a result, no detection signal is generated. However, if a biological entity derived from a cancer sample (e.g., a prostate cancer sample) expresses target biomarker 1 and target biomarker 2, and the target biomarkers are present within a sufficiently short distance from each other in the same biological entity (e.g., extracellular vesicles), a detection signal is generated.

[0065] [Figure 3]3 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein. The diagram shows an exemplary triple target entity detection system, in which, in some embodiments, three or more detection probes for each target biomarker can be added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each include a target binding moiety (e.g., an affinity agent, such as an antibody agent for the target biomarker) coupled to an oligonucleotide domain, which includes a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. A detection signal is generated when all corresponding single-stranded overhangs of the three or more detection probes hybridize to each other to form a linear double-stranded complex, allowing ligation of at least one strand of the double-stranded complex to occur, thus allowing the resulting ligated product to be detected.

[0066] [Figure 4] FIG. 4 is a non-limiting example of a double-stranded complex containing four detection probes connected to each other in a linear arrangement by hybridization of their respective single-stranded overhangs.

[0067] [Figure 5]5 is a schematic diagram illustrating a target entity detection assay according to an exemplary embodiment described herein. In some embodiments, multiple detection probes, each for a distinct target, are added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each contain a target-binding moiety (e.g., an antibody agent) coupled to an oligonucleotide domain, which contains a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. When all the detection probes are localized in close proximity to the same biological entity (e.g., extracellular vesicles or analytes), the corresponding single-stranded overhangs hybridize to form a linear double-stranded complex, ligating at least one strand of the resulting linear double-stranded complex and allowing the ligated product to be detected, a detection signal is generated.

[0068] [Figure 6] Figure 6 presents a bar graph showing 5-year relative survival by stage at diagnosis of prostate cancer taken from SEER Summary Stage 2000, SEER 18 2010-2016, All Races, Both Sexes.

[0069] [Figure 7] Figure 7 shows a pie chart showing the percentages of prostate cancer diagnoses (localized, regional, distant metastasis, and unknown). Diagnosis is generally made at the distant metastasis stage, when cancer is most lethal. SEER 18 2010-2016 by SEER Summary Stage 2000, All Races, Both Sexes.

[0070] [Figure 8-1]8 shows Ct values ​​from the characterization of certain exemplary biomarker combinations using the methods and / or assays described herein (e.g., the target entity detection system described herein) in prostate cancer-specific cell lines expressing at least two surface biomarkers and in negative control groups. Panel A shows the biomarker combination of EPCAM and T antigen, panel B shows the biomarker combination of LEY and VWA1, panel C shows the biomarker combination of MARCKSL1 and T antigen, panel D shows the biomarker combination of T antigen and Tn antigen, panel E shows the biomarker combination of ABCC4 and TSPAN1, panel F shows the biomarker combination of FOLH1 and TSPAN1, and panel G shows the biomarker combination of MUC1 and TSPAN1. [Figure 8-2] Same as above.

[0071] [Figure 9] Figure 9 shows MIF RT-PCR signals (45-Ct) after EPCAM-targeted immunoaffinity capture for OVCAR-3 (positive cell line) and SK-MEL-1 (negative cell line) EVs. Multiple detergent (Tween®-20) concentrations were evaluated, with 0% Tween® showing larger delta values. DETAILED DESCRIPTION OF THE INVENTION

[0072] A specific definition Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of the agent itself or the agent contained in the composition to a target site or site to be treated. Those of skill in the art will be aware of various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may comprise only a single dose. In some embodiments, administration may comprise the application of a fixed number of doses. In some embodiments, administration may comprise dosing that is intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart in time) dosing. In some embodiments, administration may comprise continuous dosing (e.g., perfusion) for at least a selected period of time.

[0073] Affinity agent: The term "affinity agent," as used herein, refers to an entity that is or includes a target-binding moiety described herein and thus binds to a target of interest (e.g., a molecular target of interest, such as a biomarker or epitope). In many embodiments, an affinity agent according to the present disclosure specifically binds to a biomarker described herein. In many embodiments, an affinity agent according to the present disclosure specifically binds to a surface protein biomarker described herein. In some embodiments, an affinity agent according to the present disclosure specifically binds to a carbohydrate-dependent marker described herein. In some embodiments, an affinity agent may be or include an antibody agent (e.g., an antibody, or other entity that is or includes an antigen-binding portion thereof). Alternatively, or in addition, in some embodiments, an affinity agent may be selected from the group consisting of an affimer, an aptamer, a lectin, a sialic acid-binding immunoglobulin-type lectin (siglec), and combinations thereof, and / or another binding agent that may be considered a ligand. In some embodiments, the target of the affinity agent (eg, a biomarker target) is or includes one or more polypeptide, nucleic acid, carbohydrate, and / or lipid moieties and / or entities.

[0074] Agent: Generally, the term "agent," as used herein, is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof [e.g., a cell, tissue, organ]), or a phenomenon (e.g., heat, an electric current or field, a magnetic force or field, etc.). In appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be utilized to refer to an entity that is or includes a cell or organ, or a fraction, extract, or component thereof. Alternatively, or in addition, as the context will make clear, the term may be used to refer to a natural product found in nature and / or obtained from nature. In some instances, again as will be clear from the context, the term may be used to refer to one or more entities that are artificially created, in that they have been designed, engineered, and / or produced by the human hand and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or purified form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as a collection or library that can be screened, for example, to identify or characterize active agents therein. In some cases, the term "agent" may refer to a compound or entity that is or includes a polymer, and in some cases, the term may refer to a compound or entity that includes one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymer moieties.

[0075] Amplification: The terms "amplification" and "amplifying" refer to a temperature-dependent process that results in an increase in the amount and / or level of a nucleic acid molecule compared to its initial amount and / or level.The temperature-dependent process generally involves the temperature-dependent extension of a primer molecule, and the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, JD et al., In: Molecular Biology of the Gene, 4th Ed., WA Benjamin, Inc., Menlo Park, Calif. (1987); for the purposes described herein, it is incorporated herein by reference).

[0076] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, an antibody agent refers to a polypeptide containing sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally occurring intact antibodies are approximately 150 kD tetrameric agents composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other in a commonly referred to "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domains, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding characteristics of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains that have been modified or engineered, such as glycosylated. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes a sufficient immunoglobulin domain sequence found in a natural antibody can be referred to and / or used as an "antibody," whether such polypeptide is naturally produced (e.g., made by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of rabbit, rodent (e.g., mouse, rat, hamster, etc.), camelid (e.g., llama, alpaca), ovine, caprine, bovine, equine, chicken, donkey, shark, primate, human, or in vitro-derived (e.g., yeast, phage) antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Additionally, the term "antibody," as used herein, can, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations.For example, in some embodiments, the antibody utilized in accordance with the present invention is in a format selected from, but not limited to, an IgA, IgG, IgE, or IgM antibody; a bi- or multispecific antibody (e.g., Zybodies®, etc.); an antibody fragment, such as a Fab fragment, a Fab fragment, a F(ab')2 fragment, an Fd fragment, and an isolated CDR or set thereof; a single-chain Fv; a polypeptide-Fc fusion; a single domain antibody, an alternative scaffold, or an antibody mimetic (e.g., anticalins, FN3 monobodies, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Fynomers, Im7, VLR, VNAR, Trimab, CrossMab, Trident); a nanobody, a binanobody, a di-sdFv, a single domain antibody, a trifunctional antibody, a diabody, and a minibody. In some embodiments, the relevant format may be or include Adnectins®; Affibodies®; Affilins®; Anticalins®; Avimers®; BiTEs®; camelized antibodies; Centyrins®; ankyrin repeat proteins or DARPINs®; dual affinity retargeting (DART) agents; Fynomers®; shark single domain antibodies, e.g., IgNAR; immune mobilizing monoclonal T cell receptors against cancer (ImmTACs); KALBITOR®; MicroProteins; Nanobodies® minibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); TCR-like antibodies; Trans-bodies®; TrimerX®; VHHs. In some embodiments, the antibody may lack covalent modifications (eg, glycan attachments) that it would have if produced in nature.In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.) or other pendant group (e.g., polyethylene glycol, etc.).

[0077] Antigen: As used herein, the term "antigen" refers to an entity (e.g., a molecule or molecular structure, such as a peptide or protein, carbohydrate, lipid particle, oligonucleotide, chemical molecule, or combination thereof) that contains one or more epitopes and thus recognizes and binds to an affinity agent (e.g., an antibody, affimer, or aptamer).

[0078] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. Generally, a person skilled in the art familiar with the context will recognize the relevant degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0079] Aptamer: As used herein, the term "aptamer" typically refers to a nucleic acid or peptide molecule that binds to a specific target molecule (e.g., an epitope). In some embodiments, nucleic acid aptamers may be described by their nucleotide sequence and are typically about 15-60 nucleotides in length. Nucleic acid aptamers may be or include single-stranded and / or double-stranded structures. In some embodiments, nucleic acid aptamers may be or include DNA. In some embodiments, nucleic acid aptamers may be or include RNA. While not wishing to be bound by any theory, it is contemplated that the strands of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, which allows the aptamer to bind tightly to the surface of its target molecule. In some embodiments, peptide aptamers may be described as having one or more peptide loops of variable sequence displayed by a protein scaffold. Peptide aptamers can be isolated from combinatorial libraries and often subsequently improved by rounds of directed mutation or variable region mutagenesis and selection. Given the extraordinary diversity of molecular shapes present within the universe of all possible nucleotide and / or peptide sequences, aptamers can be obtained for a wide range of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers typically have very high affinity for their targets (e.g., picomolar to low nanomolar range for proteins or polypeptides). Because aptamers are typically synthetic molecules, they are amenable to various modifications, which can optimize their function for specific applications.

[0080] Associated with: As used herein, two events or entities are "associated" with each other when the presence, level, and / or form of one correlates with that of the other. For example, a particular biological phenomenon (e.g., expression of a specific biomarker) is considered to be associated with prostate cancer (e.g., a specific type of prostate cancer (e.g., prostate adenocarcinoma) and / or a stage of prostate cancer) if its presence correlates with the occurrence and / or susceptibility of prostate cancer (e.g., an entire relevant population).

[0081] Biological entity: Where appropriate, as will be clear from context to one of skill in the art, the term "biological entity" may, in some embodiments, be or include a cell or organism, e.g., an animal or human, or, in some embodiments, may be or include a biological tissue or biological fluid. For example, in some embodiments, the term may be utilized to refer to an entity or component present in a biological sample derived from or obtained from a subject. In some embodiments, the biological entity is or includes a cell or microorganism, or a fraction, extract, or component thereof (e.g., including intracellular components and / or molecules secreted by the cell or microorganism). For example, in some embodiments, the biological entity is or includes a cell. In some embodiments, the biological entity is or includes a nanoparticle having a size in the range of about 30 nm to about 1000 nm, which, in some embodiments, is obtained from a subject's bodily fluid sample (e.g., without limitation, a blood sample, a urine sample, etc.). In some embodiments, such nanoparticles may be or include, for example, protein aggregates, including, in some embodiments, glycans and / or extracellular vesicles. In some embodiments, such nanoparticles may have a size within the range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. In some embodiments, the biological entity is or comprises an extracellular vesicle. In some embodiments, the biological entity is or comprises a biological analyte (e.g., a metabolite, carbohydrate, protein or polypeptide, enzyme, lipid, organelle, cytokine, receptor, ligand, and any combination thereof). In some embodiments, the biological entity present in the sample is in its native state (e.g., a protein or polypeptide is retained in a naturally occurring conformational structure). In some embodiments, the biological entity is processed, for example, by isolating it from the sample or by derivatizing it from a naturally occurring biological entity.For example, the biological entity can be treated with one or more chemical agents to make it more desirable for detection using the techniques provided herein. By way of example only, the biological entity may be a cell or extracellular vesicle that has been contacted with a fixative (e.g., but not limited to, methanol and / or formaldehyde) to cause crosslinking of proteins and / or peptides present in the cell or extracellular vesicle. In some embodiments, the biological entity is in an isolated or pure form (e.g., isolated from a body fluid sample, such as a blood, serum, plasma, or urine sample). In some embodiments, the biological entity may be present in a complex matrix (e.g., a body fluid sample, such as a blood, serum, plasma, or urine sample).

[0082] Biomarker: The term "biomarker" typically refers to an entity, event, or characteristic whose presence, level, degree, type, and / or form correlates with a particular biological event or state of interest and is therefore considered to be a "marker" for that event or state. To give a few examples, in some embodiments, a biomarker may be or include a marker for a particular disease state or the likelihood that a particular disease, disorder, or condition will occur, occur, or recur. In some embodiments, a biomarker may be or include a marker for a particular disease or treatment outcome or the likelihood thereof. In some embodiments, a biomarker may be or include a marker for a particular tissue (e.g., without limitation, brain, breast, colon, pancreas, prostate and / or other tissues associated with the male reproductive system, liver, lung, and skin). Such markers for a particular tissue may, in some embodiments, be specific to healthy tissue, specific to diseased tissue, or, in some embodiments, present in normal, healthy, and diseased tissue (e.g., tumors); one of skill in the art reading this disclosure will recognize the appropriate context for each such type of biomarker. In some embodiments, a biomarker may be or include a cancer-specific marker (e.g., a marker that is specific for a particular cancer). In some embodiments, a biomarker may be or include a non-specific cancer marker (e.g., a marker that is present in at least two or more cancers). A non-specific cancer marker may, in some embodiments, be or include a general marker for cancer (e.g., a marker that is typically present in cancers regardless of tissue type), or, in some embodiments, a marker for cancer of a specific tissue (e.g., but not limited to, the brain, breast, colon, pancreas, prostate and / or other tissues associated with the male reproductive system, liver, lung, and skin).Thus, in some embodiments, biomarkers are predictive, in some embodiments, biomarkers are prognostic, and in some embodiments, biomarkers are diagnostic for relevant biological events or conditions of interest. Biomarkers may be or include any chemical class of entity, or may be or include a combination of entities. For example, in some embodiments, biomarkers may be or include nucleic acids, polypeptides, lipids, carbohydrates, small molecules, inorganic agents (e.g., metals or ions), or combinations thereof. In some embodiments, biomarkers are or include portions of specific molecules, complexes, or structures; for example, in some embodiments, biomarkers may be or include epitopes. In some embodiments, biomarkers are surface markers (e.g., surface protein markers) of extracellular vesicles associated with prostate cancer (e.g., prostate adenocarcinoma). In some embodiments, biomarkers are intravesicular (e.g., protein or RNA markers present in extracellular vesicles). In some embodiments, biomarkers may be or include genetic or epigenetic signatures. In some embodiments, a biomarker may be or include a gene expression signature. In some embodiments, a "biomarker" suitable for use in accordance with the present disclosure may refer to the presence, level, and / or form of a molecular entity (e.g., an epitope) present on a target marker. For example, in some embodiments, two or more "biomarkers" (e.g., epitopes) as molecular entities may be present on the same target marker (e.g., a marker protein, e.g., a surface protein present on extracellular vesicles).

[0083] Blood-derived sample: As used herein, the term "blood-derived sample" refers to a sample derived from a blood sample (i.e., a whole blood sample) of a subject in need thereof. Examples of blood-derived samples include, but are not limited to, plasma (including, e.g., fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood fractions including red blood cells (RBCs), platelets, white blood cells, etc., and cell lysates comprising the fractions (e.g., cells, e.g., red blood cells, white blood cells, etc., can be collected and lysed to obtain cell lysates). In some embodiments, the blood-derived sample used in the methods, systems, and / or kits described herein is a plasma sample.

[0084] Cancer: The term "cancer" is used herein generally to refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer can include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure provides techniques for the detection of prostate cancer (including, e.g., prostate adenocarcinoma).

[0085] Capture assay: As used herein, the term "capture assay" refers to a process for isolating or separating a biological entity of interest from a sample (e.g., in some embodiments, a bodily fluid-derived sample). In some embodiments, the biological entity of interest is isolated or separated from a sample (e.g., in some embodiments, a bodily fluid-derived sample) using a capture probe described herein. In some embodiments, the biological entity of interest that binds to a capture probe described herein is subjected to a detection assay described herein. In some embodiments, the biological entity of interest suitable for the capture assay described herein is or comprises a nanoparticle having a size range of interest, including extracellular vesicles. In some embodiments, such nanoparticles may have a size within a range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. In some embodiments, the biological entity of interest suitable for the capture assay described herein is or comprises an extracellular vesicle of interest (e.g., in some embodiments, an exosome).

[0086] Capture probe: As used herein, the term "capture probe" refers to a capture agent for capturing a biological entity of interest from a sample (e.g., in some embodiments, a bodily fluid-derived sample). In many embodiments described herein, the capture agent comprises at least one target capture moiety that binds to a surface polypeptide of the biological entity of interest. In some embodiments, such a biological entity of interest is or comprises a nanoparticle having a size range of interest, including extracellular vesicles. In some embodiments, such nanoparticles may have a size within a range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. In some embodiments, such a biological entity of interest comprises an extracellular vesicle (e.g., in some embodiments, an exosome). In some embodiments, the capture agent comprises at least one target moiety that binds to a surface biomarker (e.g., as described herein) of a nanoparticle having a size within a range of about 30 nm to about 1000 nm, including extracellular vesicles (e.g., in some embodiments, an exosome). In some embodiments, the target capture moiety of the capture agent is or comprises an affinity agent described herein. In some embodiments, the target capture moiety of the capture agent is or comprises an antibody agent. In some embodiments, the target capture moiety of the capture agent is or comprises a lectin or a sialic acid-binding immunoglobulin-type lectin. In some embodiments, the capture agent may comprise a solid substrate to which the target capture moiety is immobilized. In some embodiments, an exemplary solid substrate is a bead (e.g., a magnetic bead). In some embodiments, the capture probe is or comprises a population of magnetic beads comprising a target capture moiety that specifically binds to a surface biomarker described herein.

[0087] Classification cutoff: As used herein, the term "classification cutoff" refers to a level, value, or score, or set of values, or index used to predict a subject's risk for a disease or condition (e.g., prostate adenocarcinoma), for example, by defining one or more boundary lines among two or more subsets of a population (e.g., normal healthy subjects and subjects with an inflammatory condition versus subjects with prostate adenocarcinoma). In some embodiments, the classification cutoff may be determined with reference to at least one reference threshold level (e.g., reference cutoff) for a target biomarker signature described herein, optionally in combination with other appropriate variables, such as the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition. In some embodiments where classification is based on a single target biomarker signature (e.g., as described herein), the classification cutoff may be the same as a predetermined reference threshold (e.g., cutoff) for the single target biomarker signature. In some embodiments where the classification is based on two or more (e.g., 2, 3, 4, or more) target biomarker signatures, the classification cutoff may refer to two or more reference thresholds (e.g., cutoffs) each individually predetermined for the corresponding target biomarker signatures, and may optionally incorporate one or more appropriate variables, e.g., the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition. In some embodiments, the classification cutoff may be determined via computer algorithm-mediated analysis that references at least one reference threshold level (e.g., reference cutoff) for the target biomarker signatures described herein, in combination with other appropriate variables, e.g., the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition.

[0088] Proximity: As used herein, the term "proximity" refers to a distance between two detection probes (e.g., two detection probes in a pair) that is sufficiently close that interaction between the detection probes (e.g., via their individual oligonucleotide domains) is likely to occur. For example, in some embodiments, the probability that two detection probes will interact with each other (e.g., via their individual oligonucleotide domains) over a period of time is at least 50% or higher, including, for example, at least 60%, at least 70%, at least 80%, at least 90%, or higher, when they are sufficiently close to each other under defined conditions (e.g., when the detection probes bind to their respective targets in extracellular vesicles). In some embodiments, the distance between two detection probes may range from approximately 0.1 to 1000 nm, or from 0.5 to 500 nm, or from 1 to 250 nm, when they are sufficiently close to each other. In some embodiments, the distance between two detection probes may range from approximately 0.1 to 10 nm, or from approximately 0.5 to 5 nm, when they are sufficiently close to each other. In some embodiments, the distance between two detection probes, when they are sufficiently close to each other, may be less than 100 nm or less, including, for example, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, less than 1 nm, or less than 100 nm. In some embodiments, the distance between two detection probes, when they are sufficiently close to each other, may range from approximately 40 to 1000 nm, or between 40 nm and 500 nm.

[0089] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, such that a person of ordinary skill in the art would recognize that they could reasonably draw conclusions based on the differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by one or a fewer number of a number of substantially identical traits and a variety of traits. A person of ordinary skill in the art will understand, in context, the degree of identity required for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable in any given situation. For example, a person of ordinary skill in the art will recognize that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and variety of substantially identical traits to warrant a reasonable conclusion that differences in results obtained under, or phenomena observed using, different sets of circumstances, individuals, or populations are caused by or indicate variations in the altered traits.

[0090] Complementary: As used herein, the term "complementary" in the context of nucleic acid base pairing refers to oligonucleotide hybridization related by the base-pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA." Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term "complementary," provided that the partial complementarity allows for oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to the base-pairing rules. Total or complete complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base-pairing rules. In the context of identifying a combination of biomarkers for the detection of a particular cancer, the term "complementary" is used herein in reference to sets of biomarkers that have different information content (e.g., the ability to detect cancer in distinct, substantially non-overlapping subgroups of subjects). For example, two sets of biomarkers, Set 1 and Set 2, are said to be "complementary" to one another if, for example, Set 1 detects cancer in a group of subjects in a population (e.g., Group A) and Set 2 detects cancer in a substantially separate and non-overlapping group of subjects in the same population (e.g., Group B), but not in Group A. Similarly, Set 1 does not detect cancer in a significant number of subjects in Group B.

[0091] Detecting: The term "detecting" is used broadly herein to include any suitable means for determining the presence or absence of extracellular vesicles expressing a target biomarker signature for prostate cancer (e.g., prostate adenocarcinoma) or any form of measurement indicative of such extracellular vesicles. Thus, "detecting" can include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) corresponding to a portion of the target biomarker signature in any manner. In some embodiments, "detecting" can include determining, measuring, assessing, or quantifying a form of measurement indicative of the entity of interest (e.g., a ligated template indicative of a surface biomarker and / or an intravesicular biomarker, or a PCR amplification product indicative of intravesicular mRNA). Quantitative and qualitative determinations, measurements, or assessments are included, including semi-quantitative determinations, measurements, or assessments. Such determination, measurement, or assessment can be relative, for example, when the entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof is detected relative to a control reference, or absolute. Thus, the term "quantifying," when used in the context of quantifying an entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof, can refer to absolute or relative quantification. Absolute quantification can be achieved by correlating the detected level of, or a form of measurement indicative of, the entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) to a known control standard (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the levels or amounts detected between two or more different entities of interest (e.g., different surface biomarkers, intravesicular biomarkers, or intravesicular RNA biomarkers) to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0092] Detection Label: The term "detection label," as used herein, refers to any element, molecule, functional group, compound, fragment, or moiety that is detectable. In some embodiments, a detection label is provided or utilized alone. In some embodiments, a detection label is provided and / or utilized in association with (e.g., conjugated to) another agent. Examples of detection labels include, but are not limited to, various ligands, radionuclides (e.g., 3 H, 14 C. 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (such as acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0093] Detection probe: The term "detection probe" typically refers to a probe intended for the detection and / or quantification of a specific target. In some embodiments, a detection probe is a quantification probe that provides an indication of the level of a specific target. According to the present disclosure, a detection probe refers to a composition comprising a target-binding entity coupled directly or indirectly to an oligonucleotide domain, where the target-binding entity specifically binds to an individual target (e.g., a molecular target), and at least a portion of the oligonucleotide domain is designed to allow hybridization with a portion of the oligonucleotide domain of another detection probe for a distinct target. In many embodiments, oligonucleotide domains suitable for use according to the present disclosure comprise a double-stranded portion and at least one single-stranded overhang. In some embodiments, the oligonucleotide domain may comprise a double-stranded portion and a single-stranded overhang at each end of the double-stranded portion. In some embodiments, the target-binding entity of the detection probe is or comprises an affinity agent described herein. In some embodiments, the target-binding entity of the detection probe is or comprises an antibody agent. In some embodiments, the target binding entity of the detection probe is or comprises a lectin or a sialic acid-binding immunoglobulin-type lectin (siglec).

[0094] Double-stranded: As used herein, the term "double-stranded" in the context of an oligonucleotide domain is understood by those skilled in the art to mean that a pair of oligonucleotides exists in a hydrogen-bonded helical configuration, typically in association with a nucleic acid such as, for example, DNA. In addition to 100% complementary forms of double-stranded oligonucleotides, the term "double-stranded," as used herein, is also meant to refer to those forms that contain mismatches (e.g., partial complementarity) and / or structural features such as bulges, loops, or hairpins.

[0095] Double-stranded complex: As used herein, the term "double-stranded complex" typically refers to a complex comprising at least two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) detection probes (e.g., as provided and / or utilized herein) each directed to a target (which may be the same target or distinct targets) connected or coupled to each other in a linear arrangement by hybridization of complementary single-stranded overhangs of the detection probes. In some embodiments, such double-stranded complexes may comprise extracellular vesicles, and the individual target-binding portions of the detection probes are simultaneously bound to the extracellular vesicles.

[0096] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized by an affinity agent (for example, but not limited to, an antibody, affimer, and / or aptamer). In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms that are groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0097] Extracellular vesicles: As used herein, the term "extracellular vesicles" typically refers to vesicles outside cells, e.g., secreted by cells. Examples of secreted vesicles include, but are not limited to, exosomes, microvesicles, microparticles, ectosomes, oncosomes, and apoptotic bodies. Without wishing to be bound by theory, exosomes are nanometer-sized vesicles (e.g., 40 nm to 120 nm) of intracellular origin that can be formed by budding of multivesicular endosomes (MVEs) into the inner side of the limiting membrane, while microvesicles typically bud from the cell surface and their size can vary from 50 nm to 1000 nm. In some embodiments, extracellular vesicles are or include exosomes and / or microvesicles. In some embodiments, a sample containing extracellular vesicles is substantially free of apoptotic bodies. In some embodiments, a sample containing extracellular vesicles may include extracellular vesicles shed from or derived from one or more tissues (e.g., cancerous tissue and / or non-cancerous or healthy tissue). In some embodiments, the extracellular vesicles in the sample may be shed from or derived from a prostate adenocarcinoma tumor, and in some embodiments, the extracellular vesicles are shed from or derived from a non-prostate adenocarcinoma tumor. In some embodiments, the extracellular vesicles are shed from or derived from healthy tissue. In some embodiments, the extracellular vesicles are shed from or derived from a benign prostate tumor. In some embodiments, the extracellular vesicles are shed from or derived from tissue of a subject with symptoms associated with prostate adenocarcinoma (e.g., non-specific symptoms).

[0098] Extracellular vesicle-associated membrane-bound polypeptide: As used herein, this term refers to a polypeptide present in the membrane of an extracellular vesicle. In some embodiments, such a biomarker may be associated with the extracellular side of the membrane. In some embodiments, such a polypeptide may be tumor-specific. In some embodiments, such a polypeptide may be tissue-specific (e.g., prostate tissue-specific). In some embodiments, such a polypeptide may be non-specific, e.g., it is present in one or more non-target tumors and / or one or more non-target tissues.

[0099] Hybridization: As used herein, the terms "hybridizing," "hybridizing," "hybridization," "annealing," or "annealing" are used interchangeably to refer to the pairing of complementary nucleic acids using any process in which one strand of nucleic acid is joined to a complementary strand by base pairing to form a hybridized complex. Hybridization and the strength of hybridization (e.g., the strength of the association between nucleic acids) are affected by various factors, including, for example, the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the melting temperature (T) of the hybridization complex formed, and the G:C ratio within the nucleic acids.

[0100] Intravesicular protein biomarker: As used herein, the term "intravesicular protein biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a polypeptide present within a biological entity (e.g., a cell or extracellular vesicle). In many embodiments, the intravesicular protein biomarker is associated with or present within an extracellular vesicle. In many embodiments, the intravesicular protein biomarker may be post-translationally modified in a reversible (e.g., phosphorylation) or irreversible (e.g., cleavage) manner. In some embodiments, the intravesicular protein biomarker may be or comprise a phosphorylated polypeptide. In some embodiments, the intravesicular protein biomarker may be or comprise a mutant polypeptide.

[0101] Intravesicular RNA biomarker: As used herein, the term "intravesicular RNA biomarker" refers to a marker that indicates the state (e.g., presence and / or level) of RNA present in a biological entity (e.g., a cell or extracellular vesicle). In many embodiments, the intravesicular RNA biomarker is associated with or present within an extracellular vesicle. In some embodiments, the intravesicular RNA biomarker is associated with or specific to cancer. In some embodiments, the intravesicular RNA biomarker is or comprises an mRNA transcript. In some embodiments, the intravesicular RNA biomarker is or comprises a non-coding RNA. Exemplary non-coding RNAs may include, but are not limited to, small nuclear RNAs, microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), small non-coding RNAs, piwi-interacting RNAs, etc. Certain RNA biomarkers for cancer are described in the prior art, for example, Xi et al. "RNA Biomarkers: Frontier of Precision Medicine for Cancer" Noncoding RNA (2017) 3:9, the contents of which are incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or includes an orphan noncoding RNA (oncRNA).Certain cancer-specific oncRNAs have been identified and described in references, for example, Teng et al. "Orphan noncoding RNAs: novel regulators and cancer biomarkers" Ann Transl Med (2019) 7:S21; Fish et al. "Cancer cells exploit an orphan RNA to drive metastatic progression" Nature Medicine (2018) 24: 1743-1751; and International Patent Publication No. WO2019 / 094780, each of which is incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or includes a long noncoding RNA. Certain non-coding RNA biomarkers for cancer have been described in the art, for example, as described in Qian et al. "Long Non-coding RNAs in Cancer: Implications for Diagnosis, Prognosis, and Therapy" Front. Med. (2020) Volume 7, Article 612393, the contents of which are incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or comprises a piwiRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a miRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a snoRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a circRNA.

[0102] Ligase: As used herein, the term "ligase" or "nucleic acid ligase" refers to an enzyme for use in ligating nucleic acids. In some embodiments, the ligase is an enzyme for use in ligating the 3' end of a polynucleotide to the 5' end of a polynucleotide. In some embodiments, the ligase is an enzyme for use in performing sticky end ligation. In some embodiments, the ligase is an enzyme for use in performing blunt end ligation. In some embodiments, the ligase is or comprises a DNA ligase.

[0103] Life history-related risk factors: As used herein, the term "life history risk factors" refers to the activities, experiences, medical history, and / or exposures of an individual in their life that may directly or indirectly increase the individual's risk of a condition, such as cancer, for example, prostate adenocarcinoma, compared to individuals who do not have such activities, experiences, medical history, and / or exposures in their lives. In some embodiments, non-limiting examples of life history-related risk factors include smoking, alcohol, drugs, carcinogens, diet, obesity, diabetes, physical activity, sun exposure, radiation exposure, bitumen smoke exposure, exposure to infectious agents such as viruses and bacteria, and / or occupational hazards (Reid et al., 2017; which is incorporated herein by reference for purposes described herein). Those skilled in the art will recognize that the above list of life history-related risk factors that contribute to cancer (e.g., prostate adenocarcinoma) susceptibility is not exhaustive and is constantly evolving.

[0104] Ligation: As used herein, the terms "ligate," "ligating," or "ligation" refer to methods or compositions known in the art for joining two oligonucleotides or polynucleotides. Ligation may be or include sticky end ligation or blunt end ligation. In some embodiments, the ligation involved in the provided techniques is or includes sticky end ligation. In some embodiments, ligation refers to joining the 3' end of a polynucleotide to the 5' end of a polynucleotide. In some embodiments, ligation is facilitated by the use of a nucleic acid ligase.

[0105] Nanoparticles: The term "nanoparticles," when used in the context of a sample for a detection assay (e.g., as described herein), refers to particles having a size ranging from about 30 nm to about 1000 nm. In some embodiments, nanoparticles have a size ranging from about 30 nm to about 750 nm. In some embodiments, nanoparticles have a size ranging from about 50 nm to about 750 nm. In some embodiments, nanoparticles have a size ranging from about 30 nm to about 500 nm. In some embodiments, nanoparticles have a size ranging from about 50 nm to about 500 nm. In some embodiments, nanoparticles are obtained from a subject's bodily fluid sample, for example, in some embodiments, by a size-exclusion-based method (e.g., in some embodiments, size-exclusion chromatography). In some embodiments, nanoparticles are or comprise analyte aggregates, which in some embodiments may be or comprise protein or mucin aggregates. In some embodiments, nanoparticles are or comprise protein multimers. In some embodiments, nanoparticles are or comprise extracellular vesicles.

[0106] Non-cancer subject: As used herein, the term "non-cancer subject" generally refers to a subject that does not have non-benign prostate cancer, more specifically, prostate adenocarcinoma. For example, in some embodiments, the non-cancer subject is a healthy subject. In some embodiments, the non-cancer subject is a healthy subject under the age of 55. In some embodiments, the non-cancer subject is a healthy subject aged 55 or older. In some embodiments, the non-cancer subject is a subject with a non-prostate-related health disease, disorder, or condition. In some embodiments, the non-cancer subject is a subject with benign prostatic hyperplasia.

[0107] Nucleic Acid / Oligonucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof).In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid is isolated from a natural source, enzymatically synthesized (e.g., in vivo or in vivo). It may be prepared in vitro, for example, by polymerization based on a complementary template, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 20 In some embodiments, the amino acid sequence may be 00, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0108] Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used, for example, as an indicator of the size of an oligonucleotide, a particular number of nucleotides refers, for example, to the number of nucleotides on a single strand of the oligonucleotide.

[0109] Patient: As used herein, the term "patient" refers to any organism suffering from or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition amenable to the provided techniques is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy for diagnosing and / or treating the disease, disorder, or condition.

[0110] Polypeptide: As used herein, the term "polypeptide" typically has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will recognize that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). In some embodiments, polypeptides may comprise L-amino acids, D-amino acids, or both, and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, glycosylation, methylation, and the like. In some embodiments, polypeptides may comprise (e.g., be or include) natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0111] Prevent or Prevention: As used herein, "prevent" or "prevention," when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of the disease, disorder, and / or condition occurring and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition has been delayed for a pre-specified period of time.

[0112] Primer: As used herein, the term "primer" refers to an oligonucleotide that can act as a point of initiation of synthesis when placed under conditions that induce the synthesis of a primer extension product that is complementary to a nucleic acid strand (e.g., in the presence of nucleotides and an inducing agent, such as DNA polymerase, and at an appropriate temperature and pH). The primer is preferably single-stranded for maximum efficiency in amplification. The primer must be sufficiently long to prime the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer can depend on many factors, such as the desired annealing temperature.

[0113] Reference: As used herein, "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is, optionally, a historical reference or control embodied in a tangible medium. In some embodiments, a reference or control in the context of a target reference level refers to the level of the target in a normal, healthy subject or a population of normal, healthy subjects. In some embodiments, a reference or control in the context of a target reference level refers to the level of the target in a subject before treatment. Typically, as will be understood by one of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being assessed. In some embodiments, cell line-derived extracellular vesicles are used as a reference or control. One of skill in the art will recognize when there is sufficient similarity to justify the reliability of and / or comparison to a particular possible reference or control.

[0114] Risk: As will be understood from the context, "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, up to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is from an individual comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher.

[0115] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a sample is obtained from or derived from a biological source of interest (e.g., a tissue or organism or cell culture). In some embodiments, the source of interest may be or include a cell or organism, e.g., an animal or human. In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, stool, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravesicular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological tissue or biological sample may be obtained by, for example, an aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or irrigation (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage or irrigation). In some embodiments, the biological sample is or comprises a bodily fluid sample or a bodily fluid-derived sample. Examples of bodily fluids include, but are not limited to, amniotic fluid, bile, blood, breast milk, bronchoalveolar lavage (BAL), cerebrospinal fluid, dialysate, stool, saliva, semen, synovial fluid, tears, urine, etc. In some embodiments, the biological sample is or comprises a liquid biopsy. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means.In some embodiments, as is expected to be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or adding one or more agents). For example, a sample is a preparation that has been processed by using a semipermeable membrane or an affinity-based method, such as an antibody-based method, to separate a biological entity of interest from other non-target entities. Such a "processed sample" may, for example, in some embodiments, include extracellular vesicles, while in some embodiments, it may also include nucleic acids and / or proteins extracted from the sample. In some embodiments, a processed sample may be obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc.

[0116] Selective or specific: The terms "selective" or "specific," as used herein with respect to an active agent, are understood by those skilled in the art to mean that the agent discriminates between potential target entities, conditions, or cells. For example, in some embodiments, an agent is said to "specifically" bind to a target if it preferentially binds to that target in the presence of one or more competing surrogate targets. In many embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of the target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding moiety. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding moiety. In some embodiments, the target-binding moiety does not detectably bind to a competing surrogate target under conditions that bind to the target entity. In some embodiments, the target binding moiety binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to a competing surrogate target.

[0117] Small molecule: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not comprise a polymer moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is biologically active. In some embodiments, suitable small molecules are selected from a wide variety of methods, including screening of large libraries of compounds (Beck-Sickinger & Weber (2001) Combinational Strategies in Biology and Chemistry (John Wiley & Sons, Chichester, Sussex); structure-activity relationships by nuclear magnetic resonance (Shuker et al. (1996) "Discovering high-affinity ligands for proteins: SAR by NMR." Science 274: 1531-1534); encoded self-assembling chemical libraries (Melkko et al. (2004) "Encoded self-assembling chemical libraries." Nature Biotechnol. 22: 568-574); DNA-templated chemistry (Gartner et al.(2004) "DNA-templated organic synthesis and selection of a library of macrocycles." Science 305: 1601-1605); dynamic combinatorial chemistry (Ramstrom & Lehn (2002) "Drug discovery by dynamic combinatorial libraries." Nature Rev. Drug Discov. 1: 26-36); tethering (Arkin & Wells (2004) "Small-molecule inhibitors of protein-protein interactions: progressing towards the dream." Nature Rev. Drug Discov. 3: 301-317); and speed screening (Muckenschnabel et al. (2004) "SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands." Anal. Biochem. 324: 241-249). In some embodiments, the small molecule may have a dissociation constant for the target in the nanomolar range. .

[0118] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in an environment in which binding occurs. A target-binding moiety that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the extent of association between the target-binding moiety and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the target-binding moiety-partner complex; and in some embodiments, specific binding is assessed by detecting or determining the ability of the target-binding moiety to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0119] Cancer stage: As used herein, the term "cancer stage" refers to a qualitative or quantitative assessment of the level of progression of a cancer (e.g., prostate adenocarcinoma). In some embodiments, the criteria used to determine the stage of a cancer may include, but are not limited to, one or more of the following: where the cancer is located in the body; tumor size; whether the cancer has spread to lymph nodes; whether the cancer has spread to one or more different body parts; and the like. In some embodiments, cancer may be staged using the AJCC staging system. The AJCC staging system is a classification system developed by the American Joint Committee on Cancer to describe the degree of disease progression in cancer patients, which utilizes, in part, the TNM scoring system: tumor size, affected lymph nodes, and metastases. In some embodiments, cancer may be staged using a classification system that includes, in part, the TNM scoring system, where T refers to the size and extent of the main tumor, usually called the primary tumor; N refers to the number of nearby lymph nodes that have cancer; and M refers to whether the cancer has metastasized. In some embodiments, cancer may be referred to as Stage 0 (abnormal cells are present but have not spread to nearby tissues, also known as intramucosal carcinoma or CIS; CIS is not cancer but may become cancer), Stage I-III (cancer is present; the higher the number, the larger the tumor and the more it has spread to nearby tissues), or Stage IV (cancer has spread to distant parts of the body). In some embodiments, cancer may be designated a stage selected from the group consisting of in situ (abnormal cells are present but have not spread to nearby tissues); localized (cancer is limited to where it started and there are no signs of spread); regional (cancer has spread to nearby lymph nodes, tissues, or organs); distant metastasis (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage). In some embodiments, prostate cancer (e.g., prostate adenocarcinoma) may be assigned a Gleason score, which is a measure of how likely the cancer is to grow and spread.In some embodiments, the Gleason score is determined from a prostate biopsy or surgery. In some embodiments, the Gleason score may include a score of 1 to 5, and those skilled in the art will know how to assign a Gleason score to prostate cancer (e.g., prostate adenocarcinoma) based on histological patterns.

[0120] Subject: As used herein, the term "subject" refers to an organism from which a sample is obtained, e.g., for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject, e.g., a human male subject. In some embodiments, the subject is afflicted with prostate adenocarcinoma. In some embodiments, the subject is predisposed to prostate adenocarcinoma. In some embodiments, the subject exhibits one or more symptoms or characteristics of prostate adenocarcinoma. In some embodiments, the subject exhibits one or more non-specific symptoms of prostate adenocarcinoma. In some embodiments, the subject does not exhibit any symptoms or characteristics of prostate adenocarcinoma. In some embodiments, the subject is a subject with one or more features characteristic of susceptibility to or risk for prostate adenocarcinoma. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnostic and / or therapy is to be administered. In some embodiments, the subject is an asymptomatic subject. Such asymptomatic subjects may be those at average population risk or those at genetic risk. For example, such asymptomatic subjects may be those with a family history of cancer, those previously treated for cancer, those at risk of cancer recurrence after cancer treatment, those in remission after cancer treatment, and / or those previously or periodically screened for the presence of at least one cancer biomarker. Alternatively, in some embodiments, asymptomatic subjects may be those who have not previously been screened for cancer, those who have not been diagnosed with cancer, and / or those who have not previously undergone cancer therapy. In some embodiments, subjects suitable for the provided technology are individuals selected based on one or more characteristics, such as age, race, geographic location, genetic history, medical history, personal history (e.g., smoking, alcohol, drugs, carcinogens, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and / or occupational hazards).

[0121] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0122] Surface analyte: As used herein, "surface analyte" refers to an analyte present on the surface of a biological entity (e.g., a cell or nanoparticle from a biological sample). In some embodiments, the surface analyte is or comprises a surface polypeptide or surface protein. In some embodiments, the surface analyte is or comprises a glycan.

[0123] Surface biomarker: As used herein, "surface biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a surface analyte (e.g., as described herein) of a biological entity (e.g., a cell or nanoparticle, which in some embodiments includes analyte aggregates (e.g., protein or mucin aggregates) and / or extracellular vesicles). In some embodiments, the surface biomarker is or comprises a surface protein biomarker. In some embodiments, the surface biomarker is or comprises a carbohydrate-dependent marker.

[0124] Surface polypeptide or surface protein: As used interchangeably herein, the terms "surface polypeptide" and "surface protein" refer to a polypeptide or protein that is present in and / or on the surface of a biological entity (e.g., a cell or nanoparticle, including, in some embodiments, analyte aggregates (e.g., protein or mucin aggregates) and / or extracellular vesicles, etc.) through direct or indirect interactions. As will be understood by one of skill in the art, a surface protein may, in some embodiments, include post-translational modifications, including, for example, but not limited to, glycosylation. In some embodiments, a surface polypeptide or protein may be or include a membrane-bound polypeptide. In some embodiments, a membrane-bound polypeptide refers to a polypeptide or protein having one or more domains or regions that are present in or on the surface of the membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, a membrane-bound polypeptide may include one or more domains or regions that span and / or are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions that span and / or are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.) and project into the intracellular and / or intravesicular space. In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.) via, for example, one or more non-peptide linkages (e.g., by a glycosylphosphatidylinositol (GPI) anchor or lipidation, or by non-covalent interactions). In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions anchored on either side of the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, the surface protein is associated with or present on the surface of a nanoparticle (e.g., as described herein).In some embodiments, the surface protein is associated with or present within an extracellular vesicle. In some embodiments, the surface protein may be associated with or present within a prostate adenocarcinoma-associated extracellular vesicle (e.g., an extracellular vesicle obtained from or derived from a body fluid-derived sample (e.g., but not limited to, a blood-derived sample, a urine-derived sample, etc.) of a subject suffering from or susceptible to prostate adenocarcinoma). As will be understood by those skilled in the art, detecting the presence of at least a portion of a surface polypeptide or surface protein on / in an extracellular vesicle can facilitate separation and / or isolation of prostate adenocarcinoma-associated extracellular vesicles from a biological sample (e.g., a blood sample or a blood-derived sample) from a subject. In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detection of an intravesicular portion (e.g., an intravesicular epitope) of such a surface polypeptide or surface protein. In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detection of a membrane-spanning portion of such a surface polypeptide or surface protein. In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detecting the extravesicular portion of such a surface polypeptide or surface protein.

[0125] Surface protein biomarker: As used herein, the term "surface protein biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a surface protein (e.g., as described herein) of a biological entity (e.g., a cell or nanoparticle, which in some embodiments comprises an analyte aggregate (e.g., a protein or mucin aggregate) and / or an extracellular vesicle). In some embodiments, a surface protein refers to a polypeptide or protein having one or more domains or regions located in or on the surface of the membrane of a biological entity (e.g., a cell or extracellular vesicle). In some embodiments, a surface protein biomarker may be or include an epitope present on the inside (intravesicular) or outside (extravesicular) of a membrane. In some embodiments, a surface protein biomarker is associated with or present within an extracellular vesicle. In some embodiments, a surface protein biomarker may be or include a mutated polypeptide. In some embodiments, a surface protein biomarker may be post-translationally modified (e.g., without limitation, glycosylation, phosphorylation, etc.). In some embodiments, the surface protein biomarker may be post-translationally processed and may exist in the form of a truncated polypeptide, for example, as a result of proteolytic cleavage. In some embodiments, the surface protein biomarker may be or include an epitope present on the exterior surface of the nanoparticle.

[0126] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than a member of the general population. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition in the future. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition in the future.

[0127] Target-binding moiety: In general, the terms "target-binding moiety" and "binding moiety" are used interchangeably herein to refer to any entity or moiety that binds to a target of interest (e.g., a molecular target of interest, e.g., a biomarker or epitope). In many embodiments, a target-binding moiety of interest is one that specifically binds to its target (e.g., a target biomarker) in the context of a particular interaction, such that it distinguishes that target from other potential binding partners. In general, a target-binding moiety may be or include an entity or moiety of any chemical class (e.g., polymeric, non-polymeric, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.). In some embodiments, a target-binding moiety is a single chemical entity. In some embodiments, a target-binding moiety is a complex of two or more distinct chemical entities associated with each other under relevant conditions by non-covalent interactions. For example, one skilled in the art will recognize that in some embodiments, a target-binding moiety can include a "general" binding moiety (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamer to a particular molecular target) linked to a generic binding moiety partner. In some embodiments, such an approach can allow for modular assembly of multiple target-binding moieties by linking different specific binding moieties to the generic binding moiety partner.

[0128] Target biomarker signature: The term "target biomarker signature," as used herein, refers to a combination of biomarkers (e.g., at least two or more, including at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) that correlates with a particular biological event or condition of interest, such that one of skill in the art will recognize that this may appropriately be considered a "signature" of that event or condition. To give a few examples, in some embodiments, a target biomarker signature may correlate with a particular disease or disease state and / or the likelihood that a particular disease, disorder, or condition will develop, occur, or recur. In some embodiments, a target biomarker signature may correlate with a particular disease or treatment outcome, or likelihood thereof. In some embodiments, the target biomarker signature may be correlated with a specific cancer and / or stage thereof. In some embodiments, the target biomarker signature may be correlated with prostate adenocarcinoma, and / or its stage and / or subtype.In some embodiments, the target biomarker signature includes a combination of biomarkers (e.g., at least 2 or more, including at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) that are together specific for prostate adenocarcinoma, or a subtype and / or disease state thereof, although one or more biomarkers in such a combination may be directed to a target that is not specific to prostate adenocarcinoma (e.g., a surface biomarker, an intravesicular biomarker, and / or an intravesicular RNA). For example, in some embodiments, a target biomarker signature may include at least one biomarker specific to prostate adenocarcinoma, or a stage and / or subtype thereof (i.e., a prostate adenocarcinoma-specific target), and may further include biomarkers that are not necessarily or completely specific to prostate adenocarcinoma (e.g., that may also be found in some or all biological entities, such as cells, extracellular vesicles, etc., that are not cancerous, are not of related cancers, and / or are not of the particular stage and / or subtype of interest). That is, as will be recognized by one of skill in the art upon reading this specification, so long as the combination of biomarkers utilized in a target biomarker signature is or includes a number of biomarkers that together are specific for the relevant target biological entity of interest (e.g., prostate adenocarcinoma cells of interest, or extracellular vesicles secreted by prostate adenocarcinoma cells) (i.e., sufficiently distinguish the relevant target biological entity for detection (e.g., prostate adenocarcinoma cells of interest, or extracellular vesicles secreted by prostate adenocarcinoma cells) from other biological entities that are not of interest for detection), then such a combination of biomarkers is a useful target biomarker signature according to certain embodiments of the present disclosure.

[0129] Therapeutic Agent: As used interchangeably herein, the phrases "therapeutic agent" or "therapy" refer to an agent or intervention that, when administered to a subject or patient, has a therapeutic effect and / or induces a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0130] Threshold level (e.g., cutoff): As used herein, the term "threshold level" refers to a level used as a reference for obtaining information on and / or classifying the results of a measurement, for example, the results of a measurement obtained in an assay. For example, in some embodiments, the threshold level (e.g., cutoff) refers to a value measured in an assay that defines the boundary between two subsets of a population (e.g., normal and / or non-prostate adenocarcinoma vs. prostate adenocarcinoma). Thus, a value equal to or higher than the threshold level defines one subset of the population, and a value lower than the threshold level defines the other subset of the population. The threshold level can be determined based on one or more control samples, or the entire population of control samples. The threshold level can be determined before, simultaneously with, or after obtaining the measurement of interest. In some embodiments, the threshold level can be a range of values.

[0131] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later stage of a disease, disorder, and / or condition.

[0132] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art, or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for purposes described herein. Detailed Description of Certain Embodiments

[0133] Prostate cancer was responsible for an estimated 191,930 deaths in the United States in 2020 (new case and death estimates for 2020 are projections made by the American Cancer Society (ACS) based on previously reported data; this is incorporated herein by reference for purposes described herein). The majority of these deaths are due to delayed diagnosis; the 5-year overall survival rate for prostate cancer in the United States from 2010 to 2016 was 30.2% for metastatic stage cases. Patients with localized disease at the time of diagnosis had a 100% 5-year survival rate, and 76% of prostate cancers were detected at the localized stage (Figures 6 and 7). In addition, 13% of prostate cancers were detected at the regional stage, and the 5-year survival rate was 100% from 2010 to 2016. This means that any case detected earlier than the metastatic stage virtually guarantees a 5-year survival rate for that individual. Therefore, it is essential to use the techniques disclosed herein for early cancer detection, and more specifically, prostate cancer detection.

[0134] Prostate adenocarcinoma (PRAD) refers to cancer in the prostate, a walnut-shaped organ responsible for producing semen in men. Prostate cancer varies widely in the rate at which it spreads, making it difficult to predict which men will benefit from aggressive treatment. Symptoms of prostate cancer include incontinence and erectile dysfunction.

[0135] A common type of screening for prostate cancer includes a digital rectal examination (DRE), in which a doctor inserts his finger into a patient's rectum to check the feel, shape, and size of the prostate.Another common type of screening is performing prostate-specific antigen (PSA) quantification from blood samples, where an increase in PSA can indicate prostate infection, enlargement, and / or cancer.Testing for prostate cancer-specific markers (such as PCA3) in urine is also used to help diagnose patients for prostate cancer.

[0136] In addition, common post-initial screening techniques and diagnostic methods may include ultrasound, magnetic resonance imaging (MRI), and / or biopsy.Compared with common / existing screening methods, this technology has higher precision and accuracy (e.g., higher signal-to-noise ratio) in early screening, which can reduce the number of false positives and thereby reduce the use of expensive imaging systems.

[0137] The present disclosure identifies the origin of certain prior technology challenges, including, among other things, certain conventional approaches to the detection and diagnosis of prostate cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as digital rectal examination (DRE), urinalysis, and / or CT scanning, can be time-consuming, expensive, and / or lack sufficient sensitivity and / or specificity to provide a reliable, comprehensive diagnostic assessment. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that address such challenges by, among other things, identifying combinations of biomarkers that are predicted to exhibit high sensitivity and specificity for prostate cancer based on bioinformatics analysis. In some embodiments, the present disclosure provides techniques (including systems, compositions, and methods) that solve such problems by detecting co-localization of target biomarker signatures for prostate cancer in individual extracellular vesicles (e.g., identified by bioinformatics analysis), which include at least one extracellular vesicle-associated surface biomarker and at least one target biomarker selected from the group consisting of surface biomarkers, internal protein biomarkers, and RNA biomarkers present on extracellular vesicles associated with prostate cancer. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems by detecting such target biomarker signatures of prostate cancer using, among other things, a target entity detection approach developed by the applicant and described in U.S. Patent Application No. 16 / 805,637 (published as US2020 / 0299780; issued as US11,085,089) and International Application No. PCT / US2020 / 020529 (published as WO2020180741), both filed February 28, 2020, entitled "Systems, Compositions, and Methods for Target Entity Detection," based on the interaction and / or co-localization of target biomarker signatures in individual extracellular vesicles. The contents of each of the foregoing disclosures are incorporated herein by reference in their entirety.

[0138] In some embodiments, extracellular vesicles for detection as described herein can be isolated from a subject's bodily fluids by size-exclusion-based methods. As will be understood by those skilled in the art, in some embodiments, size-exclusion-based methods can provide samples containing nanoparticles of a desired size range, including extracellular vesicles. Thus, in some embodiments, the provided techniques of the present disclosure encompass the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for prostate cancer in individual nanoparticles of a desired size range (e.g., in some embodiments, about 30 nm to about 1000 nm), including extracellular vesicles. Those skilled in the art who read this disclosure will understand that various embodiments described herein in the context of "extracellular vesicles" (e.g., assays for detecting individual extracellular vesicles and / or the provided "extracellular vesicle-associated surface biomarkers") may also be applicable in the context of "nanoparticles" as described herein.

[0139] The present disclosure provides insights and techniques for achieving effective prostate cancer screening, for example, for the early detection of prostate cancer, including, but not limited to, prostate adenocarcinoma, among others. In some embodiments, the present disclosure provides techniques for the early detection of prostate cancer in subjects who may be experiencing another symptom associated with prostate cancer. In some embodiments, the present disclosure provides techniques for the early detection of prostate cancer in subjects who have a genetic risk for prostate cancer. In some embodiments, the present disclosure provides techniques for the early detection of prostate cancer in subjects who may be at genetic risk for prostate cancer and / or who may be experiencing one or more symptoms associated therewith. In some embodiments, the present disclosure provides techniques for the early detection of prostate cancer in subjects who may have lifestyle risk factors. In some embodiments, the present disclosure provides techniques for screening individuals, for example, individuals who have a certain risk (e.g., genetic risk, lifestyle-related risk, or average risk) for early-stage prostate cancer (e.g., prostate adenocarcinoma). In some embodiments, the provided techniques are effective for detecting early-stage prostate cancer (e.g., prostate adenocarcinoma). In some embodiments, the provided techniques are effective when applied to a population that includes or consists of individuals with one or more symptoms that may be associated with prostate cancer.In some embodiments, the provided techniques are effective even when applied to a population that includes or consists of asymptomatic or symptomatic individuals (e.g., due to sufficiently high sensitivity and / or low false positive and / or false negative rates).In some embodiments, the provided techniques are effective when applied to a population that includes or consists of individuals without a genetic risk and / or lifestyle-related risk of developing prostate cancer (e.g., asymptomatic or symptomatic individuals).In some embodiments, the provided techniques are effective when applied to a population that includes or consists of individuals with a genetic risk of developing prostate cancer (e.g., asymptomatic or symptomatic individuals).In some embodiments, the provided technology is effective when applied to populations that include or consist of individuals who are predisposed to prostate cancer (e.g., individuals with known genetic, environmental, or experiential risk, etc.). In some embodiments, the provided technology may be or may include one or more compositions (e.g., molecular complexes, systems, collections, combinations, kits, etc.) and / or methods (e.g., methods of making, using, assessing, etc.), as would be apparent to one of skill in the art upon reading the disclosure provided herein.

[0140] In some embodiments, the provided techniques achieve detection (e.g., early detection, e.g., in asymptomatic individuals and / or populations) of one or more features of prostate cancer (e.g., incidence, progression, response to therapy, recurrence, etc.) with sensitivity and / or specificity (e.g., resulting false positive and / or false negative rates) appropriate to enable useful application of the provided techniques to single and / or regular (e.g., periodic) assessments. In some embodiments, the provided techniques are useful in conjunction with an individual's regular screening, including, but not limited to, physical exams, general practitioner visits, cholesterol / lipid blood tests, urinalysis, diabetes (type 2) screening, colonoscopy, blood pressure screening, thyroid function tests, prostate cancer screening, and / or vaccinations. In some embodiments, the provided techniques are useful in conjunction with treatment regimens, and in some embodiments, the provided techniques may improve one or more characteristics (e.g., success rate by accepted parameters) of such treatment regimens.

[0141] In some embodiments, the present disclosure provides, among other things, insight that screening asymptomatic individuals, e.g., periodic screening before the onset of symptoms or otherwise in their absence, may be beneficial and even important for the effective management (e.g., successful treatment) of prostate cancer. In some embodiments, the present disclosure provides prostate cancer screening systems that can be implemented to detect prostate cancer, including early stage cancers, in some embodiments, in asymptomatic individuals (e.g., without genetic and / or lifestyle-related risk for prostate cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals (e.g., with or without genetic risk for prostate cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of symptomatic individuals (e.g., with or without genetic and / or lifestyle-related risk for prostate cancer). The present disclosure provides compositions (e.g., reagents, kits, components, etc.), including, for example, strategies involving periodic testing of one or more individuals (e.g., asymptomatic individuals), as well as methods of providing and / or using them. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them. I. Prostate Cancer Detection

[0142] Prostate cancer accounts for 10.6% of all new cancer cases in the United States each year, ranking third in new case counts in 2020. In the United States, the annual new case rate for prostate cancer was 109.8 per 100,000 men from 2013 to 2017. The mortality rate was 19 per 100,000 men from 2014 to 2018. The lifetime risk for men developing prostate cancer at any point in their lifetime was 12.1% from 2015 to 2017. Fortunately, mortality rates have decreased by approximately 30% over the past 30 years. However, prostate cancer still accounts for 7.0% of all cancer-related deaths. In 2017, there were an estimated 3,170,339 men living with prostate cancer in the United States (data obtained from seer.cancer.gov, incorporated herein by reference in its entirety).

[0143] Surveillance, Epidemiology and End Results (SEER) data from 2000 to 2017 provide extensive information on the prevalence and epidemiology of prostate cancer in the United States. SEER reported that in 2017, there were 615.3 cases of prostate cancer per 100,000 men aged 65 years or older in the United States. In addition, there were 252.5 cases per 100,000 men aged 50–64 years and 3.7 cases per 100,000 men under 50 years (SEER * Explorer: An interactive website for SEER cancer statistics. Surveillance Research Program, National Cancer Institute. [Cited September 14, 2020]. Available at https: / / seer.cancer.gov / explorer / , which is incorporated herein by reference in its entirety. The technology disclosed herein is designed to address current shortcomings in prostate cancer screening technology.

[0144] According to the CDC and the American Cancer Society, risk factors for prostate cancer (e.g., prostate adenocarcinoma) include age (e.g., risk increases after age 50), race (e.g., prostate cancer occurs more frequently in men of African American descent and is less common in men of Asian or Hispanic descent), geographic location (e.g., prostate cancer is more common in North America, Europe, and Australia and less common in Asia, Africa, and Central and South America), diet (e.g., consumption of high amounts of red meat and a high-fat diet increases risk), obesity, and family history.

[0145] In general, the ingestion of tobacco and tobacco smoke increases the rate of all cancer types. The International Agency for Research on Cancer (IARC) has identified at least 50 known carcinogens in tobacco smoke. Examples of such carcinogens include, but are not limited to, tobacco-specific N-nitrosamines (TSNAs), formed by the nitrosation of nicotine during tobacco processing and smoking. The chemical 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK) is known to induce cancer in experimental animals. NNK is known to bind to DNA, create DNA adducts, and cause DNA damage. Failure to repair this damage can lead to permanent mutations. NNK is associated with DNA mutations that result in the activation of the K-RAS oncogene, which has been detected in humans.

[0146] In some embodiments, the present disclosure provides techniques for effective screening of prostate cancer in individuals with genetic risk or lifestyle-related risk. In some embodiments, the present disclosure provides techniques for effective screening of prostate cancer in average-risk individuals. In some embodiments, the present disclosure provides techniques for effective screening of prostate cancer in individuals with one or more symptoms associated with prostate cancer. In some embodiments, the present disclosure provides techniques for effective screening of prostate cancer in asymptomatic individuals. Despite its relative prevalence in men, there are currently no recommended non-invasive prostate cancer screening tools based on subject blood samples intended for screening asymptomatic and / or average-risk individuals (e.g., individuals under 50 years of age or over 50 years of age). This is due, in part, to cost, limited availability, potential side effects, and / or the poor performance (e.g., high false-positive rates or ineffectiveness) of existing prostate cancer and prostate cancer screening technologies. Given the incidence of prostate cancer in average-risk individuals, inadequate test specificity (<99.5%) can result in false-positive results that exceed the number of true-positive results by more than an order of magnitude. This places a significant burden on the healthcare system and on individuals who are screened with false-positive results, resulting in additional testing, unnecessary surgery, and emotional / physical distress (Wu et al., 2016). In some embodiments, the present disclosure provides the insight that a particularly useful prostate cancer screening test would be characterized by (1) ultra-high specificity (>99%), minimizing the number of false-positives, and (2) high sensitivity (>40%) for stages I and II, with the ability to distinguish between low-grade (e.g., Gleason score <7) and high-grade (e.g., Gleason score ≥7) prostate cancer (i.e., where the prognosis is most favorable and treatment is indicated).

[0147] In some embodiments, the present disclosure provides insight that a particularly useful prostate cancer screening test may be characterized by (1) ultra-high specificity (>98%), minimizing the number of false positives, and (2) high sensitivity (>40%) for stage I and II (Gleason score <7) prostate cancer (i.e., when the prognosis is most favorable). For example, in some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of >98% and a sensitivity of >50%, for example, for stage I and II (Gleason score <7) prostate cancer. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of >98% and a sensitivity of >60%, for example, for stage I and II (Gleason score <7) prostate cancer. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of >98% and a sensitivity of >70%, for example, for stage I and II (Gleason score <7) prostate cancer. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >65%, for example, for stage I and II (Gleason score <7) prostate cancer. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >60%, for example, for stage I and II (Gleason score <7) prostate cancer. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of 99% or higher and a sensitivity of >10% or higher (e.g., including >15%, >20%, >25%). In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of 99% or higher and a sensitivity of 50% or higher. In some embodiments, a particularly useful prostate cancer screening test may be characterized by a specificity of 90% or higher and a sensitivity of 50% or higher.

[0148] In some embodiments, the present disclosure provides insight that prostate cancer screening tests comprising two or more sets of biomarker combinations (e.g., a combination of at least two orthogonal biomarkers described herein) can increase the specificity and / or sensitivity of such assays compared to that achieved by a single set of biomarker combinations. For example, in some embodiments, a prostate cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve at least 98% specificity and at least 50% sensitivity. In some embodiments, a prostate cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve at least 98% specificity and at least 60% sensitivity. In some embodiments, a prostate cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve 99% specificity and 50% or higher sensitivity.

[0149] In some embodiments, the present disclosure provides insight that a particularly useful prostate cancer screening test can be characterized by an acceptable positive predictive value (PPV) at an economically justifiable cost. PPV is the probability that a patient has the disease after a positive test, and is affected by sensitivity, specificity, and / or disease prevalence. In some embodiments, the assays described herein can be useful for early prostate cancer detection, achieving a PPV of more than 10% or higher, including, for example, more than 15%, more than 20%, or more than 25%, or higher, with a specificity cutoff of at least 70%, including, for example, at least 75%, at least 80%, at least 85%, or higher. In some embodiments, the assays described herein are particularly useful for early prostate cancer detection, achieving a PPV of greater than 10% or higher, including, for example, greater than 15%, greater than 20%, or greater than 25%, or higher, at a specificity cutoff of at least 85%, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for subjects at genetic risk for prostate cancer, or a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with prostate cancer).

[0150] In some embodiments, the assays described herein may be useful for early prostate cancer detection, achieving a PPV of greater than 30%, including, for example, greater than 35%, greater than 40%, or greater than 45%, or higher, at a specificity cutoff of at least 70%, including, for example, at least 75%, at least 80%, at least 85%, or higher. In some embodiments, the assays described herein are particularly useful for early prostate cancer detection, achieving a PPV of greater than 30%, including, for example, greater than 35%, greater than 40%, or greater than 45%, or higher, at a specificity cutoff of at least 85%, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for subjects at genetic risk for prostate cancer, or a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with prostate cancer).

[0151] In some embodiments, the assay described herein is particularly useful as a first screening test for detecting early prostate cancer.In some embodiments, subjects who receive a positive test result from the assay described herein are recommended to undergo a follow-up test, such as a digital rectal examination (DRE).In some such embodiments, the assay described herein can be useful for detecting early prostate cancer, achieving a PPV of more than 20%, or more than 25%, or more than 30%, or more than 35%, or more than 40%, or even higher.In some embodiments, the assay described herein can achieve a specificity cutoff of at least 70% or higher, including, for example, at least 75%, at least 80%, at least 85%, or even higher. In some such embodiments, the assays described herein can achieve a specificity cutoff of at least 85% or higher, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for subjects at genetic risk for prostate cancer, or a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with prostate cancer). In some embodiments, the assays described herein can achieve a sensitivity cutoff of at least 75% or higher, including, for example, at least 80%, at least 85%, at least 90%, or higher.

[0152] Several different biomarker classes, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), bulk proteins, and extracellular vesicles (EVs), are being investigated for prostate cancer liquid biopsy assays. EVs are particularly promising due to their abundance and stability in the bloodstream compared to ctDNA and CTCs, suggesting improved sensitivity for early-stage cancer. EVs also contain cargo (i.e., proteins, RNA, and metabolites) of the same cellular origin, providing superior specificity over bulk protein measurements. While the diagnostic utility of EVs has been investigated, much of this work has focused on bulk EV measurements or low-throughput single-EV analysis. II. PROVIDED BIOMARKER AND / OR TARGET BIOMARKER SIGNATURES FOR THE DETECTION OF PROSTATE CANCER

[0153] The present disclosure provides, among other things, various target biomarkers for prostate cancer or combinations thereof (for example, target biomarker signatures). Such target biomarker signatures that are predicted to show high sensitivity and specificity for prostate cancer are discovered through a wide range of bioinformatics analysis and biological approaches, for example, in some embodiments, machine learning and / or computer computational modeling, for example, in some embodiments, computer analysis of a diverse set of data, including one or more of sequencing data, expression data, mass spectrometry, histology, post-translational modification data, and / or in vitro and / or in vivo experimental data.

[0154] In some embodiments, the target biomarker signature for prostate cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., surface polypeptides present in extracellular vesicles associated with prostate cancer; "extracellular vesicle-associated surface biomarkers") and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) selected from the group consisting of surface biomarkers, intravesicular biomarkers, and intravesicular RNA biomarkers. and (7, 8, or more) target biomarkers, such that the combination of such surface biomarkers and such target biomarkers presents a targeted biomarker signature for prostate cancer that provides (a) high specificity (e.g., greater than 98% or higher, e.g., greater than 99%, or greater than 99.5%) that minimizes the number of false positives, and (b) high sensitivity (e.g., greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%) for stage I and II prostate cancer, which have the most favorable prognosis.

[0155] In some embodiments, the present disclosure recognizes that, in certain embodiments, the sensitivity and specificity ratios for subjects with different prostate cancer risk levels may vary depending on the risk tolerance guidelines set forth by the attending physician and / or the medical association of interest. In some embodiments, lower specificity and / or sensitivity may be used to screen patients at higher risk of prostate cancer (e.g., patients with lifestyle-related risk factors, symptomatic patients, patients with a family history of prostate cancer, or patients with abnormal serum PSA, etc.) compared to those for patients at lower risk for prostate cancer. For example, in some embodiments, the combination of biomarkers described herein useful for detecting prostate cancer may provide a specificity of at least 70%, including, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99.5%, or higher. Additionally or alternatively, in some embodiments, a combination of biomarkers described herein useful for detecting prostate cancer may provide a sensitivity of at least 50%, including, for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99.5%, or higher.

[0156] In certain embodiments, subjects at risk for prostate cancer may be tested at a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, subjects with lifestyle-related risk factors may be tested at a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, symptomatic subjects may be administered with a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, asymptomatic subjects may be administered with a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, subjects at risk for prostate cancer may be administered with a specificity rate of 99.5% and a sensitivity of 70%, or a specificity rate of 98% and a sensitivity of 80%. In certain embodiments, subjects with lifestyle-related risk factors may be subjected to a 99.5% specificity rate and a 70% sensitivity, or a 98% specificity rate and a 80% sensitivity. In some embodiments, the assays described herein for detecting prostate cancer in at-risk subjects (e.g., with lifestyle-related risk factors) may have a set sensitivity rate that is less than 80% sensitivity, including, for example, a sensitivity rate of less than 70%, less than 60%, less than 50%, or even lower. In certain embodiments, asymptomatic subjects may be subjected to a 99.5% specificity rate and a 70% sensitivity, or a 98% specificity rate and a 80% sensitivity.In some embodiments, the assays described herein for detecting prostate cancer in asymptomatic subjects may have a set sensitivity ratio that is less than 80% sensitivity, including, for example, a sensitivity ratio of less than 70%, less than 60%, less than 50%, or even lower. In some embodiments, the techniques and / or assays described herein for detecting prostate cancer in symptomatic subjects may have lower sensitivity and / or specificity requirements than those for detecting prostate cancer in asymptomatic subjects. In some embodiments, the assays described herein for detecting prostate cancer in symptomatic subjects may have a set specificity ratio that is less than 99.5% specificity, including, for example, a sensitivity ratio of less than 99%, less than 95%, less than 90%, or less than 85% specificity. In some embodiments, the assays described herein for detecting prostate cancer in symptomatic subjects may have a set sensitivity ratio that is less than 80% sensitivity, including, for example, a sensitivity ratio of less than 70% or less than 60%.

[0157] In some embodiments, the present disclosure recognizes that a biomarker signature for prostate cancer that provides, among other things, a positive predictive value (PPV) of 30% or higher is useful for screening individuals at risk for prostate cancer. In some embodiments, the target biomarker signature for prostate cancer includes at least one surface biomarker (e.g., a surface biomarker present on the surface of extracellular vesicles associated with prostate cancer) and at least one target biomarker selected from the group consisting of a surface biomarker, an intravesicular biomarker, and an intravesicular RNA biomarker, such that the combination of such surface biomarkers and such target biomarkers presents a target biomarker signature for prostate cancer that provides a positive predictive value (PPV) of at least 25% or higher in a high-risk population, including, for example, at least 30% or higher, at least 35% or higher, at least 40% or higher, and / or at least 45% or higher.

[0158] Generally, gene identifiers as used herein refer to Gene Identifications as classified by the UniProt Consortium (UniProt.org), and one of skill in the art will understand that a particular gene may be known by multiple names and will readily recognize such multiple names.

[0159] Generally, the carbohydrate identifiers used herein refer to the Kegg Cancer-associated Carbohydrates database (genome.jp / kegg / disease / br08441.html), and one of skill in the art will understand that a particular carbohydrate may be known by multiple names and will readily recognize such multiple names.

[0160] In some embodiments, the target biomarkers included in the target biomarker signature for prostate cancer are ATP-binding cassette subfamily C member 4 (ABCC4) polypeptide, alpha / beta hydrolase domain-containing protein 17C (ABHD17C) polypeptide, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (ADI1) polypeptide, angiotensin II receptor-associated protein type 1 (AGTRAP) polypeptide, AP-1 complex subunit mu-2 (AP1M2) polypeptide, MICOS Complex subunit MIC26 (APOO) polypeptide, brefeldin A-inhibitory guanine nucleotide exchange protein 3 (ARFGEF3) polypeptide, calcium-transporting ATPase type 2C member 1 (ATP2C1) polypeptide, basal cell adhesion molecule (BCAM) polypeptide, cell adhesion molecule 4 (CADM4) polypeptide, soluble calcium-activated nucleotidase 1 (CANT1) polypeptide, cadherin-1 (CDH1) polypeptide, charged multivesicular body protein 4c (CHMP4C) polypeptide, claudin-3 (CLDN3) polypeptide Claudin-4 (CLDN4) polypeptide, calmegin (CLGN) polypeptide, ceroid lipofuscinosis neuronal protein 5 (CLN5) polypeptide, transmembrane ascorbate-dependent reductase CYB561 (CYB561) polypeptide, DnaJ homolog subfamily C member 30, mitochondrial (DNAJC30) polypeptide, ectonucleotide pyrophosphatase / phosphodiesterase family member 5 (ENPP5) polypeptide, epithelial cell adhesion molecule (EPCAM) polypeptide, endoplasmic reticulum-Golgi intermediate compartment ment protein 1 (ERGIC1) polypeptide, fatty acid amide hydrolase 1 (FAAH) polypeptide, glutamate carboxypeptidase 2 (FOLH1) polypeptide, polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) polypeptide, guanine nucleotide-binding protein G(I) / G(S) / G(O) subunit gamma-4 (GNG4) polypeptide, glucosamine 6-phosphate N-acetyltransferase (GNPNAT1) polypeptide, Golgi membrane protein 1 (GOLM1) polypeptide,Grainy head-like protein 2 homolog (GRHL2) polypeptide, protein HID1 (HID1) polypeptide, Homer protein homolog 2 (HOMER2) polypeptide, serine protease hepsin (HPN) polypeptide, plastin-2 (LCP1) polypeptide, leucine-rich repeat and immunoglobulin-like domain protein 1 (LRIG1) polypeptide, ensconsin (MAP7) polypeptide, MARCKS-related protein (MARCKSL1) polypeptide, MARVEL domain-containing protein 2 (MARVELD2) polypeptide, lysophospholipid acyltransferase 2 (MBOAT2) polypeptide, transport and Golgi organization protein 1 homolog (MIA3) polypeptide, mucin-1 (MUC1) polypeptide, N-acylethanolamine hydrolyzing acid amidase (NAAA) polypeptide, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 (NDUFA2) polypeptide, protein TMEPAI (PMEPA1) polypeptide, podocalyxin-like protein 2 (PODXL2) polypeptide, serine / threonine-protein phosphatase 2B catalytic subunit alpha isoform (PPP3CA) polypeptide, prostasin (PRSS8) polypeptide, Ras-related protein Rab-3B (RAB3B) polypeptide, Ras-related protein Rab-3D (RAB3D) polypeptide, Rap1 GTPase-activating protein 1 (RAP1GAP) polypeptide, retinol dehydrogenase 11 (RDH11) polypeptide, lysosomal membrane protein 2 (SCARB2) polypeptide, serine incorporater 5 (SERINC5) polypeptide, sideroflexin-2 (SFXN2) polypeptide, protein Shroom2 (SHROOM2) polypeptide, protein Shroom3 (SHROOM3) polypeptide, solute carrier family 35 member F2 (SLC35F2) polypeptide, zinc transporter ZIP6 (SLC39A6) polypeptide, zinc transporter SLC39A7 (SLC39A7) polypeptide, electrogenic sodium bicarbonate cotransporter 1 (SLC4A4) polypeptide, acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3B) polypeptide,Sorbitol dehydrogenase (SORD) polypeptide, metalloreductase STEAP1 (STEAP1) polypeptide, metalloreductase STEAP2 (STEAP2) polypeptide, synaptogyrin-2 (SYNGR2) polypeptide, synaptotagmin-7 (SYT7) polypeptide, transmembrane channel-like protein 5 (TMC5) polypeptide, transmembrane emp24 domain-containing protein 3 (TMED3) polypeptide, transmembrane protein 141 (TMEM141) polypeptide, transmembrane protein 192 (TMEM1 92) polypeptide, proton transporting V-type ATPase complex assembly regulator TMEM9 (TMEM9) polypeptide, transmembrane protease serine 2 (TMPRSS2) polypeptide, transient receptor potential cation channel subfamily M member 4 (TRPM4) polypeptide, tetraspanin-1 (TSPAN1) polypeptide, protein unc-13 homolog B (UNC13B) polypeptide, von Willebrand factor A domain-containing protein 1 (VWA1) polypeptide, protein YIPF1 (YIPF1) polypeptide a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) polypeptide, CC motif chemokine 2 (CCL2) polypeptide, programmed cell death 1 ligand 1 (CD274) polypeptide, ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1 (CD38) polypeptide, C-type lectin domain family 2 member D (CLEC2D) polypeptide, receptor tyrosine-protein kinase erbB-2 (ERBB2) polypeptide, filamin-A (FLNA) polypeptide Filamin-B (FLNB) polypeptide, glypican-1 (GPC1) polypeptide, interleukin-6 (IL6) polypeptide, integrin alpha-V (ITGAV) polypeptide, prostate-specific antigen (KLK3) polypeptide, plasma kallikrein (KLKB1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, protein phosphatase 1 regulatory subunit 3A (PPP1R3A) polypeptide, prostate stem cell antigen (PSCA) polypeptide, poliovirus receptor (PVR) polypeptide,Choline transporter-like protein 4 (SLC44A4) polypeptide, TGF beta type 2 receptor (TGFBR2) polypeptide, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) polypeptide, tumor necrosis factor ligand superfamily member 11 (TNFSF11) polypeptide, vascular endothelial growth factor C (VEGFC) polypeptide, zinc-alpha-2-glycoprotein (AZGP1) polypeptide, beta-galactosidase-1-like protein 2 (GLB1L2) polypeptide, polyadenylate-binding protein 1-like 2 (PABC1L2A) polypeptide, testican-1 (SPOCK1) polypeptide, protein-glutamine gamma-glutamyltransferase 4 (TGM4) polypeptide, (Tn) antigen, (sialyl Tn(sTn)) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), and combinations thereof.

[0161] In some embodiments, the target biomarkers included in the target biomarker signature for prostate cancer are ATP-binding cassette subfamily C member 4 (ABCC4) polypeptide, AP-1 complex subunit mu-2 (AP1M2) polypeptide, brefeldin A-inhibited guanine nucleotide exchange protein 3 (ARFGEF3) polypeptide, soluble calcium-activated nucleotidase 1 (CANT1) polypeptide, ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1 (CD38) polypeptide, cadherin-1 (CDH1) polypeptide, claudin-3 (CLDN3) polypeptide, claudin-4 (CLDN4) polypeptide, calmegin (CLGN) polypeptide, ectonucleotide pyrophosphatase / phosphodiesterase family member 5 (ENPP5) polypeptide, glutamate carboxypeptidase 2 (FOLH1) polypeptide, Golgi membrane protein 1 (GOLM1) polypeptide, grainy head-like protein 2 homolog (GRHL2) polypeptide, ensconsin (MAP7) polypeptide, MARCKS-related protein Mucin-1 (MARCKSL1) polypeptide, Mucin-1 (MUC1) polypeptide, TMEPAI (PMEPA1) polypeptide, Podocalyxin-like protein 2 (PODXL2) polypeptide, Serine / Threonine-protein phosphatase 2B catalytic subunit alpha isoform (PPP3CA) polypeptide, Prostate stem cell antigen (PSCA) polypeptide, Ras-related protein Rab-3B (RAB3B) polypeptide, Ras-related protein Rab-3D (RAB3D) polypeptide, Retinol dehydrogenase 11 (RD) polypeptide H11) polypeptide, zinc transporter ZIP6 (SLC39A6) polypeptide, electrogenic sodium bicarbonate cotransporter 1 (SLC4A4) polypeptide, acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3B) polypeptide, sorbitol dehydrogenase (SORD) polypeptide, metalloreductase STEAP1 (STEAP1) polypeptide, metalloreductase STEAP2 (STEAP2) polypeptide, synaptotagmin-7 (SYT7) polypeptide, transmembrane protease serine 2 (TMPRSS2) polypeptide,The surface biomarker is or comprises a surface biomarker selected from the group consisting of a transient receptor potential cation channel subfamily M member 4 (TRPM4) polypeptide, a tetraspanin-1 (TSPAN1) polypeptide, a protein unc-13 homolog B (UNC13B) polypeptide, a Lewis Y antigen, a sialyl Tn (sTn) antigen, a sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), a T antigen, a Tn antigen, and combinations thereof.

[0162] In some embodiments, the target biomarker signature is an ABCC4 polypeptide, an ABHD17C polypeptide, an ADI1 polypeptide, an AGTRAP polypeptide, an AP1M2 polypeptide, an APOO polypeptide, an ARFGEF3 polypeptide, an ATP2C1 polypeptide, a BCAM polypeptide, a CADM4 polypeptide, a CANT1 polypeptide, a CDH1 polypeptide, a CHMP4C polypeptide, a CLDN3 polypeptide, a CLDN4 polypeptide, a CLGN polypeptide, a CLN5 polypeptide, a CYB561 polypeptide, a DNAJC30 polypeptide, an ENPP5 polypeptide, an EPCAM polypeptide, an ERGIC1 polypeptide, a FAAH polypeptide, a FOLH1 polypeptide, a GALNT3 polypeptide, a GNG4 polypeptide, a GNPNAT1 polypeptide, a GOLM1 polypeptide, a GRHL2 polypeptide, a HID1 polypeptide, a HOMER2 polypeptide, an HPN polypeptide, an LCP1 polypeptide, a LRIG1 polypeptide, a MAP7 polypeptide, a MARCKSL1 polypeptide, a MARVELD2 polypeptide, a MBOAT2 polypeptide, a MIA3 polypeptide, a peptide, MUC1 polypeptide, NAAA polypeptide, NDUFA2 polypeptide, PMEPA1 polypeptide, PODXL2 polypeptide, PPP3CA polypeptide, PRSS8 polypeptide, RAB3B polypeptide, RAB3D polypeptide, RAP1GAP polypeptide, RDH11 polypeptide, SCARB2 polypeptide, SERINC5 polypeptide, SFXN2 polypeptide, SHROOM2 polypeptide, SHROOM3 polypeptide, SLC35F2 polypeptide, SLC39A6 polypeptide, SLC39A7 polypeptide, SLC4A4 polypeptide, SMPDL3B polypeptide, SORD polypeptide, STEAP1 polypeptide, STEAP2 polypeptide, SYNGR2 polypeptide, SYT7 polypeptide, TMC5 polypeptide, TMED3 polypeptide, TMEM141 polypeptide, TMEM192 polypeptide, TMEM9 polypeptide, TMPRSS2 polypeptide, TRPM4 polypeptide, TSPAN1 polypeptide, UNC13B polypeptide, VWA1 polypeptide, YIPF1 polypeptide, ADAM17 polypeptide, CCL2 polypeptide,CD274 polypeptide, CD38 polypeptide, CLEC2D polypeptide, ERBB2 polypeptide, FLNA polypeptide, FLNB polypeptide, GPC1 polypeptide, IL6 polypeptide, ITGAV polypeptide, KLK3 polypeptide, KLKB1 polypeptide, PLAC1 polypeptide, PPP1R3A polypeptide, PSCA polypeptide, PVR polypeptide, SLC44A4 polypeptide, TGFBR2 polypeptide, TNFRSF4 polypeptide, TNFSF11 polypeptide, VEGFC polypeptide, AZGP1 polypeptide, GLB1L2 polypeptide, PABPC1L2A polypeptide, SPOCK1 polypeptide, TGM4 polypeptide, Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Lewis Y antigen (also known as CD174) antigen, sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)) antigen, and combinations thereof.

[0163] In some embodiments, the target biomarker signature is an ABCC4 polypeptide, an AP1M2 polypeptide, an ARFGEF3 polypeptide, a CANT1 polypeptide, a CD38 polypeptide, a CDH1 polypeptide, a CLDN3 polypeptide, a CLDN4 polypeptide, a CLGN polypeptide, an ENPP5 polypeptide, a FOLH1 polypeptide, a GOLM1 polypeptide, a GRHL2 polypeptide, a MAP7 polypeptide, a MARCKSL1 polypeptide, a MUC1 polypeptide, a PMEPA1 polypeptide, a PODXL2 polypeptide, a PPP3CA polypeptide, a PSCA polypeptide, a RAB3B polypeptide, a RAB3D polypeptide, a RDH11 polypeptide, a SLC39A6 polypeptide, a SLC4A4 polypeptide, a SMPDL3B polypeptide, a SORD polypeptide, a STEAP1 polypeptide, a STEAP2 polypeptide, a SYT7 polypeptide, a TMPRSS2 polypeptide, a TRPM4 polypeptide, a TSPAN1 polypeptide, an UNC13B polypeptide, a Lewis Y antigen, a sialyl-Tn (sTn) antigen, a sialyl-Lewis The extracellular vesicle-associated surface biomarkers and / or one or more surface biomarkers each independently selected from the list consisting of an X (sLex) antigen (also known as sialyl SSEA-1 (SLX)) antigen, a T antigen, a Tn antigen, and combinations thereof.

[0164] In some embodiments, the target biomarker signature comprises one or more extracellular vesicle-associated surface biomarkers and / or one or more surface biomarkers, each selected from the list consisting of a CLDN3 polypeptide, an ABCC4 polypeptide, a RAB3B polypeptide, a FOLH1 polypeptide, a PMEPA1 polypeptide, a TMPRSS2 polypeptide, a TSPAN1 polypeptide, a PODXL2 polypeptide, a STEAP2 polypeptide, a GOLM1 polypeptide, an AP1M2 polypeptide, an ARFGEF3 polypeptide, a SORD polypeptide, a SLC39A6 polypeptide, a CLGN polypeptide, a GRHL2 polypeptide, a CANT1 polypeptide, a SYT7 polypeptide, a SMPDL3B polypeptide, a PPP3CA polypeptide, an ENPP5 polypeptide, a CDH1 polypeptide, a RDH11 polypeptide, a SLC4A4 polypeptide, a STEAP1 polypeptide, a MARCKSL1 polypeptide, an UNC13B polypeptide, a MAP7 polypeptide, a RAB3D polypeptide, a CLDN4 polypeptide, and combinations thereof.

[0165] In some embodiments, the target biomarker signature comprises one or more extracellular vesicle-associated surface biomarkers and / or one or more surface biomarkers, each selected from the list consisting of a CLDN3 polypeptide, an ABCC4 polypeptide, a RAB3B polypeptide, a FOLH1 polypeptide, a PMEPA1 polypeptide, a TMPRSS2 polypeptide, a TSPAN1 polypeptide, a PODXL2 polypeptide, a STEAP2 polypeptide, a GOLM1 polypeptide, an AP1M2 polypeptide, an ARFGEF3 polypeptide, a SORD polypeptide, a SLC39A6 polypeptide, a CLGN polypeptide, and combinations thereof.

[0166] In some embodiments, the target biomarker signature is an ABCC4 polypeptide, an AP1M2 polypeptide, an ARFGEF3 polypeptide, a CANT1 polypeptide, a CD38 polypeptide, a CDH1 polypeptide, a CLDN3 polypeptide, a CLDN4 polypeptide, a CLGN polypeptide, an ENPP5 polypeptide, a FOLH1 polypeptide, a GOLM1 polypeptide, a GRHL2 polypeptide, a MAP7 polypeptide, a MARCKSL1 polypeptide, a MUC1 polypeptide, a PMEPA1 polypeptide, a PODXL2 polypeptide, a PPP3CA polypeptide, a PSCA polypeptide, a RAB3B polypeptide, a RAB3D polypeptide, a RDH11 polypeptide, a SLC39A6 polypeptide, a SLC4A4 polypeptide, a SMPDL3B polypeptide, a SORD polypeptide, a STEAP1 polypeptide, a STEAP2 polypeptide, a SYT7 polypeptide, a TMPRSS2 polypeptide, a TRPM4 polypeptide, a TSPAN1 polypeptide, an UNC13B polypeptide, a Lewis Y antigen, a sialyl-Tn (sTn) antigen, a sialyl-Lewis The extracellular vesicle-associated surface biomarkers and / or one or more surface biomarkers each independently selected from the list consisting of an X (sLex) antigen (also known as sialyl SSEA-1 (SLX)) antigen, a T antigen, a Tn antigen, and combinations thereof.

[0167] In some embodiments, the target biomarker signature comprises one or more extracellular vesicle-associated surface biomarkers and / or one or more surface biomarkers, each selected from the list consisting of a CLDN3 polypeptide, an ABCC4 polypeptide, a RAB3B polypeptide, a FOLH1 polypeptide, a PMEPA1 polypeptide, a TMPRSS2 polypeptide, a TSPAN1 polypeptide, a PODXL2 polypeptide, a GOLM1 polypeptide, a SORD polypeptide, a CLGN polypeptide, a GRHL2 polypeptide, a CANT1 polypeptide, a SYT17 polypeptide, a PPP3CA polypeptide, and combinations thereof.

[0168] In some embodiments, the target biomarkers in the target biomarker signature for prostate cancer are an ACP5 polypeptide, an ACPP polypeptide, an ACSM1 polypeptide, an ACTA2 polypeptide, an ACTG2 polypeptide, an ADAMTS1 polypeptide, an AGR2 polypeptide, an AIM1 polypeptide, an ALCAM polypeptide, an ALDH1A3 polypeptide, an ALOX15B polypeptide, an ANTXR1 polypeptide, an ANTXR2 polypeptide, an AP1M2 polypeptide, an AR polypeptide, an ARG2 polypeptide, an ARHGAP6 polypeptide, an ARHGEF26 polypeptide, an ARHGEF37 polypeptide, an ASAP3 polypeptide, an ATP8B1 polypeptide, an ATP9A polypeptide, a BAIAP2L1 polypeptide, a BCAM polypeptide, a BHLHA15 polypeptide, a BOK polypeptide, a C15orf48 polypeptide, a C19orf48 polypeptide, a C1S polypeptide, a C1orf116 polypeptide, a C2orf72 polypeptide, a C8orf4 polypeptide, a C9orf152 polypeptide, a CADM4 polypeptide, a CALD1 polypeptide, a CAMSAP3 polypeptide peptide, CAPN5 polypeptide, CBLC polypeptide, CD24 polypeptide, CD74 polypeptide, CDC42EP1 polypeptide, CDC42EP5 polypeptide, CGN polypeptide, CGNL1 polypeptide, CHMP4C polypeptide, CLGN polypeptide, CMTM4 polypeptide, COLCA1 polypeptide, CORO2A polypeptide, CPE polypeptide, CPNE4 polypeptide, CPQ polypeptide, CRYM polypeptide, CTSF polypeptide, CX3CL1 polypeptide, CXADR polypeptide, CXCR4 polypeptide, CYB561 polypeptide, DPYSL3 polypeptide, DSP polypeptide, EEF1A2 polypeptide, EFNA1 polypeptide, EHD2 polypeptide, EHF polypeptide, ELF3 polypeptide, EMP2 polypeptide, EPHX1 polypeptide, EPN3 polypeptide, ERBB3 polypeptide, ERG polypeptide, ESRP1 polypeptide, ESRP2 polypeptide, F3 polypeptide, FAAH polypeptide, FAM129A polypeptide, FAM129B polypeptide, FAM210B polypeptide, FAM3B polypeptide,FAM3D polypeptide, FAM83H polypeptide, FAM84A polypeptide, FAM84B polypeptide, FAT1 polypeptide, FBLIM1 polypeptide, FBP1 polypeptide, FLNC polypeptide, FNBP1L polypeptide, FOS polypeptide, FOXA1 polypeptide, GADD45G polypeptide, GATA2 polypeptide, GGT1 polypeptide, GJB1 polypeptide, GLYATL1 polypeptide, GNG4 polypeptide, GNMT polypeptide, GRHL2 polypeptide, GUCY1A3 polypeptide, HGD polypeptide, HID1 polypeptide, HIST1H2BK polypeptide, HIST3H2A polypeptide, HMGCS2 polypeptide, HOMER2 polypeptide, HOXB13 polypeptide, HSD17B6 polypeptide, HSPB6 polypeptide, HSPB8 polypeptide, ICA1 polypeptide, ID1 polypeptide, IQGAP2 polypeptide, IRF6 polypeptide, ITGA6 polypeptide, KIAA1522 polypeptide, KIF5C polypeptide, KLK2 polypeptide, KRT14 polypeptide, KRT15 polypeptide, KRT17 polypeptide Do, KRT18 polypeptide, KRT19 polypeptide, KRT5 polypeptide, KRT7 polypeptide, KRT8 polypeptide, LCP1 polypeptide, LIFR polypeptide, LIMCH1 polypeptide, LMOD1 polypeptide, LRP11 polypeptide, LRP3 polypeptide, LSR polypeptide, MAL2 polypeptide, MAOA polypeptide, MAP7 polypeptide, MARC1 polypeptide, MARCKSL1 polypeptide, MB polypeptide, MESP1 polypeptide, MLPH polypeptide, MME polypeptide, MPZL2 polypeptide, MT1G polypeptide, MYBPC1 polypeptide, MYL9 polypeptide, MYLK polypeptide, NCAPD3 polypeptide, NEDD4L polypeptide, NEFH polypeptide, NFIX polypeptide, NKX3-1 polypeptide, NPDC1 polypeptide, NUPR1 polypeptide, OBSL1 polypeptide, PALLD polypeptide, PARVA polypeptide, PCDH1 polypeptide, PCP4 polypeptide, PDIA5 polypeptide, PEBP4 polypeptide, PGM5 polypeptide, PKP1 polypeptide, PKP3 polypeptide,PPL polypeptide, PPM1H polypeptide, PPP1R1B polypeptide, PPP3CA polypeptide, PRAC1 polypeptide, PRR15L polypeptide, PSAT1 polypeptide, PTPRF polypeptide, PYCR1 polypeptide, RAB3B polypeptide, RAMP1 polypeptide, RAP1GAP polypeptide, RASEF polypeptide, RBM47 polypeptide, RCAN3 polypeptide, RDH11 polypeptide, REEP6 polypeptide, REPS2 polypeptide, RGS10 polypeptide, RHPN2 polypeptide, RNF144B polypeptide, RORC polypeptide, RPS4Y1 polypeptide, S100A16 polypeptide, SELENBP1 polypeptide, SERINC5 polypeptide, SH3BGRL2 polypeptide, SH3BP4 polypeptide, SHC2 polypeptide, SLC1A5 polypeptide, SLC22A3 polypeptide, SLC2A12 polypeptide, SLC40A1 polypeptide, SLC4A4 polypeptide, SLC7A8 polypeptide, SLC9A3R2 polypeptide, SMOC2 polypeptide, SORD polypeptide, SPDEF polypeptide, SPINT1 polypeptide, SPINT2 polypeptide, SPTBN2 polypeptide, ST14 polypeptide, STAP2 polypeptide, STK39 polypeptide, SULT2B1 polypeptide, SYNE4 polypeptide, SYT7 polypeptide, SYTL1 polypeptide, TAGLN polypeptide, TBC1D16 polypeptide, TBX3 polypeptide, TC2N polypeptide, TCEA3 polypeptide, TEAD3 polypeptide, TESC polypeptide, THBS4 polypeptide, TM4SF1 polypeptide, TMED3 polypeptide, TMEM150C polypeptide, TMEM54 polypeptide, TMEM98 polypeptide, TMSB15A polypeptide, TP53I11 polypeptide, TPD52 polypeptide, TPM1 polypeptide, TSPAN13 polypeptide, TSPAN6 polypeptide, TSPAN8 polypeptide, VAMP8 polypeptide, VSTM2L polypeptide, WLS polypeptide, WWC1 polypeptide, YAP1 polypeptide, ZBTB42 polypeptide, ZC3H11A polypeptide, ZNF217 polypeptide,and combinations thereof. In some embodiments, the vesicular biomarkers described herein may include at least one post-translational modification.

[0169] ABCC4 RNA, ACP5 RNA, ACP5 RNA, ACP5 RNA, ACTA RNA, 12 RNA、ACTG2 RNA、ADAMTS1 RNA、AGR2 RNA、AIM1 RNA、ALCAM RNA、ALDH1A3 RNA、ALOX15B RNA、ANO7 RNA、ANPEP RNA、ANTXR1 RNA、ANTXR2 RNA、APCD1 RNA、AQP3 RNA、AR RNA、ARG2 RNA、ARHGAP6 RNA、ARHGEF26 RNA、ARHGEF37 RNA、ASAP3 RNA、ASTN2 RNA、ATP8B1 RNA、ATP9A RNA、LBC2 RNA RNA、BHLHA15 RNA、BIK RNA、BMPR1B RNA、BOK RNA、C15orf48 RNA、C19orf48 RNA、C1S RNA、C1orf116 RNA、C2orf72 RNA、C844 RNA、C2049CDMRNA RNA、CALD1 RNA、CAMSAP3 RNA、CAPN5 RNA、CBLC RNA、CD24 RNA、CD74 RNA、CDC42EP1 RNA、CDC42EP5 RNA、CDH1 RNA、CGN RNA、CGNL1 RNACCHRM1 RNA、CHRNA2 RNA、CLDN3 RNA、CLDN4 RNA、CLDN7 RNA、CLDN8 RNA、CLGN RNA、CMTM4 RNA、COLCA1 RNA、COLEC12 RNA、CORO2A RNA、CPENE RNA4CRNA3 RNA、CREB3L1 RNA、CREB3L4 RNA、CRYM RNA、CTSF RNA、CWH43 RNA、CX3CL1 RNA、CXADR RNA、CXCR4 RNA、CYB561 RNA、DPP4 RNA3DPP4 RNADSP RNA、EEF1A2 RNA、EHD2 RNA、EHF RNA、ELF3 RNA、ELOVL7 RNA、EMB RNA、EMP2 RNA、ENPP5 RNA、EPCAM RNA、EPHX1 RNA、EPCAM RNA、GPN3GRNARNA、ESRP1 RNA、ESRP2 RNA、F3 RNA、FAAH RNA、FAM129A RNA、FAM129B RNA、FAM189A2 RNA、FAM210B RNA、FAM3B RNA、FAM3D RNA、FAM83H RNA、FAM84A RNA、FAM84B RNA、FAT1 RNA、FBLIM1 RNA、FBP1 RNA、FLNC RNA、FNBP1L RNA、FOLH1 RNA、FOS RNA、FOXA1 RNA、FXYD3 RNA、GADD45G RNA、GALNT3 RNA、GALNT7 RNA、GATA2 RNA、GCNT1 RNA、GGT1 RNA、GJB1 RNA、GLYATL1 RNA、GNG4 RNA、GNMT RNA、GOLM1 RNA、GPR160 RNA、GREB1 RNA、GRHL2 RNA、GUCY1A3 RNA、HGD RNA、HID1 RNA、HIST1H2BK RNA、HIST3H2A RNA、HMGCS2 RNA、HOMER2 RNA、HOXB13 RNA、HPN RNA、HSD17B6 RNA、HSPB6 RNA、HSPB8 RNA、ICA1 RNA、ID1 RNA、IQGAP2 RNA、IRF6 RNA、ITGA6 RNA、KCNN4 RNA、KIAA1324 RNA、KIAA1522 RNA、KIF5C RNA、KLK2 RNA、KRT14 RNA、KRT15 RNA、KRT17 RNA、KRT18 RNA、KRT19 RNA、KRT5 RNA、KRT7 RNA、KRT8 RNA、KRTCAP3 RNA、LCP1 RNA、LIFR RNA、LIMCH1 RNA、LMOD1 RNA、LPAR3 RNA、LRP11 RNA、LRP3 RNA、LRRC26 RNA、LSR RNA、MAL2 RNA、MAOA RNA、MAP7 RNA、MARC1 RNA、MARCKSL1 RNA、MB RNA、MBOAT2 RNA、MESP1 RNA、MLPH RNA、MME RNA、MPZL2 RNA、MT1G RNA、MYBPC1 RNA、MYL9 RNA、MYLK RNA、NCAPD3 RNA、NEDD4L RNA、NEFH RNA、NFIX RNA、NKX3-1 RNA、NPDC1 RNA、NUPR1 RNA、OBSL1RNA、OR51E1 RNA、OR51E2 RNA、PALLD RNA、PARM1 RNA、PARVA RNA、PCDH1 RNA、PCP4 RNA、PDIA5 RNA、PDLIM5 RNA、PDZK1IP1 RNA、PDZRN3 RNA、PEBP4 RNA、PGM5 RNA、PIGR RNA、PKP1 RNA、PKP3 RNA、PLA2G2A RNA、PMEPA1 RNA、PODXL2 RNA、PPL RNA、PPM1H RNA、PPP1R1B RNA、PPP3CA RNA、PRAC1 RNA、PROM2 RNA、PRR15L RNA、PRSS8 RNA、PSAT1 RNA、PSCA RNA、PTPRF RNA、PTPRN2 RNA、PYCR1 RNA、RAB25 RNA、RAB27B RNA、RAB3B RNA、RAMP1 RNA、RAP1GAP RNA、RASEF RNA、RBM47 RNA、RCAN3 RNA、RDH11 RNA、REEP6 RNA、REPS2 RNA、RGS10 RNA、RHPN2 RNA、RNF144B RNA、RORC RNA、RPS4Y1 RNA、S100A16 RNA、SCNN1A RNA、SDC1 RNA、SELENBP1 RNA、SERINC2 RNA、SERINC5 RNA、SEZ6L2 RNA、SH3BGRL2 RNA、SH3BP4 RNA、SHC2 RNA、SLC15A2 RNA、SLC1A5 RNA、SLC22A3 RNA、SLC2A10 RNA、SLC2A12 RNA、SLC30A4 RNA、SLC35F2 RNA、SLC40A1 RNA、SLC44A4 RNA、SLC45A3 RNA、SLC4A4 RNA、SLC7A8 RNA、SLC9A3R2 RNA、SMIM22 RNA、SMOC2 RNA、SORD RNA、SPDEF RNA、SPINT1 RNA、SPINT2 RNA、SPTBN2 RNA、ST14 RNA、STAP2 RNA、STEAP1 RNA、STEAP2 RNA、STEAP4 RNA、STK39 RNA、SULT2B1 RNA、SYNE4 RNA、SYT7 RNA、SYTL1 RNA、TACSTD2 RNA、TAGLN RNA、TBC1D16 RNA、TBX3 RNA、TC2N RNA、TCEA3the biomarkers comprise one or more intravesicular RNA biomarkers selected from the list consisting of RNA, TEAD3 RNA, TESC RNA, THBS4 RNA, TM4SF1 RNA, TMC4 RNA, TMC5 RNA, TMED3 RNA, TMEFF2 RNA, TMEM125 RNA, TMEM150C RNA, TMEM30B RNA, TMEM45B RNA, TMEM54 RNA, TMEM98 RNA, TMPRSS2 RNA, TMSB15A RNA, TP53I11 RNA, TPD52 RNA, TPM1 RNA, TRPM4 RNA, TRPM8 RNA, TRPV6 RNA, TSPAN1 RNA, TSPAN13 RNA, TSPAN6 RNA, TSPAN8 RNA, TUSC3 RNA, UPK3A RNA, VAMP8 RNA, VSIG2 RNA, VSTM2L RNA, WLS RNA, WWC1 RNA, YAP1 RNA, ZBTB42 RNA, ZC3H11A RNA, ZNF217 RNA, and combinations thereof.

[0170] In some embodiments, the target biomarker signature for prostate cancer includes at least two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., those described herein) present on the surface of nanoparticles having a size range of interest, including extracellular vesicles, for example, in some embodiments, nanoparticles having a size within the range of about 30 nm to about 1000 nm. In some embodiments, the two or more surface biomarkers are the same. In some embodiments, the two or more surface biomarkers are distinct.

[0171] In some embodiments, the target biomarker signature for prostate cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., those described herein). In some embodiments, at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker are the same.

[0172] In some embodiments, at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker of the target biomarker signature for prostate cancer are distinct. For example, in some embodiments, the target biomarker signature for prostate cancer comprises at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker.

[0173] In some embodiments, the target biomarker signature for prostate cancer includes at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., those described herein) present on the surface of nanoparticles having a size range of interest, including extracellular vesicles, e.g., in some embodiments, nanoparticles having a size within the range of about 30 nm to about 1000 nm. In some such embodiments, the surface biomarkers and intravesicular biomarkers may be encoded by the same gene, but the former are present on the surface of the nanoparticles and the latter are contained within the extracellular vesicles (e.g., cargo). In some such embodiments, the surface biomarkers and intravesicular biomarkers may be encoded by different genes.

[0174] In some embodiments, the target biomarker signature for prostate cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular biomarkers (e.g., those described herein). In some such embodiments, the extracellular vesicle-associated surface biomarkers and intravesicular biomarkers may be encoded by the same gene, but the former are expressed in the membrane of the extracellular vesicle, and the latter are contained within the extracellular vesicle (e.g., cargo). In some such embodiments, the extracellular vesicle-associated surface biomarkers and intravesicular biomarkers may be encoded by different genes.

[0175] In some embodiments, the target biomarker signature for prostate cancer includes at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular RNA (e.g., mRNA) biomarkers (e.g., those described herein). In some such embodiments, the surface biomarkers and intravesicular RNA (e.g., including but not limited to, mRNA, and non-coding RNA, such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers may be encoded by the same gene. In some such embodiments, the surface biomarkers and intravesicular RNA (e.g., including but not limited to, mRNA, and non-coding RNA, such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers may be encoded by different genes.

[0176] In some embodiments, the target biomarker signature for prostate cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular RNA (e.g., mRNA) biomarkers (e.g., those described herein). In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular RNA (e.g., mRNA) biomarkers may be encoded by the same gene. In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular RNA (e.g., mRNA) biomarkers may be encoded by different genes.

[0177] In some embodiments, any one of the provided biomarkers can be detected and / or measured by protein and / or RNA (eg, mRNA) expression levels of the wild-type form.

[0178] In some embodiments, any one of the provided biomarkers can be detected and / or measured by protein and / or RNA (e.g., mRNA) expression levels of mutant forms. Thus, some embodiments can include mutant-specific detection of a provided biomarker (e.g., protein and / or RNA, such as, e.g., mRNA).

[0179] As described herein, in some embodiments, a biomarker is or comprises a particular form of one or more polypeptides or proteins (e.g., proforms, truncated forms, modified forms, e.g., glycosylated, phosphorylated, acetylated, methylated, ubiquitinated, lipidated forms, etc.). In some embodiments, detection of such forms detects a majority (and in some embodiments, substantially all) of the polypeptides present in that form (e.g., containing a particular modification, e.g., a particular glycosylation, e.g., sialyl-Tn (sTn) glycosylation, e.g., a truncated O-glycan containing sialic acid α-2,6 linked to GalNAc α-O-Ser / Thr, etc.).

[0180] Thus, in some embodiments, a surface biomarker may be or include a glycosylated moiety (e.g., an sTn antigen moiety, a Tn antigen moiety, or a T antigen moiety). The Thomsen-Noubel (Tn) antigen is an O-linked glycan that is thought to be associated with a wide range of tumors. Tn is a single alpha-linked GalNAc attached to Ser or Thr as the first step in the major O-linked glycosylation pathway. Those skilled in the art will understand that in certain embodiments, T antigen typically refers to an O-linked glycan having the structure Galβ1-3GalNAc-.

[0181] In some embodiments, the surface protein biomarker may be or may include tumor-associated post-translational modifications. In some embodiments, such post-translational modifications may be or may include tumor-specific glycosylation patterns, such as mucins with abnormally truncated glycans at the first GalNAc (e.g., Tn), or a combination thereof. In some embodiments, the surface protein biomarker may be or may include tumor-specific proteoforms of mucins resulting from altered splicing and / or translation (isoforms) or proteolysis (cancer-specific protease activity occurring in abnormal cleavage products).

[0182] In some embodiments, the target biomarker signature comprises a combination of at least two biomarkers, the combination being: an ABCC4 polypeptide and a RAB3B polypeptide; or an ABCC4 polypeptide and a FOLH1 polypeptide; or an ABCC4 polypeptide and a GOLM1 polypeptide; or an ABCC4 polypeptide and a PODXL2 polypeptide; or a FOLH1 polypeptide and a TSPAN1 polypeptide; or a GOLM1 polypeptide and a RAB3B polypeptide; or an ABCC4 polypeptide and an ARFGEF3 polypeptide; or an ABCC4 polypeptide and a TMPRSS2 polypeptide; or an ABCC4 polypeptide and a CLGN polypeptide; or an ABCC4 polypeptide and a SYT7 polypeptide; or a FOLH1 polypeptide and a GOLM1 polypeptide; or an ABCC4 polypeptide and a PMEPA1 polypeptide; or a FOLH1 polypeptide and a SMPDL3B polypeptide; or a FOLH1 polypeptide and a PODXL2 polypeptide; or P a MEPA1 polypeptide and a TSPAN1 polypeptide; or a CLDN4 polypeptide and a FOLH1 polypeptide; or a RAB3B polypeptide and a SLC4A4 polypeptide; or a PODXL2 polypeptide and a RAB3B polypeptide; or an ABCC4 polypeptide and an RDH11 polypeptide; or a CANT1 polypeptide and a FOLH1 polypeptide; or a FOLH1 polypeptide and a PPP3CA polypeptide; or an ARFGEF3 polypeptide and a RAB3B polypeptide; or an AP1M2 polypeptide and a FOLH1 polypeptide; or an ENPP5 polypeptide and a RAB3B polypeptide; or a FOLH1 polypeptide and a SLC4A4 polypeptide; or a RAB3B polypeptide and an SMPDL3B polypeptide; or a MARCKSL1 polypeptide and a RAB3B polypeptide; or a PPP3CA polypeptide and a RAB3B polypeptide; or a CLGN polypeptide and GOLM1 polypeptide; or a RAB3B polypeptide and TSPAN1 polypeptide;or a GOLM1 polypeptide and a SORD polypeptide; or an SLC39A6 polypeptide and a TSPAN1 polypeptide; or a PPP3CA polypeptide and a TSPAN1 polypeptide; or a STEAP2 polypeptide and a TSPAN1 polypeptide; or a CLGN polypeptide and a TSPAN1 polypeptide; or a CLDN3 polypeptide and a PMEPA1 polypeptide; or an SLC39A6 polypeptide and a TMPRSS2 polypeptide; or a CLGN polypeptide and an SLC4A4 polypeptide; or a HOMER2 polypeptide and a TSPAN1 polypeptide; or a CLGN polypeptide and a PODXL2 polypeptide; or a PPP3CA polypeptide and a TMPRSS2 polypeptide; or a PODXL2 polypeptide and a TMPRSS2 polypeptide; or an ARFGEF3 polypeptide and a CLGN polypeptide; or a CLN5 polypeptide and a STEAP1 polypeptide; or a CLDN3 polypeptide and a SLC39A6 polypeptide; or a CLGN polypeptide and an SMPDL3B polypeptide a polypeptide; or a PODXL2 polypeptide and a SORD polypeptide; or a GOLM1 polypeptide and a TMPRSS2 polypeptide; or an APOO polypeptide and a YIPF1 polypeptide; or an APOO polypeptide and a SYNGR2 polypeptide; or a SYT7 polypeptide and a TSPAN1 polypeptide; or a GOLM1 polypeptide and a GRHL2 polypeptide; or an HPN polypeptide and a PMEPA1 polypeptide; or an ENPP5 polypeptide and a TMPRSS2 polypeptide; or a STEAP1 polypeptide and a TSPAN1 polypeptide; or a CLDN3 polypeptide and a PPP3CA polypeptide; or a CLDN3 polypeptide and a STEAP2 polypeptide; or an APOO polypeptide and an MIA3 polypeptide; or a MARCKSL1 polypeptide and a TMPRSS2 polypeptide; or a SYNGR2 polypeptide and a YIPF1 polypeptide; or an ADI1 polypeptide and an SMPDL3B polypeptide; or a GOLM1 polypeptide and an RDH11 polypeptide;or an AP1M2 polypeptide and a CLGN polypeptide; or a RAP1GAP polypeptide and an SLC39A7 polypeptide; or a GOLM1 polypeptide and a PMEPA1 polypeptide; or a STEAP1 polypeptide and a TMPRSS2 polypeptide; or an APOO polypeptide and a TMEM192 polypeptide; or a PMEPA1 polypeptide and a PODXL2 polypeptide; or a CADM4 polypeptide and a MARCKSL1 polypeptide; or a CLGN polypeptide and a PPP3CA polypeptide; or a GOLM1 polypeptide and an SLC39A6 polypeptide; or a PODXL2 polypeptide and a STEAP2 polypeptide; or an ARFGEF3 polypeptide and a SORD polypeptide; or a GRHL2 polypeptide and an SLC4A4 polypeptide; or an ARFGEF3 polypeptide and a TMPRSS2 polypeptide; or a SLC39A7 polypeptide and an SMPDL3B polypeptide; or a PODXL2 polypeptide and a STEAP1 polypeptide; or an SLC 4A4 polypeptide and SORD polypeptide; or CANT1 polypeptide and CLGN polypeptide; or SLC4A4 polypeptide and TMPRSS2 polypeptide; or STEAP1 polypeptide and STEAP2 polypeptide; or APOO polypeptide and TMEM9 polypeptide; or MARCKSL1 polypeptide and PMEPA1 polypeptide; or AGTRAP polypeptide and SMPDL3B polypeptide; or SMPDL3B polypeptide and SORD polypeptide; or MARCKSL1 polypeptide and STEAP2 polypeptide; or HPN polypeptide and SLC39A6 polypeptide; or HPN polypeptide and STEAP2 polypeptide; or ARFGEF3 polypeptide and CLDN3 polypeptide; or AP1M2 polypeptide and CANT1 polypeptide; or APOO polypeptide and BCAM polypeptide; or APOO polypeptide and SMPDL3B polypeptide; or HPN polypeptide and PPP3CA polypeptide;or an SMPDL3B polypeptide and a SYNGR2 polypeptide; or an ARFGEF3 polypeptide and a PMEPA1 polypeptide; or an EPCAM polypeptide and a STEAP1 polypeptide; or an ARFGEF3 polypeptide and an HPN polypeptide; or a GNPNAT1 polypeptide and a RAP1GAP polypeptide; or an APOO polypeptide and a CLN5 polypeptide; or an APOO polypeptide and an ATP2C1 polypeptide; or an ABCC4 polypeptide and a CLDN3 polypeptide; or an ABCC4 polypeptide and a TSPAN1 polypeptide; or a CLDN3 polypeptide and a FOLH1 polypeptide; or a PODXL2 polypeptide and a TSPAN1 polypeptide; or a GOLM1 polypeptide and a PODXL2 polypeptide; or a PODXL2 polypeptide and a PPP3CA polypeptide; or a CLGN polypeptide and a MARCKSL1 polypeptide; or a CLGN polypeptide and a PMEPA1 polypeptide; or a PMEPA1 polypeptide and a PPP3CA polypeptide ; or a PPP3CA polypeptide and a STEAP1 polypeptide; or a PMEPA1 polypeptide and a SMPDL3B polypeptide; or a GRHL2 polypeptide and an HPN polypeptide; or an AGTRAP polypeptide and an HPN polypeptide; or a CANT1 polypeptide and an SLC39A6 polypeptide; or an SLC39A6 polypeptide and an SLC4A4 polypeptide; or a CADM4 polypeptide and a STEAP1 polypeptide; or a CLDN3 polypeptide and an ENPP5 polypeptide; or a CLDN3 polypeptide and a MARCKSL1 polypeptide; or an SLC39A6 polypeptide and a SYNGR2 polypeptide; or an APOO polypeptide and a HOMER2 polypeptide; or an APOO polypeptide and a SCARB2 polypeptide; or a CANT1 polypeptide and a SORD polypeptide; or a MARCKSL1 polypeptide and a STEAP1 polypeptide; or an APOO polypeptide and a SLC39A6 polypeptide; or a LRIG1 polypeptide and a PRSS8 polypeptide;or an ABCC4 polypeptide and a SORD polypeptide; or an ABCC4 polypeptide and a PPP3CA polypeptide; or an ABCC4 polypeptide and an ENPP5 polypeptide; or a FOLH1 polypeptide and a PMEPA1 polypeptide; or a FOLH1 polypeptide and a TRPM4 polypeptide; or an ENPP5 polypeptide and a FOLH1 polypeptide; or a CLDN3 polypeptide and a CLDN4 polypeptide; or a CLDN3 polypeptide and a GOLM1 polypeptide; or a CDH1 polypeptide and a FOLH1 polypeptide; or an EPCAM polypeptide and a PMEPA1 polypeptide; or a CLDN4 polypeptide and a GOLM1 polypeptide; or a GOLM1 polypeptide and a PPP3CA polypeptide; or a CDH1 polypeptide and a GOLM1 polypeptide; or an ENPP5 polypeptide and a PMEPA1 polypeptide; or an ADI1 polypeptide and a TSPAN1 polypeptide; or an ENPP5 polypeptide and a PPP3CA polypeptide; or an RDH11 polypeptide peptide and a TSPAN1 polypeptide; or a SORD polypeptide and a TSPAN1 polypeptide; or an EPCAM polypeptide and a PPP3CA polypeptide; or a GOLM1 polypeptide and a TSPAN1 polypeptide; or a BCAM polypeptide and a TSPAN1 polypeptide; or a CLGN polypeptide and an ENPP5 polypeptide; or a CLDN4 polypeptide and a PPP3CA polypeptide; or a CLDN4 polypeptide and an HPN polypeptide; or a CLDN3 polypeptide and a SCARB2 polypeptide; or a CLDN3 polypeptide and an LCP1 polypeptide; or a CDH1 polypeptide and a CLGN polypeptide; or a BCAM polypeptide and a CLDN3 polypeptide; or a BCAM polypeptide and a CLGN polypeptide; or an HID1 polypeptide and a RAP1GAP polypeptide; or a BCAM polypeptide and an EPCAM polypeptide; or a CLDN4 polypeptide and a CLGN polypeptide; or an EPCAM polypeptide and an RDH11 polypeptide;Or a CLGN polypeptide and a TRPM4 polypeptide; or a RAP1GAP polypeptide and a SORD polypeptide; or an ENPP5 polypeptide and a PRSS8 polypeptide; or an ENPP5 polypeptide and a RAP1GAP polypeptide; or an ENPP5 polypeptide and an EPCAM polypeptide; or an EPCAM polypeptide and an HPN polypeptide; or an ENPP5 polypeptide; a CADM4 polypeptide and a TRPM4 polypeptide; or an EPCAM polypeptide and a SORD polypeptide; or a BCAM polypeptide and a PRSS8 polypeptide; or a CDH1 polypeptide and an HPN polypeptide; or a SCARB2 polypeptide and a SORD polypeptide; or a RAP1GAP polypeptide and a SCARB2 polypeptide; or a CLDN4 polypeptide and a RAP1GAP polypeptide; or a CLDN4 polypeptide and a PRSS8 polypeptide; or a CLDN4 polypeptide and an RDH11 polypeptide; or a CDH1 polypeptide and an RDH11 polypeptide; or a RAP1GAP polypeptide and a TRPM4 polypeptide; or a CLDN4 polypeptide and a TRPM4 polypeptide; or a CDH1 polypeptide and a RAP1GAP polypeptide; or a RAP1GAP polypeptide and an RDH11 polypeptide; or a CADM4 polypeptide and a PRSS8 polypeptide; or a BCAM polypeptide and an HPN polypeptide; or an HPN polypeptide and an RDH11 polypeptide ; or a CDH1 polypeptide and a SCARB2 polypeptide; or an ADI1 polypeptide and a PRSS8 polypeptide; or an LCP1 polypeptide and a RAP1GAP polypeptide; or an HPN polypeptide and a PRSS8 polypeptide; or a CADM4 polypeptide and a TRPM4 polypeptide; or a SORD polypeptide and a TRPM4 polypeptide; or a CADM4 polypeptide and a CDH1 polypeptide; or a CDH1 polypeptide and a TRPM4 polypeptide; or a BCAM polypeptide and a CDH1 polypeptide; or a BCAM polypeptide and an RDH11 polypeptide; or an RDH11 polypeptide and a TRPM4 polypeptide; or a CADM4 polypeptide and an HPN polypeptide; or an HID1 polypeptide and an HPN polypeptide; or a PRSS8 polypeptide and a SCARB2 polypeptide; or an FAAH polypeptide and a RAP1GAP polypeptide; or a CADM4 polypeptide and an RDH11 polypeptide; or an ADI1 polypeptide and an HID1 polypeptide;or an LCP1 polypeptide and a TRPM4 polypeptide; or an HPN polypeptide and an LCP1 polypeptide; or combinations thereof. In some embodiments, the target biomarkers in the aforementioned combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0183] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a GOLM1 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and an ARFGEF3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a TMPRSS2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a CLGN polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a SYT7 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a PMEPA1 polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a CLDN3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and an RDH11 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a SORD polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a PPP3CA polypeptide and a TSPAN1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLDN4 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ABCC4 polypeptide and an ENPP5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLGN polypeptide and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLGN polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and a TRPM4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ENPP5 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLDN3 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLDN3 polypeptide and a CLDN4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a GOLM1 polypeptide and a SORD polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CLDN3 polypeptide and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a PODXL2 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a RAB3B polypeptide and an SLC4A4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CANT1 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a RAB3B polypeptide and an SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ENPP5 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FOLH1 polypeptide and an SLC4A4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a SLC39A6 polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ARFGEF3 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an AP1M2 polypeptide and a FOLH1 polypeptide. In some embodiments, the target biomarkers in the above combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0184] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ABCC4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an APIM2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ARFGEF3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CANT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CD38 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CDH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CLDN3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CLDN4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CLGN polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ENPP5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a FOLH1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a GRHL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a MAP7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a MARCKSL1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a PSCA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a RAB3D polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a RDH11 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an SLC39A6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an SLC4A4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a SORD polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a STEAP1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a STEAP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a SYT7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a TMPRSS2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a TRPM4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an UNC13B polypeptide.In some embodiments, the target biomarkers in the aforementioned combinations may be used as targets for capture probes and / or targets for detection probes in the assays described herein.

[0185] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ABCC4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an APIM2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ARFGEF3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CANT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CD38 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CDH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CLDN3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CLDN4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CLGN polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ENPP5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a GOLM1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a GRHL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a MAP7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a MARCKSL1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a PSCA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a RAB3D polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an RDH11 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an SLC39A6 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an SLC4A4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a SORD polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a STEAP1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a STEAP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a SYT7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a TMPRSS2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a TRPM4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an UNC13B polypeptide. In some embodiments, the target biomarkers in the above combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0186] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ABCC4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an APIM2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ARFGEF3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CANT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CD38 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CDH1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CLDN3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CLDN4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CLGN polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ENPP5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a FOLH1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a GRHL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a MAP7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a MARCKSL1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a PSCA polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a RAB3D polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an RDH11 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an SLC39A6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an SLC4A4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a SORD polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a STEAP1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a STEAP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a SYT7 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a TMPRSS2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a TRPM4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an UNC13B polypeptide.In some embodiments, the target biomarkers in the aforementioned combinations may be used as targets for capture probes and / or targets for detection probes in the assays described herein.

[0187] In some embodiments, the target biomarker signature comprises a combination of at least three biomarkers, the combination being: an ABCC4 polypeptide, a CLDN3 polypeptide, and a PMEPA1 polypeptide; or an ABCC4 polypeptide, an APOO polypeptide, and a RAB3B polypeptide; or an ABCC4 polypeptide, a MARCKSL1 polypeptide, and a RAB3B polypeptide; or an ABCC4 polypeptide, a GOLM1 polypeptide, and a PODXL2 polypeptide; or an ABCC4 polypeptide, a RAB3B polypeptide, and a SMPDL3B polypeptide; or an ABCC4 polypeptide, a GOLM1 polypeptide, and a RAB3B polypeptide; or an ABCC4 polypeptide, a CLDN3 polypeptide, and a SLC39A6 polypeptide; or an ABCC4 polypeptide, a CLDN3 polypeptide, and a HOMER2 polypeptide; or an ABCC4 polypeptide, an AP1M2 polypeptide, and a PMEPA1 polypeptide; or an ABCC4 polypeptide, a CLDN3 polypeptide, and a PPP3CA polypeptide. or a PMEPA1 polypeptide, a PPP3CA polypeptide, and a TSPAN1 polypeptide; or a PMEPA1 polypeptide, an SLC39A6 polypeptide, and a TSPAN1 polypeptide; or a PPP3CA polypeptide, a RAB3B polypeptide, and an SMPDL3B polypeptide; or a CLDN4 polypeptide, a FOLH1 polypeptide, and a PODXL2 polypeptide; or a FOLH1 polypeptide, a PODXL2 polypeptide, and a TSPAN1 polypeptide; or a GOLM1 polypeptide, a PODXL2 polypeptide, and a SORD polypeptide; or a FOLH1 polypeptide, a MARCKSL1 polypeptide, and a TSPAN1 polypeptide; or an AP1M2 polypeptide, a FOLH1 polypeptide, and a PODXL2 polypeptide; or a CLN5 polypeptide, a FOLH1 polypeptide, and a TSPAN1 polypeptide; or a PMEPA1 polypeptide, a PODXL2 polypeptide, and a TSPAN1 polypeptide; or a GOLM1 polypeptide, a PPP3CA polypeptide, and a RAB3B polypeptide;or a GOLM1 polypeptide, a PODXL2 polypeptide, and a RAB3B polypeptide; or a CLDN4 polypeptide, a FOLH1 polypeptide, and a MARCKSL1 polypeptide; or a PODXL2 polypeptide, a SLC39A6 polypeptide, and a TSPAN1 polypeptide; or an ENPP5 polypeptide, a RAB3B polypeptide, and a SMPDL3B polypeptide; or a CLDN3 polypeptide, a PODXL2 polypeptide, and a SLC39A6 polypeptide; or a GOLM1 polypeptide, a MARCKSL1 polypeptide, and a RAB3B polypeptide; or a GOLM1 polypeptide, a RAB3B polypeptide, and a SLC39A6 polypeptide; or a FOLH1 polypeptide, an MBOAT2 polypeptide, and a SMPDL3B polypeptide; or a FOLH1 polypeptide, a PODXL2 polypeptide, and a SMPDL3B polypeptide; or a PPP3CA polypeptide, a SLC39A6 polypeptide, and TSPAN1 polypeptide; or a FOLH1 polypeptide and an HPN polypeptide. and an SMPDL3B polypeptide; or a PMEPA1 polypeptide, a RAB3D polypeptide, and a TSPAN1 polypeptide; or a MARCKSL1 polypeptide, a SLC39A6 polypeptide, and a TMPRSS2 polypeptide; or a GOLM1 polypeptide, a SLC39A6 polypeptide, and a TMPRSS2 polypeptide; or an ENPP5 polypeptide, a MARCKSL1 polypeptide, and a TMPRSS2 polypeptide; or a MARCKSL1 polypeptide, a PPP3CA polypeptide, and a TMPRSS2 polypeptide; or a GOLM1 polypeptide, a MARCKSL1 polypeptide, and a SORD polypeptide; or a CLGN polypeptide, a GOLM1 polypeptide, and a PPP3CA polypeptide; or an APOO polypeptide, a SLC39A6 polypeptide, and a TMPRSS2 polypeptide; or a MARCKSL1 polypeptide, a STEAP1 polypeptide, and a TMPRSS2 polypeptide; or an AP1M2 polypeptide, a CLGN polypeptide, and a PPP3CA polypeptide;or a CLGN polypeptide, a PPP3CA polypeptide, and an SMPDL3B polypeptide; or an AP1M2 polypeptide, an HPN polypeptide, and a STEAP2 polypeptide; or a CLGN polypeptide, an SLC39A6 polypeptide, and an SMPDL3B polypeptide; or a GRHL2 polypeptide, an HPN polypeptide, and a PPP3CA polypeptide; or a CLGN polypeptide, a GRHL2 polypeptide, and a MARCKSL1 polypeptide; or an AP1M2 polypeptide, a CLGN polypeptide, and an SLC4A4 polypeptide; or a CLGN polypeptide, a GRHL2 polypeptide, and an SMPDL3B polypeptide; or a CANT1 polypeptide, a CLGN polypeptide, and an SLC4A4 polypeptide; or a CANT1 polypeptide, a GRHL2 polypeptide, and an HPN polypeptide; or an ARFGEF3 polypeptide, an HPN polypeptide, and a TRPM4 polypeptide; or an EPCAM polypeptide, a HOMER2 polypeptide, and a STEAP2 polypeptide; or an EPCAM polypeptide, a SORD polypeptide or a GALNT3 polypeptide, an HPN polypeptide, and a STEAP2 polypeptide; or an ARFGEF3 polypeptide, an SLC4A4 polypeptide, and a SORD polypeptide; or a GRHL2 polypeptide, an SLC4A4 polypeptide, and a STEAP1 polypeptide; or a GALNT3 polypeptide, an HPN polypeptide, and a STEAP2 polypeptide; or a GRHL2 polypeptide, an LCP1 polypeptide, and a SLC4A4 polypeptide; or an HPN polypeptide, an MBOAT2 polypeptide, and an RDH11 polypeptide; or a GALNT3 polypeptide, a GNG4 polypeptide, and an HPN polypeptide; or a CANT1 polypeptide, an SLC4A4 polypeptide, and a SYT7 polypeptide; or an EPCAM polypeptide, a HOMER2 polypeptide, and a STEAP1 polypeptide; or a CLDN4 polypeptide, a STEAP1 polypeptide, and a SYT7 polypeptide; or a CANT1 polypeptide, an RDH11 polypeptide, and a SLC4A4 polypeptide; or an MBOAT2 polypeptide, an RDH11 polypeptide, and a SLC4A4 polypeptide;or a GNG4 polypeptide, an HPN polypeptide, and an MBOAT2 polypeptide; or an MBOAT2 polypeptide, an SLC4A4 polypeptide, and a SYT7 polypeptide; or an HPN polypeptide, an RDH11 polypeptide, and an SLC35F2 polypeptide; or a CLDN4 polypeptide, an ENPP5 polypeptide, and an RDH11 polypeptide; or a CDH1 polypeptide, an ENPP5 polypeptide, and a SYT7 polypeptide; or a CANT1 polypeptide, a CDH1 polypeptide, and an ENPP5 polypeptide; or an EPCAM polypeptide, a GNG4 polypeptide, and a HOMER2 polypeptide; or an ENPP5 polypeptide, an EPCAM polypeptide, and an RDH11 polypeptide; or a CLDN4 polypeptide, an ENPP5 polypeptide, and a GNG4 polypeptide; or a CLDN4 polypeptide, an ENPP5 polypeptide, and a STEAP1 polypeptide; or an ENPP5 polypeptide, a MARVELD2 polypeptide, and RDH11 polypeptide; or a CLDN4 polypeptide, an RDH11 polypeptide or a CLDN4 polypeptide, a GNG4 polypeptide, and a STEAP1 polypeptide; or a GALNT3 polypeptide, a GNG4 polypeptide, and a STEAP1 polypeptide; or an EPCAM polypeptide, a HOMER2 polypeptide, and an LCP1 polypeptide; or a HOMER2 polypeptide, an NAAA polypeptide, and a TMC5 polypeptide; or a CADM4 polypeptide, a SERINC5 polypeptide, and a TMC5 polypeptide; or an MIA3 polypeptide, a SERINC5 polypeptide, and a TMC5 polypeptide; or a BCAM polypeptide, a FAAH polypeptide, and a TMC5 polypeptide; or a RAP1GAP polypeptide, a SERINC5 polypeptide, and an SLC35F2 polypeptide; or a SERINC5 polypeptide, a TMC5 polypeptide, and a TMEM192 polypeptide; or a SERINC5 polypeptide, a TMC5 polypeptide, and a VWA1 polypeptide; or a BCAM polypeptide, a SERINC5 polypeptide, and a TMC5 polypeptide;or a RAP1GAP polypeptide, a SHROOM2 polypeptide, and a SLC35F2 polypeptide; or a BCAM polypeptide, an NAAA polypeptide, and a PRSS8 polypeptide; or an ATP2C1 polypeptide, a RAP1GAP polypeptide, and a SLC35F2 polypeptide; or a BCAM polypeptide, an FAAH polypeptide, and a PRSS8 polypeptide; or an APOO polypeptide, a CYB561 polypeptide, and a FAAH polypeptide; or a BCAM polypeptide, a CYB561 polypeptide, and a PRSS8 polypeptide; or a CYB561 polypeptide, a RAP1GAP polypeptide, and a SLC35F2 polypeptide; or a CYB561 polypeptide, a FAAH polypeptide, and a SERINC5 polypeptide; or a BCAM polypeptide, an MIA3 polypeptide, and a PRSS8 polypeptide; or a FAAH polypeptide, a RAP1GAP polypeptide, and a SLC35F2 polypeptide; or an APOO polypeptide, a FAAH polypeptide, and a SERINC5 polypeptide; or a BCAM polypeptide, a PRSS8 polypeptide, and a SERINC5 polypeptide; or and combinations thereof. In some embodiments, at least one target biomarker in the above combination may be used as a target for a capture probe, and at least two biomarkers may be used as targets for a detection probe;

[0188] In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, an APOO polypeptide, and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a MARCKSL1 polypeptide, and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a GOLM1 polypeptide, and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a RAB3B polypeptide, and a SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a GOLM1 polypeptide, and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and a SLC39A6 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and a HOMER2 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, an AP1M2 polypeptide, and a PMEPA1 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and a PPP3CA polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a PODXL2 polypeptide, and a RAB3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a PMEPA1 polypeptide, a PPP3CA polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a PMEPA1 polypeptide, a SLC39A6 polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a PPP3CA polypeptide, a RAB3B polypeptide, and a SMPDL3B polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CLDN4 polypeptide, a FOLH1 polypeptide, and a PODXL2 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FOLH1 polypeptide, a PODXL2 polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a GOLM1 polypeptide, a PODXL2 polypeptide, and a SORD polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FOLH1 polypeptide, a MARCKSL1 polypeptide, and a TSPAN1 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CLN5 polypeptide, a FOLH1 polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a PMEPA1 polypeptide, a PODXL2 polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a PMEPA1 polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a FOLH1 polypeptide, and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLGN polypeptide, and a GOLM1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and a FOLH1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a GOLM1 polypeptide, and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CDH1 polypeptide, and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLDN3 polypeptide, and an ENPP5 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ABCC4 polypeptide, a CLGN polypeptide, and a TSPAN1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPCAM polypeptide, a FOLH1 polypeptide, and a PMEPA1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CLGN polypeptide, a PPP3CA polypeptide, and a TSPAN1 polypeptide. In some embodiments, at least one target biomarker in the above combination may be used as a target for a capture probe, and at least two biomarkers may be used as targets for a detection probe. III. Exemplary Methods of Detecting Provided Markers and / or Target Biomarker Signatures for Prostate Cancer

[0189] In general, the present disclosure provides techniques whereby a target biomarker signature is analyzed and / or assessed in a bodily fluid-derived sample (e.g., but not limited to, a blood-derived sample, a urine-derived sample, etc.) containing extracellular vesicles from a subject in need thereof, and in some embodiments, a diagnostic or therapeutic decision is made based on such analysis and / or assessment.

[0190] In some embodiments, methods of detecting a target biomarker signature include methods for detecting one or more provided markers of the target biomarker signature as proteins, glycans, or proteoglycans (e.g., including but not limited to, proteins having carbohydrate or glycan moieties). Exemplary protein-based methods for detecting one or more provided markers include, but are not limited to, proximity ligation assays, immunoassays such as mass spectrometry (MS) and immunoprecipitation, Western blots, ELISAs, immunohistochemistry, immunocytochemistry, flow cytometry, and immuno-PCR. In some embodiments, the immunoassay may be a chemiluminescent immunoassay. In some embodiments, the immunoassay may be a high-throughput and / or automated immunoassay platform.

[0191] In some embodiments, methods for detecting one or more provided markers as proteins, glycans, or proteoglycans (e.g., including but not limited to, proteins having carbohydrate or glycan moieties) in a sample include contacting the sample with one or more antibody agents directed to the provided markers of interest. In some embodiments, such methods also include contacting the sample with one or more detection labels. In some embodiments, the antibody agents are labeled with one or more detection labels.

[0192] In some embodiments, detecting binding between a biomarker of interest and an antibody agent for the biomarker of interest involves determining an absorbance or luminescence value for one or more detection agents. For example, the absorbance or luminescence value indicates the amount and / or concentration of the biomarker of interest expressed by the extracellular vesicles (e.g., a higher absorbance indicates a higher level of the biomarker of interest expressed by the extracellular vesicles). In some embodiments, the absorbance or luminescence value for the detection agent exceeds a threshold value. In some embodiments, the absorbance or luminescence value for the detection agent is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 3.5 times higher than the threshold value. In some embodiments, the threshold value is determined for the entire control or reference group population (e.g., non-cancer subjects).

[0193] In some embodiments, methods for detecting one or more provided markers include methods for detecting one or more provided markers as nucleic acids. Exemplary nucleic acid-based methods for detecting one or more provided markers include, but are not limited to, nucleic acid amplification methods, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, nucleic acid-based methods for detecting one or more provided markers include detecting hybridization between one or more nucleic acid probes and one or more nucleotide sequences encoding the biomarkers of interest. In some embodiments, the nucleic acid probes are each complementary to at least a portion of one of the one or more nucleotide sequences encoding the biomarkers of interest. In some embodiments, the nucleotide sequence encoding the biomarkers of interest comprises DNA (e.g., cDNA). In some embodiments, the nucleotide sequence encoding the biomarkers of interest comprises RNA. In some embodiments, the nucleotide sequence encoding the biomarkers of interest may be or comprise mRNA. In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a microRNA. In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a non-coding RNA, which in some embodiments may be or comprise an orphan non-coding RNA (oncRNA). In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a long non-coding RNA (lncRNA). In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a piwi-interacting RNA (piwiRNA).In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a circular RNA (circRNA). In some embodiments, the nucleotide sequence encoding the biomarker of interest may be or comprise a small nucleolar RNA (snoRNA).

[0194] In some embodiments, the method for detecting one or more provided markers comprises proximity ligation-immunoquantitative polymerase chain reaction (pliq-PCR). Pliq-PCR may have certain advantages over other techniques for profiling EVs. For example, pliq-PCR may have a sensitivity three orders of magnitude higher than other standard immunoassays, such as ELISA (Darmanis et al., 2010; which is incorporated herein by reference for purposes described herein). In some embodiments, pliq-PCR reactions can be designed to have an extremely low LOD, allowing for the detection of trace levels of tumor-derived EVs, for example, up to 1,000 EVs per mL.

[0195] In some embodiments, methods for detecting one or more provided markers include, for example, detecting approximately 10 3 and about 10 4 LODs for EVs have been reported (Shao et al., 2018; which is incorporated herein by reference for purposes described herein), and other technologies for detecting EVs may include the Nanoplasmic Exosome (nPLEX) sensor (Im et al., 2014; which is incorporated herein by reference for purposes described herein) and the Integrated Magnetic-Electrochemical Exosome (iMEX) sensor (Jeong et al., 2016; which is incorporated herein by reference for purposes described herein).

[0196] In some embodiments, methods for detecting one or more provided biomarkers in extracellular vesicles can be based on bulk EV sample analysis.

[0197] In some embodiments, methods for detecting one or more provided biomarkers in extracellular vesicles can be based on profiling individual EVs (e.g., single-EV profiling assays), which are further discussed below in the section entitled "Exemplary Methods for Profiling Individual Nanoparticles with a Size Range of Interest, Including Extracellular Vesicles (EVs)."

[0198] Those skilled in the art reading this disclosure will understand that the assays described herein for detecting or profiling individual EVs can also be used to detect combinations of biomarkers on the surface of nanoparticles (e.g., as described herein) having a size range of interest, including extracellular vesicles (e.g., as described herein).

[0199] In some embodiments, nanoparticles having a desired size range, including extracellular vesicles, in a sample may be captured or immobilized on a solid substrate prior to detecting one or more provided biomarkers according to the present disclosure. In some embodiments, nanoparticles having a desired size range, including extracellular vesicles, may be captured on the surface of a solid substrate by non-specific interactions, including adsorption, for example. In some embodiments, nanoparticles having a desired size range, including extracellular vesicles, may be selectively captured on the surface of a solid substrate. For example, in some embodiments, the surface of the solid substrate may be coated with an agent that specifically binds to nanoparticles having a desired size range, including extracellular vesicles (e.g., an antibody agent that specifically targets such nanoparticles, e.g., nanoparticles associated with prostate cancer). In some embodiments, the surface of the solid substrate may be coated with a member of an affinity binding pair, and the captured entity of interest (e.g., extracellular vesicles) may be conjugated to a complementary member of the affinity binding pair. In some embodiments, exemplary affinity binding pairs include, but are not limited to, biotin and avidin-like molecules, such as streptavidin. As will be understood by those skilled in the art, other suitable affinity binding pairs can also be used to facilitate capture of the entity of interest on the solid substrate surface. In some embodiments, the entity of interest can be captured on the solid substrate surface by application of an electric current, as described, for example, in Ibsen et al. ACS Nano., 11: 6641-6651 (2017) and Lewis et al. ACS Nano., 12: 3311-3320 (2018), both of which are incorporated herein by reference for purposes described herein, and which describe the use of an alternating current electrokinetic microarray chip device to isolate extracellular vesicles from undiluted human blood or plasma samples.

[0200] The solid substrate may be provided in a form suitable for capturing nanoparticles having a desired size range, including extracellular vesicles, that do not interfere with downstream handling, processing, and / or detection. For example, in some embodiments, the solid substrate may be or include beads (e.g., magnetic beads). In some embodiments, the solid substrate may be or include a surface. For example, in some embodiments, such a surface may be the capture surface of an assay chamber (e.g., including a tube, well, microwell, plate, filter, membrane, matrix, etc.). Thus, in some embodiments, the methods described herein include capturing or immobilizing nanoparticles having a desired size range, including extracellular vesicles, on a solid substrate prior to detecting biomarkers provided in a sample.

[0201] In some embodiments, a sample may be processed to remove undesirable entities, such as cellular debris or cells, before capturing nanoparticles having a desired size range, including extracellular vesicles, on a solid substrate surface. For example, in some embodiments, such a sample may be subjected to centrifugation, for example, to remove cellular debris, cells, and / or other particles. Additionally or alternatively, in some embodiments, such a sample may be subjected to size-exclusion-based purification or filtration. Various size-exclusion-based purification or filtration methods are known in the art, and those skilled in the art will recognize that in some cases, a sample may be subjected to spin-column purification based on a specific molecular weight or particle size cutoff. Those skilled in the art will also recognize that an appropriate molecular weight or particle size cutoff for purification purposes can be selected based on, for example, the size of the extracellular vesicles. For example, in some embodiments, a size-exclusion separation method may be applied to a sample containing extracellular vesicles to isolate a fraction of nanoparticles containing extracellular vesicles of a certain size (e.g., greater than 30 nm and less than or equal to 1000 nm, or greater than 70 nm and less than or equal to 200 nm). Typically, extracellular vesicles may range in diameter from 30 nm to several micrometers. See, for example, Chuo et al., "Imaging extracellular vesicles: current and emerging methods," Journal of Biomedical Sciences 25: 91 (2018), which provides size information for different extracellular vesicle (EV) subtypes: migrasomes (0.5-3 μm), microvesicles (0.1-1 μm), oncosomes (1-10 μm), exosomes (<50 nm), small exosomes (60-80 nm), and large exosomes (90-120 nm). In some embodiments, nanoparticles having a size range of approximately 30 nm to 1000 nm may be isolated by one or more size-exclusion separation methods, e.g., for detection assays in some embodiments.In some embodiments, specific EV subtypes may be isolated by one or more size exclusion separation methods, e.g., in some embodiments, for detection assays.

[0202] In some embodiments, nanoparticles having a desired size range, including extracellular vesicles, in a sample may be processed before detecting one or more provided biomarkers of a target biomarker signature for prostate cancer. Different sample processing and / or preparation can be performed, for example, to stabilize the target (e.g., target biomarker) in the extracellular vesicles to be detected, and / or to facilitate the exposure of the target (e.g., intravesicular proteins and / or RNA such as mRNA) to a detection assay (e.g., as described herein), and / or to reduce non-specific binding. Examples of such sample processing and / or preparation are known in the art and include, but are not limited to, crosslinking (e.g., fixation) of molecular targets, permeabilization of biological entities (e.g., nanoparticles having a desired size range, including cells or extracellular vesicles), and / or blocking of non-specific binding sites.

[0203] In one aspect, the present disclosure provides a method for detecting the presence or absence of a target biomarker signature for prostate cancer in a biological sample from a subject in need thereof, which may, in some embodiments, be a biological sample (e.g., a blood-derived sample, a urine-derived sample, etc.) containing nanoparticles having a desired size range, including extracellular vesicles. In some embodiments, such a method includes: (a) detecting a biological entity of interest having the target biomarker signature for prostate cancer (e.g., containing nanoparticles having a desired size range, including extracellular vesicles) in a biological sample from the subject, such as a blood-derived sample (e.g., a plasma sample); and (b) comparing sample information indicating the level of the target biomarker signature-expressing biological entity of interest (e.g., nanoparticles having a desired size range, including extracellular vesicles) in the biological sample (e.g., a blood-derived sample) with reference information including a reference threshold level. In some embodiments, the reference threshold level corresponds to the level of the biological entity of interest expressing such target biomarker signature (e.g., nanoparticles having a desired size range, including extracellular vesicles) in a comparable sample from a reference subject, e.g., a population of non-cancer subjects. In some embodiments, exemplary non-cancer subjects include healthy subjects (e.g., healthy subjects within a predetermined age range, such as under or over 55 years of age), subjects with a non-prostate-related health disease, disorder, or condition (including, e.g., subjects with a non-prostate cancer such as lung cancer, ovarian cancer, etc., or subjects with symptoms of an inflammatory disease or disorder), subjects with benign prostatic hyperplasia, and combinations thereof.

[0204] In some embodiments, samples are pre-screened for certain characteristics before use in the assays described herein. In some embodiments, samples that meet certain pre-screening criteria are more suitable for diagnostic applications than samples that do not meet the pre-screening criteria. For example, in some embodiments, samples are visually inspected for appearance using known standards, such as normal, hemolyzed (red), icteric (yellow), and / or fatty (whitish / cloudy) samples. In some embodiments, the samples can then be graded on a known standard scale (e.g., 1, 2, 3, 4, 5), and the results are recorded. In some embodiments, samples are visually inspected for hemolysis (e.g., heme) and graded on a scale of 1 to 5, where visual inspection correlates with known concentrations, e.g., 1 indicates approximately 0 mg / dL, 2 indicates approximately 50 mg / dL, 3 indicates approximately 150 mg / dL, 4 indicates approximately 250 mg / dL, and 5 indicates approximately 525 mg / dL. In some embodiments, samples are visually inspected for jaundice levels (e.g., bilirubin) and graded on a scale of 1 to 5, where visual inspection correlates to known concentrations, e.g., 1 indicates approximately 0 mg / dL, 2 indicates approximately 1.7 mg / dL, 3 indicates approximately 6.6 mg / dL, 4 indicates approximately 16 mg / dL, and 5 indicates approximately 30 mg / dL. In some embodiments, samples are visually inspected for turbidity (e.g., lipids) and graded on a scale of 1 to 5, where visual inspection correlates to known concentrations, e.g., 1 indicates approximately 0 mg / dL, 2 indicates approximately 125 mg / dL, 3 indicates approximately 250 mg / dL, 4 indicates approximately 500 mg / dL, and 5 indicates approximately 1000 mg / dL.

[0205] In some embodiments, samples scoring below a certain level in one or more metrics, e.g., equal to or less than a score of 4, may be utilized in the assays described herein. In some embodiments, samples scoring below a certain level in one or more metrics, e.g., equal to or less than a score of 3, may be utilized in the assays described herein. In some embodiments, samples scoring below a certain level in one or more metrics, e.g., equal to or less than a score of 2, may be utilized in the assays described herein. In some embodiments, samples scoring below a certain level in all three metrics (e.g., hemolysis, icterus, and lipemia), e.g., equal to or less than a score of 2, may be utilized in the assays described herein. In some embodiments, low visual inspection scores (e.g., equal to or less than a score of 2) in pre-screening criteria such as hemolysis, bilirubin, and / or lipemia may not have a clear effect on (e.g., may not correlate with) the diagnostic characteristics (e.g., Ct values) generated in the assays described herein.

[0206] In some embodiments, a sample is determined to be positive for the presence of a target biomarker signature (e.g., as described herein) if the sample shows an elevated level of nanoparticles (having a size range of interest, including extracellular vesicles) that display the target biomarker signature on their surface compared to a reference threshold level (e.g., as described herein). In some embodiments, a sample is determined to be positive for the presence of a target biomarker signature (e.g., as reflected by the level of target biomarker signature-expressing extracellular vesicles) if the level is at least 30% or higher, including, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher, compared to the reference threshold level. In some embodiments, a sample is determined to be positive for the presence of a target biomarker signature (e.g., as reflected by the level of target biomarker signature-expressing extracellular vesicles) if the level is at least 2-fold or higher, including, for example, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 2500-fold, at least 5000-fold, or higher, compared to a reference threshold level.

[0207] In some embodiments, a binary classification system may be used to determine whether a sample is positive for the presence of a target biomarker signature. For example, in some embodiments, a sample is determined to be positive for the presence of a target biomarker signature when its level is at or above a reference threshold level, e.g., a cutoff value (e.g., as reflected by the level of target biomarker signature-expressing extracellular vesicles). In some embodiments, such a reference threshold level (e.g., a cutoff value) may be determined by selecting a certain number of standard deviations from the mean value obtained from control subjects, so that the desired sensitivity and / or specificity of the prostate cancer detection assay (e.g., that described herein) can be achieved. In some embodiments, such a reference threshold level (e.g., a cutoff value) may be determined by selecting a certain number of standard deviations from the maximum assay signal obtained from control subjects, so that the desired sensitivity and / or specificity of the prostate cancer detection assay (e.g., that described herein) can be achieved. In some embodiments, such a reference threshold level (e.g., cut-off value) may be determined by selecting the less restrictive of (i) a certain number of standard deviations from the mean value obtained from control subjects, or (ii) a certain number of standard deviations from the maximum assay signal obtained from control subjects, so that the desired sensitivity and / or specificity of the prostate cancer detection assay (e.g., as described herein) can be achieved. In some embodiments, the control subjects for determining the reference threshold level (e.g., cut-off value) may include, but are not limited to, healthy subjects, subjects with inflammatory conditions (e.g., benign prostatic hyperplasia, prostatitis, etc.), subjects with benign tumors, and combinations thereof. In some embodiments...

Claims

A method for obtaining the amount of extracellular vesicles as an indicator for screening prostate cancer, the method comprising: contacting a biological sample derived from a subject with a capture agent, wherein the capture agent specifically binds to a first prostate cancer biomarker present on the surface of the extracellular vesicles; contacting the captured extracellular vesicles with a first oligonucleotide probe, wherein the first oligonucleotide probe specifically binds to a second prostate cancer biomarker; contacting the captured extracellular vesicles with a second oligonucleotide probe, wherein the second oligonucleotide probe specifically binds to a third prostate cancer biomarker; measuring the amount of extracellular vesicles to which the capture agent, the first oligonucleotide probe, and the second oligonucleotide probe simultaneously bind; and comparing the amount with a threshold value, wherein an amount exceeding the threshold value indicates that the subject has prostate cancer A method comprising. Claim 2. The second and third biomarkers are selected from the group consisting of surface biomarkers and intravesicular biomarkers. wherein the surface biomarker is (i) a polypeptide encoded by the following human genes: ABCC4, ABHD17C, ADI1, AGTARP, AP1M2, APOO, ARFGEF3, ATP2C1, BCAM, CADM4, CANT1, CDH1, CHMP4C, CLDN3, CLDN4, CLGN, CLN5, CYB561, DNAJC30, ENPP5, EPCAM, ERGIC1, FAAH, FOLH1, GALNT3, GNG4, GNPNAT1, GOLM1, GRHL2, HID1, HOMER2, HPN, LCP1, LRIG1, MAP7, MARCKSL1, MARVELD2, MBOAT2, MIA3, MUC1, NAAA, NDUFA2, PMEPA1, PODXL2, PPP3CA, PRSS8, RAB3B, RAB3D, RAP1GAP, RDH11, SCARB2, SERINC5, SFXN2, SHROOM2, SHROOM3, SLC35F2, SLC39A6, SLC39A7, SLC4A4, SMPDL3B, SORD, STEAP1, STEAP2, SYNGR2, SYT7, TMC5, TMED3, TMEM141, TMEM192, TMEM9, TMPRSS2, TRPM4, TSPAN1, UNC13B, VWA1, YIPF1, ADAM17, CCL2, CD274, CD38, CLEC2D, ERBB2, FLNA, FLNB, GPC1, IL6, ITGAV, KLK3, KLKB1, PLAC1, PPP1R3A, PSCA, PVR, SLC44A4, TGFBR2, TNFRSF4, TNFSF11, VEGFC, AZGP1, GLB1L2, PABPC1L2A, SPOCK1, TGM4; and / or (ii) a carbohydrate-dependent marker selected from Tn antigen, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T, TF) antigen, Lewis Y antigen (also known as CD174), sialyl Lewis X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), and combinations thereof; and The intracellular biomarker is the following human gene: ACPP, ACSM1, ACTA2, ACTG2, ADAMTS1, AGR2, AIM1, ALCAM, ALDH1A3, ALOX15B, ANTXR1, ANTXR2, AP1M2, AR, ARG2, ARHGAP6, ARHGEF26, ARHGEF37, ASAP3, ATP8B1, ATP9A, BAIAAP2L1, BCAM, BHLHA15, BOK, C15orf48, C19orf48, C1S, C1orf116, C2orf72, C8orf4, C9orf152, CADM4, CALD1, CAMSAP3, CAPN5, CBLC, CD24, CD74, CDC42EP1, CDC42EP5, CGN, CGNL1, CHMP4C, CLGN, CMTM4, COLCA1, CORO2A, CPE, CPNE4, CPQ, CRYM, CTSF, CX3CL1, CXADR, CXCR4, CYB561, DPYSL3, DSP, EEF1A2, EFNA1, EHD2, EHF, ELF3, EMP2, EPHX1, EPN3, ERBB3, ERG, ESRP1, ESRP2, F3, FAAH, FAM129A, FAM129B, FAM210B, FAM3B, FAM3D, FAM83H, FAM84A, FAM84B, FAT1, FBLIM1, FBP1, FLNC, FNBPL1, FOS, FOXA1, GADD45G, GATA2, GGT1, GJB1, GLYATL1, GNG4, GNMT, GRHL2, GUCY1A3, HGD, HID1, HIST1H2BK, HIST3H2A, HMGCS2, HOMER2, HOXB13, HSD17B6, HSPB6, HSPB8, ICA1, ID1, IQGAP2, IRF6, ITGA6, KIAA1522, KIF5C, KLK2, KRT14, KRT15, KRT17, KRT18, KRT19, KRT5, KRT7, KRT8, LCP1, LIFR, LIMCH1, LMOD1, LRP11, LRP3, LSR, MAL2, MAOA, MAP7, MARC1, MARCKSL1, MB, MESP1, MLPH, MME, MPZL2, MT1G, MYBPC1, MYL9, MYLK, NCAPD3, NEDD4L, NEFH, NFIX, NKX3-1, NPDC1, NUPR1, OBSL1, PALLD, PARVA, PCDH1, PCP4, PDIA5, PEBP4, PGM5, PKP1,The method according to claim 1, selected from polypeptides encoded by PKP3, PPL, PPM1H, PPP1R1B, PPP3CA, PRAC1, PRR15L, PSAAT1, PTPRF, PYCR1, RAB3B, RAMP1, RAP1GAP, RASEF, RBM47, RCAN3, RDH11, REEP6, REPS2, RGS10, RHP N2, RNF144B, RORC, RPS4Y1, S100A16, SELENBP1, SERINC5, SH3BGR L2, SH3BP4, SHC2, SLC1A5, SLC22A3, SLC2A12, SLC40A1, SLC4A4, SLC7A8, SLC9A3R2, SMOC2, SORD, SPDEF, SPINT1, SPINT2, SPTBN2, ST14, STAP2, STK39, SULT2B1, SYNE4, SYT7, SYTL1, TAGLN, TBC1D16, TBX3, TC2N, TCEA3, TEAD3, TESC, THBS4, TM4SF1, TMED3, TMEM150C, TMEM54, TMEM98, TMSB15A, TP53I11, TPD52, TPM1, TSPAN13, TSPAN6, TSPAN8, VAMP8, VSTM2L, WLS, WWC1, YAP1, ZBTB42, ZC3H11A, ZNF217, ACSL3, CADH7, KCNN2, OPRK1, PDE9A, PLPP1, SPN2, TMEM121B, and combinations thereof., **Claim 3**: The method according to claim 1, wherein the first biomarker is a polypeptide encoded by the following human genes: CLDN3, ABCC4, RAB3B, FOLH1, PMEPA1, TMPRSS2, TSPAN1, PODXL2, GOLM1, SORD, CLGN, GRHL2, CANT1, SYT17, PPP3CA, or a combination thereof, or comprises the same. **Claim 4**: The method according to claim 1, wherein the capture agent comprises an antibody. **Claim 5**: The method according to claim 4, wherein the antibody is attached to a solid substrate. **Claim 6**: The method according to claim 5, wherein the solid substrate is magnetic beads. **Claim 7**: The method according to claim 1, wherein the sample is subjected to size exclusion chromatography to isolate the extracellular vesicles. **Claim 8**: The method according to claim 1, wherein the second biomarker is a surface biomarker, and the surface biomarker and the first biomarker are different. **Claim 9**: The second and third biomarkers are in the following combinations: - at least two distinct surface biomarkers; - at least two distinct intra-vesicular biomarkers; and - a surface biomarker and an intra-vesicular biomarker; The method according to claim 1, comprising one of the above. **Claim 10**: The method according to claim 1, wherein the threshold value is determined by the amount of extracellular vesicles to which the capture agent, the first oligonucleotide probe, and the second oligonucleotide probe simultaneously bind, as observed in comparable samples from a population of non-cancer subjects. **Claim 11**: The method according to claim 10, wherein the population of non-cancer subjects comprises one or more of the following subject populations: healthy subjects, subjects diagnosed with benign tumors, subjects having colon-related diseases, and subjects having non-colon-related diseases, disorders, and / or conditions. **Claim 12**: The method according to claim 1, wherein when the first and / or second biomarker comprises at least one surface biomarker and / or intra-vesicular biomarker, the detection assay comprises an immunoassay. **Claim 13**: The method according to claim 12, wherein the detection assay comprises a proximity ligation assay. **Claim 14**: The method according to claim 1, wherein the first and second oligonucleotide probes target the same biomarker. The method according to claim 14, wherein the oligonucleotides of the first and second oligonucleotide probes are different. The method according to claim 1, wherein the method is performed to obtain an amount of extracellular vesicles as an indicator for screening for early prostate cancer, advanced prostate cancer, or recurrent prostate cancer in the subject. The subject has the following characteristics: (i) An asymptomatic subject prone to prostate cancer; (ii) A subject with a family history of prostate cancer; (iii) A subject who is a smoker or who was a smoker; (iv) An obese subject; (v) A subject who consumes an excessive amount of alcohol; (vi) A subject 40 years of age or older; (vii) A subject having one or more non-specific symptoms of prostate cancer, wherein, optionally, at least one of the non-specific symptoms resembles one or more general gastrointestinal symptoms associated with a non-cancerous disease, disorder, or condition; (viii) A subject for whom imaging, such as an X-ray, CT scan, or low-dose CT scan, is recommended; (ix) A subject diagnosed with a prostate tumor confirmed by imaging; (x) A subject having a benign colon tumor; (xi) A subject previously treated for prostate cancer; (xii) A subject determined to have an inflammatory bowel disease; (xiii) A subject determined to have ulcerative colitis or Crohn's disease; (xiv) A subject having a large amount of current or past alcohol consumption; (xv) A subject having a hereditary mutation in a gene associated with hereditary polyposis syndrome and / or a gene associated with hereditary colorectal cancer syndrome; and (xvi) A subject exposed to radiotherapy and / or chemotherapy The method according to claim 1, having at least one or more of the above. The method includes the following health evaluations and / or diagnostic assays: (i) An annual health check-up of the subject; (ii) An imaging examination; (iii) A digital rectal examination; (iv) A genetic assay for screening plasma for gene mutations in circulating tumor DNA and / or cancer-associated protein biomarkers; (v) An assay including immunofluorescence staining to identify cell phenotypes and marker expression, followed by amplification and analysis by next-generation sequencing; (vi) An immunochemical fecal occult blood test (FIT); and (vii) Serum biomarkers The method according to claim 1, used in combination with one or more of them.

19. The method according to claim 1, wherein the prostate cancer is prostate adenocarcinoma.

20. The method according to claim 1, wherein the method is performed to obtain the amount of extracellular vesicles as an indicator for monitoring prostate cancer patients with respect to the response to treatment of anti-prostate cancer therapy (e.g., surgery, radiotherapy, chemotherapy, radiosurgery, targeted drug therapy, immunotherapy) and / or for cancer recurrence / metastasis.

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