Compositions and methods for the detection of colorectal cancer

Abstract

In one aspect, the present disclosure provides a technique for detecting colorectal cancer, for example, for early detection of colorectal cancer.In another aspect, the technique provided herein is useful for selecting and / or monitoring and / or evaluating the effectiveness of treatments administered to subjects who have been determined to have or be susceptible to colorectal cancer.In some embodiments, the technique provided herein is useful for developing companion diagnostics, for example, by measuring tumor burden and changes in tumor burden 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 / 224,378, 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 colorectal cancer, still lack either effective screening recommendations or patient compliance with such recommendations. Typical challenges for cancer screening tests include limited sensitivity and specificity. High false positive results can be particularly concerning, as they 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 results will miss patients who need therapy, resulting in delayed treatment and consequently reduced chances of success, thus failing to 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 colorectal 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 colorectal adenocarcinoma screening. In some embodiments, the provided techniques are effective for detecting early-stage colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the provided techniques are effective even when applied to populations that include or consist 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 populations that include or consist of individuals without a genetic risk for developing colorectal cancer (e.g., colorectal adenocarcinoma) (e.g., asymptomatic individuals). In some embodiments, the provided techniques are effective when applied to populations that include or consist of symptomatic individuals (e.g., individuals suffering from one or more symptoms of colorectal cancer). In some embodiments, the provided technology is effective when applied to a population that includes or consists of individuals at risk for colorectal cancer (e.g., individuals with genetic and / or lifestyle-related risk factors for colorectal 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 colorectal cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as colonoscopy, stool tests, CT scanning, and / or cell-free nucleic acid-based molecular tests, serum biomarkers, and / or bulk analysis of extracellular vesicles, 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, detecting the co-localization of a target biomarker signature for colorectal 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 colorectal cancer using, among other things, a target entity detection approach 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, 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, and entitled "Systems, Compositions, and Methods for Target Entity Detection."

[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 colorectal 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 colorectal cancer (e.g., in some embodiments, colorectal adenocarcinoma). In some embodiments, the present disclosure provides colorectal cancer screening systems that can be implemented to detect colorectal cancer (e.g., in some embodiments, colorectal 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 the same. 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 characteristics of colorectal cancer (e.g., incidence, progression, responsiveness 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, colorectal 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 colorectal cancer (e.g., in some embodiments, colorectal 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 colorectal cancer (e.g., in some embodiments, colorectal adenocarcinoma). In some embodiments, the provided methods or assays include (a) detecting extracellular vesicles expressing a target biomarker signature of colorectal cancer (e.g., in some embodiments, colorectal adenocarcinoma) in a body fluid-derived sample (e.g., without limitation, a blood-derived sample, a stool-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 stool-derived sample, etc.) with reference information including a reference threshold level; and (c) classifying the subject as having or susceptible to colorectal cancer (e.g., in some embodiments, colorectal adenocarcinoma) if the body fluid-derived sample (for example, but not limited to, a blood-derived sample, a stool-derived sample, etc.) exhibits 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: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CL IC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GP CR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCK SL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B , RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD, SSR4, ST14, STO Polypeptides encoded by ML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, and combinations thereof;and / or (ii) a carbohydrate-dependent marker selected from the following: CanAg (a glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3 (N-glycolyl GM3 ganglioside), and combinations thereof;

[0010] In some embodiments, the one or more surface biomarkers that may be included in the target biomarker signature are selected from: (i) polypeptides encoded by the following human genes: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, and combinations thereof; 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.

[0011] In some embodiments, one or more intravesicular biomarkers that may be included in the target biomarker signature are the following human genes: AGMAT, AGR2, AGR3, ANKS4B, AP1M2, ARSE, ASCL2, BSPRY, C10orf99, C15orf48, C1orf106, C9orf152, CBLC, CCL24, CDCA7, CDX1, CDX2, DDC, DSG2, EHF, ELF3, EPS8L3, ESRP1, ESRP2, ETV4, EVPL, FABP1, FAM3D, FAM83E, FAM84A, FERMT1, FOXA2, FOXA3, FOXA4, FOXA5, FOXA6, FOXA7, FOXB1, FOXB2, FOXB3, FOXB4, FOXB5, FOXB6, FOXB7, FOXB8, FOXB9, FOXB10, FOXB11, FOXB12, FOXB13, FOXB14, FOXB15, FOXB16, FOXB17, FOXB18, FOXB19, FOXB20, FOXB21, FOXB22, FOXB23, FOXB24, FOXB25, FOXB26, FOXB27, FOXB28, FOXB29, FOXB29, FOXB30, FOXB31, FOXB32, FOXB33, FOXB34, FOXB35, FOXB36, FOXB37, FOXB38, FOXB39, FOXB40, FOXB41, FOXB42, FOXB43, FOXB44, FOXB45, FOXB46, FOXB47, FOXB48, FOXB49, FOXB49, FOX 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 are selected from the following human genes: AGMAT, AGR2, AGR3, ANKS4B, ANO9, AP1M2, ARSE, ASCL2, ATP10B, B3GNT3, BIK, BSPRY, C10orf99, C15orf48, C1orf106, C1orf210, C9orf152, CA12, CBLC, CCL24, CD24, CDCA7, CDH1, CDH17, CDH3, CDHR1, CDHR5, CDX1, CDX2, CDX3, CDX4, CDX5, CDX6, CDX7, CDX8, CDX9, CDX10, CDX11, CDX12, CDX13, CDX14, CDX15, CDX16, CDX17, CDX18, CDX19, CDX19, CDX19, CDX12, CDX15, CDX16, CDX19, CDX19, CDX20, CDX21, CDX22, CDX23, CDX24, CDX30, CDX31, CDX32, CDX33, CDX34, CDX35, CDX36, CDX37, CDX38, CDX40, CDX41, CDX42, CDX43, CDX44, CDX45, CDX46, CDX47, CDX48, CDX49, CDX50, CDX51, CDX52, CDX53, CDX54, CDX55, CDX56, CDX57, CDX58, CDX59, CDX60, CDX61, CDX62, CDX63, CDX64, CDX75, CDX76, CDX77, CDX78 2, CEACAM5, CEACAM6, CEACAM7, CFTR, CLDN2, CLDN3, CLDN4, CLDN7, CLRN3, COL17A1, CRB3, CYP2S1, DDC, DPEP1, DSG2, EHF, ELF3, EPCAM, EPHB3, EPS8 L3, ERN2, ESRP1, ESRP2, ETV4, EVPL, FA2H, FABP1, FAM3D, FAM83E, FAM84A, FAT1, FERMT1, FOXA2, FOXA3, FOXQ1, FUT2, FUT3, FXYD3, GCNT3, GGT6, GJB1 , GJB3, GPA33, GPR160, GPR35, GPX2, GRB7, GUCY2C, HKDC1, HMGCS2, HNF4A, HOXB9, IHH, ITLN1, KCNN4, KIAA1324, KLK1, KRT20, KRT23, KRT8, LGALS4, LGR5, LY6G6D, MEP1A, METTL7B, MISP, MUC13, MUC2, MYB, MYBL2, MYO1A, NOX1, PDZK1IP1, PHGR1, PIGR, PITX1, PKP3, PLAC8, PLEK2, PLS1, POF1B, PPP1R 14D, PROM1, PRR15, PRSS8, PTK6, RAB25, RNF128, RNF186, RNF43, S100A14, S100P, SAPCD2, SERPINB5, SLC26A3, SLC39A5, SLC44A4, SLC5A1, SMIM22, S PDEF, ST6GALNAC1, TJP3, TM4SF5, TMC5, TMEM45B, TMPRSS2, TMPRSS4, TNS4, TRABD2A, TRIM15, TRIM31, TSPAN1, TSPAN8, UGT2B17, UGT8, USH1C, VIL1,and RNA transcripts (e.g., mRNA transcripts) encoded by the 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, extracellular vesicle-associated surface biomarkers for use in the target biomarker signatures for colorectal cancer used and / or described herein may be or include tumor-specific and / or tissue-specific biomarkers (e.g., colon and / or rectum 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 the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MA RCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RA P2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD , SSR4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2 , FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA,or any combination thereof; and / or (ii) one or more surface proteins encoded by CanAg (a glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, and combinations thereof.

[0015] In some embodiments, the extracellular vesicle-associated surface biomarker may be or may include (i) one or more of the polypeptides encoded by the human gene MUC1; and / or (ii) one or more of 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, or a combination thereof.

[0016] In some embodiments, a target biomarker signature for colorectal cancer (e.g., colorectal 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 include the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCA P31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDA R, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22 one or more polypeptides encoded by , SNTB1, SORD, SSR4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA;and / or may be or include one or more of the following carbohydrate markers: CanAg (glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, or a combination thereof;

[0017] In some embodiments, a target biomarker signature for colorectal cancer (e.g., colorectal adenocarcinoma) may include an extracellular vesicle-associated surface biomarker (e.g., those described herein) and at least one (e.g., including 1, 2, 3, or more) additional surface biomarkers, which may be derived from (i) polypeptides encoded by the following human genes: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: Lewis It is selected from 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 colorectal cancer (e.g., colorectal 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: AGMAT, AGR2, AGR3, ANKS4B, ANO9, AP1M2, ARSE, ASCL2, ATP10B, B3GNT3, BIK, BSPRY, C10orf99, C15orf48, C1orf106, C1orf210, C9orf1 52, CA12, CBLC, CCL24, CD24, CDCA7, CDH1, CDH17, CDH3, CDHR1, CDHR5, CDX1, CDX2, CEACAM5, CEACAM6, CEACAM7, CFTR, CLDN2, CLDN3, CLDN4, CLDN7, CLRN 3, COL17A1, CRB3, CYP2S1, DDC, DPEP1, DSG2, EHF, ELF3, EPCAM, EPHB3, EPS8L3, ERN2, ESRP1, ESRP2, ETV4, EVPL, FA2H, FABP1, FAM3D, FAM83E, FAM84A, FA T1, FERMT1, FOXA2, FOXA3, FOXQ1, FUT2, FUT3, FXYD3, GCNT3, GGT6, GJB1, GJB3, GPA33, GPR160, GPR35, GPX2, GRB7, GUCY2C, HKDC1, HMGCS2, HNF4A, HOXB9 , IHH, ITLN1, KCNN4, KIAA1324, KLK1, KRT20, KRT23, KRT8, LGALS4, LGR5, LY6G6D, MEP1A, METTL7B, MISP, MUC13, MUC2, MYB, MYBL2, MYO1A, NOX1, PDZK1IP 1, PHGR1, PIGR, PITX1, PKP3, PLAC8, PLEK2, PLS1, POF1B, PPP1R14D, PROM1, PRR15, PRSS8, PTK6, RAB25, RNF128, RNF186, RNF43, S100A14, S100P, SAPCD2 , SERPINB5, SLC26A3, SLC39A5, SLC44A4, SLC5A1, SMIM22, SPDEF, ST6GALNAC1, TJP3, TM4SF5, TMC5, TMEM45B, TMPRSS2, TMPRSS4, TNS4, TRABD2A, TRIM15,The gene may be or may include at least one RNA transcript (e.g., an mRNA transcript) encoded by TRIM31, TSPAN1, TSPAN8, UGT2B17, UGT8, USH1C, VIL1, or a combination thereof.

[0019] In some embodiments, a target biomarker signature for colorectal 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 may be selected from the following human genes: AGMAT, AGR2, AGR3, ANKS4B, AP1M2, ARSE, ASCL2, BSPRY, C10orf99, C15orf48, C1orf106, C9orf152, CBLC, CCL24, CDCA7, CDX1, CDX2, DDC, DSG2, EHF, ELF3, EPS8L3, ESRP1, ESRP2, ETV4, EVPL, FABP1, FAM3D, FAM83E, FAM In some embodiments, the intravesicular biomarkers described herein may be or include at least one polypeptide encoded by 84A, FERMT1, FOXA2, FOXA3, FOXQ1, GPX2, GRB7, HKDC1, HMGCS2, HNF4A, HOXB9, KCNN4, KLK1, KRT20, KRT23, KRT8, LGALS4, METTL7B, MISP, MUC2, MYB, MYBL2, MYO1A, PHGR1, PITX1, PKP3, PLAC8, PLEK2, PLS1, PPP1R14D, PRR15, PTK6, S100A14, S100P, SAPCD2, SERPINB5, SPDEF, TRIM15, TRIM31, USH1C, VIL1, 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-colorectal cancer subjects.

[0021] In some embodiments, extracellular vesicle-associated surface biomarkers included in the target biomarker signature may be detected using an affinity agent (for example, but not limited to, an antibody-based agent). In some embodiments, extracellular vesicle-associated surface biomarkers may be detected using a capture assay including 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 stool-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 stool-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 such detection probes 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 detection probes, including 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 ligated templates indicates the presence of extracellular vesicles that are positive for the target biomarker signature of colorectal cancer (e.g., colorectal adenocarcinoma). Such proximity ligation assays may perform better, e.g., with greater specificity and / or sensitivity, than other existing proximity ligation assays, although those skilled in the art reading this disclosure will recognize that other forms of proximity ligation assays known in the art may be used instead.

[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 multiple colorectal 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 susceptible to colorectal 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 colorectal cancer (e.g., colorectal adenocarcinoma), while 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 a 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 colorectal 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 co-localization of at least two surface biomarkers, the combined expression levels of which have been determined to be associated with colorectal cancer, on the surface of the nanoparticles, the combined expression levels of which have been determined to be associated with colorectal cancer, wherein the surface biomarkers are selected from the following human genes: (i) ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CI SD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH , HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, N LN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A 6, polypeptides encoded by SLC2A1, SMIM22, SNTB1, SORD, SSR4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, and combinations thereof;and / or (ii) the colocalization level is selected from the following carbohydrate-dependent markers: CanAg (a glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3 (N-glycolyl GM3 ganglioside), and combinations thereof; (c) comparing the detected colocalization level with the determined level; and (d) classifying the subject as having or susceptible to colorectal cancer if the detected colocalization level is at or above the determined level.

[0029] In some embodiments, the first surface biomarker and the second surface biomarker are each independently selected from (i) polypeptides encoded by the following human genes: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, and combinations thereof; 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.

[0030] Thus, in some embodiments, the techniques provided herein may be useful for detecting the occurrence or recurrence of colorectal 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 colorectal cancer. In some embodiments, a target biomarker signature may be selected for the detection of a specific category of colorectal cancer, for example, including but not limited to, colorectal 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) colorectal cancer, for example, including but not limited to, colorectal 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) colorectal cancer, for example, including but not limited to, colorectal 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 colorectal cancer or recurrence of colorectal cancer.

[0031] In some embodiments, subjects suitable for the techniques provided herein for detecting the occurrence or recurrence of colorectal cancer (e.g., colorectal adenocarcinoma) may be asymptomatic human subjects and / or the entire asymptomatic population. Such asymptomatic subjects may include subjects with a family history of colorectal cancer, subjects with a lifestyle history that places the subject at increased risk for colorectal cancer, subjects previously treated for colorectal cancer, subjects at risk of colorectal cancer recurrence after cancer treatment, and / or subjects in remission after colorectal cancer treatment. In some embodiments, such asymptomatic subjects may be subjects who have been determined to have normal medical diagnostic results, for example, from colonoscopy, stool tests, CT scanning, and / or molecular testing, and / or based on cell-free nucleic acid. In some embodiments, such asymptomatic subjects may be subjects who have been determined to have an abnormal medical diagnostic result, e.g., based on cell-free nucleic acid, e.g., from a colonoscopy, stool test, CT scanning, and / or molecular test, when compared to results typically observed in non-colorectal cancer subjects and / or normal healthy subjects. Alternatively, in some embodiments, asymptomatic subjects may be subjects who have not previously been screened for colorectal cancer, have not been diagnosed with colorectal cancer, and / or have not previously received a colorectal 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 colon, and / or occupational hazards).

[0033] In some embodiments, the techniques provided herein may be useful for selecting a surgery or therapy for a subject suffering from or susceptible to colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, colorectal cancer surgery, therapy, and / or adjuvant therapy can 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 colorectal 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) whose ages range from 40 to 90 years of age. 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) whose ages range from 45 to 85 years. 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 colorectal 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 (e.g., a close relative, such as a blood relative, previously diagnosed with colorectal cancer), the identification of one or more risk factors associated with colorectal 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 colorectal cancer (e.g., an abnormal medical result such as occult blood in the stool, and / or symptoms potentially indicative of colorectal 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 provides a determination of whether an individual subject has one or more indicators of the development or recurrence of colorectal 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., colorectal cancer response biomarkers). In some embodiments, the provided technology provides a determination of whether an individual subject is responsive to a current treatment, for example, based on findings of changes in the levels of one or more molecular targets associated with colorectal 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 (e.g., as described herein) for a tumor biomarker signature of colorectal 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 on extracellular vesicles associated with colorectal cancer; and (b) at least one or more detection agents directed to one or more target biomarkers of a target biomarker signature for colorectal 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 colorectal cancer. For example, in some embodiments, the provided systems and kits may include at least one set of detection probes for the detection of colorectal 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 colorectal cancer (including, for example, colorectal adenocarcinoma). In some embodiments, the two or more sets may be directed to the detection of different stages of colorectal cancer. In some embodiments, two or more sets may be directed to detecting the same stage of colorectal 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 systems or kits may include (i) a capture agent (e.g., as described herein) for a first surface biomarker of a colorectal 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 colorectal 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 colorectal 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 colorectal cancer, the first and second surface biomarkers being selected from the group consisting of (i) the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, and CEACAM6. , CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT 3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RA B25, RAP2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD, SSR 4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, and polypeptides encoded by these genes, and combinations thereof;and / or (ii) each independently selected from the following carbohydrate-dependent markers: CanAg (glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3 (N-glycolyl GM3 ganglioside), and combinations thereof.

[0047] In some embodiments, the first surface biomarker and the second surface biomarker are each independently selected from (i) polypeptides encoded by the following human genes: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, and combinations thereof; 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.

[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 colorectal cancer. In some embodiments, the provided systems and / or kits can be used for screening and / or other assessment of individuals predisposed to colorectal 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 colorectal 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 colorectal 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 colorectal cancer. In some embodiments, the provided systems and / or kits can be used to select a therapy for a subject suffering from colorectal 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 having one or more symptoms (e.g., non-specific symptoms) associated with colorectal 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: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF43, R PN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD, SSR4, ST14, STOML2, STT3B, SY at least one polypeptide encoded by AP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, or a combination thereof;and / or (ii) may be or include at least one of the following carbohydrate-dependent markers: CanAg (glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, or a combination thereof;

[0052] In some embodiments, the extracellular vesicle-associated biomarker may be or may include (i) one or more of the polypeptides encoded by the human gene MUC1; and / or (ii) one or more of 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, or a combination thereof.

[0053] In some embodiments, the surface biomarkers present on the complexed extracellular vesicles include (i) the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, C YP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, M UC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF4 3, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD, SSR4, ST14, STOML2, STT3B, at least one polypeptide encoded by SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, or a combination thereof;and / or (ii) may be or include at least one of the following carbohydrate-dependent markers: CanAg (glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, 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: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, 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: AGMAT, AGR2, AGR3, ANKS4B, AP1M2, ARSE, ASCL2, BSPRY, C10orf99, C15orf48, C1orf106, C9orf152, CBLC, CCL24, CDCA7, CDX1, CDX2, DDC, DSG2, EHF, ELF3, EPS8L3, ESRP1, ESRP2, ETV4, EVPL, FABP1, FAM3D, FAM83E, FAM84A, FERMT1, FOXA2, FOXA3, FOXQ1, GPX2, GRB7, H The vesicular biomarkers may be or include at least one polypeptide encoded by KDC1, HMGCS2, HNF4A, HOXB9, KCNN4, KLK1, KRT20, KRT23, KRT8, LGALS4, METTL7B, MISP, MUC2, MYB, MYBL2, MYO1A, PHGR1, PITX1, PKP3, PLAC8, PLEK2, PLS1, PPP1R14D, PRR15, PTK6, S100A14, S100P, SAPCD2, SERPINB5, SPDEF, TRIM15, TRIM31, USH1C, VIL1, 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: AGMAT, AGR2, AGR3, ANKS4B, ANO9, AP1M2, ARSE, ASCL2, ATP10B, B3GNT3, BIK, BSPRY, C10orf99, C15orf48, C1orf106, C1orf210, C9orf152, CA12, CBLC, CCL24, CD24, CDCA7, CDH1, CDH17, CDH3, CDHR1, CDHR5, CDX1, CDX2, CEACAM5, CEACAM6, CEAC AM7, CFTR, CLDN2, CLDN3, CLDN4, CLDN7, CLRN3, COL17A1, CRB3, CYP2S1, DDC, DPEP1, DSG2, EHF, ELF3, EPCAM, EPHB3, EPS8L3, ERN2, ESRP1, ESRP2, ETV4, EVPL, FA2H, FABP1, FAM3D, FAM83E, FAM84A, FAT1, FERMT1, FOXA2, FOXA3, FOXQ1, FUT2, FUT3, FXYD3, GCNT3, GGT6, GJB1, GJB3, GPA33, GPR160, GPR35, GPX 2, GRB7, GUCY2C, HKDC1, HMGCS2, HNF4A, HOXB9, IHH, ITLN1, KCNN4, KIAA1324, KLK1, KRT20, KRT23, KRT8, LGALS4, LGR5, LY6G6D, MEP1A, METTL7B, MISP, MUC13, MUC2, MYB, MYBL2, MYO1A, NOX1, PDZK1IP1, PHGR1, PIGR, PITX1, PKP3, PLAC8, PLEK2, PLS1, POF1B, PPP1R14D, PROM1, PRR15, PRSS8, PTK6, RAB25, at least one RNA transcript encoded by RNF128, RNF186, RNF43, S100A14, S100P, SAPCD2, SERPINB5, SLC26A3, SLC39A5, SLC44A4, SLC5A1, SMIM22, SPDEF, ST6GALNAC1, TJP3, TM4SF5, TMC5, TMEM45B, TMPRSS2, TMPRSS4, TNS4, TRABD2A, TRIM15, TRIM31, TSPAN1, TSPAN8, UGT2B17, UGT8, USH1C, VIL1, or a combination thereof (e.g.,It may be or contain an mRNA transcript.

[0057] 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 FERMT1 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 an EPHB2 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 CEACAM6 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 CEACAM5 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 CDH17 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 MARCKSL1 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 TOMM34 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 S100P 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 EPHB3 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 CDH1 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 MUC13 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 SLC12A2 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 RAB25 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 LAMC2 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 DSG2 polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or include a CASK polypeptide. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or include a LMNB2 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 having a desired size range, including extracellular vesicles, and comprising a colorectal cancer-specific biomarker signature, which comprise at least two surface biomarkers described herein, wherein the nanoparticles are immobilized on a solid substrate comprising a binding moiety targeting a first surface biomarker of the colorectal cancer-specific biomarker signature. In some embodiments, such complexes are also bound to at least two detection probes, each targeting a surface biomarker of the colorectal cancer-specific biomarker signature (which may be the same or different surface biomarkers), each detection probe binding to a respective surface biomarker and comprising: (i) a binding moiety targeting 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 and comprising at least a first surface biomarker and a second surface biomarker on their surface, the combination of which has been determined to be a target biomarker signature for colorectal cancer, wherein the first surface biomarker and the second surface biomarker are selected from the group consisting of (i) the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1 , B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1 , DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2 , KCNQ1, KEL, KPNA2, LAD1, LAMC2, LBR, LMNB1, LMNB2, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, O CIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SM Polypeptides encoded by IM22, SNTB1, SORD, SSR4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, and combinations thereof;and / or (ii) the following carbohydrate-dependent markers: CanAg (glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (Lewis (b) a solid substrate comprising a nanoparticle, each independently selected from a nucleotide sequence selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 125, 130, 132, 133, 134, 135,

[0061] In some embodiments, the first surface biomarker and the second surface biomarker are each independently selected from (i) polypeptides encoded by the following human genes: ACVR2B, B3GNT3, CD133, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CYP2S1, DLL4, EDAR, EPCAM, EPHB2, EPHB3, ERBB2, FAP, GPCR5A, IHH, ILDR1, ITGAV, KCNQ1, KEL, MARCKSL1, MST1R, MUC1, MUC5AC, NOX1, OCIAD2, RNF43, SMIM22, and combinations thereof; 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.

[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 colorectal 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 colorectal 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 (localized, regional, distant metastasis, and unknown) at which point diagnosis is made for colorectal cancer. Diagnosis is generally made at the distant metastasis stage, when the cancer is most lethal. SEER 18 2010-2016 by SEER Summary Stage 2000, All Races, Both Sexes.

[0070] [Figure 8-1]Figure 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 systems described herein) in colorectal cancer-specific cell lines expressing at least two surface biomarkers, and in negative control groups.Panel A shows the biomarker combination of BCAP31 and EPCAM, panel B shows the biomarker combination of BCAP31 and LeX antigen, panel C shows the biomarker combination of BCAP31 and sLex antigen, panel D shows the biomarker combination of CDH1 and sTn antigen, panel E shows the biomarker combination of CEACAM5 and LeX antigen, panel F shows the biomarker combination of CEACAM5 and LEY antigen, and panel G shows the biomarker combination of CEACAM5 and sLex antigen. Panel H shows a biomarker combination of CEACAM5 and sTn antigen, Panel I shows a biomarker combination of CEACAM5 and T antigen, Panel J shows a biomarker combination of CEACAM6 and LeX antigen, Panel K shows a biomarker combination of CEACAM6 and LEY antigen, Panel L shows a biomarker combination of CEACAM6 and sLex antigen, Panel M shows a biomarker combination of CEACAM6 and sTn antigen, and Panel N shows a biomarker combination of EPC Panel A shows a combination of AM and LeX antigen biomarkers, Panel B shows a combination of EPCAM and sLex antigen biomarkers, Panel C shows a combination of LeX antigen and LeX antigen biomarkers, Panel D shows a combination of LeX antigen and LeX antigen biomarkers, Panel E shows a combination of LeX antigen and sLex antigen biomarkers, Panel F shows a combination of LEY antigen and MET biomarkers, Panel G shows a combination of LEY antigen and sLex antigen biomarkers, Panel H shows a combination of LEY antigen and sTn antigen biomarkers, Panel I shows a combination of LEY antigen and sTn antigen biomarkers, Panel J ... K shows a combination of LEY antigen and MET biomarkers, Panel S shows a combination of LEY antigen and sLex antigen biomarkers, Panel T shows a combination of LEY antigen and sTn antigen biomarkers, Panel U shows a combination of LEY antigen and sTn antigen biomarkers, Panel V shows a combination of biomarkers for sLex and sTn antigens, panel W shows a combination of biomarkers for ERBB2 and MUC5A, panel X shows a combination of biomarkers for DLL4 and ITGAV, panel Y shows a combination of biomarkers for ERBB2 and ITGAV, panel Z shows a combination of biomarkers for ITGAV and MUC5A, and panel AA shows a combination of biomarkers for DLL4 and MUC5A. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] 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 Ct 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 protein biomarker described herein. In some embodiments, an affinity agent according to the present disclosure specifically binds to a carbohydrate-dependent biomarker 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 colorectal cancer (e.g., a specific type of colorectal cancer (e.g., colorectal adenocarcinoma) and / or a stage of colorectal cancer) if its presence correlates with the occurrence and / or susceptibility of colorectal cancer (e.g., an entire relevant population).

[0081] Biological entity: Where appropriate, and 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, 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 stool 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 stool 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 stool 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 likelihood thereof. In some embodiments, a biomarker may be or include a marker for a particular tissue (e.g., but not limited to, brain, breast, colon, ovaries and / or other tissues associated with the female reproductive system, 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, be present in normal healthy tissue and diseased tissue (e.g., tumors); those skilled 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 to 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). In some embodiments, a non-specific cancer marker may be or include a general marker for cancer (e.g., a marker that is typically present in cancer 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, ovary and / or other tissues associated with the female reproductive system, 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 colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, biomarkers are intravesicular (e.g., protein or RNA markers present in extracellular vesicles). In some embodiments, a biomarker may be or include a genetic or epigenetic signature. 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 colorectal cancer (including, e.g., colorectal 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., colorectal 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 colorectal adenocarcinoma subjects). 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 observed differences or similarities. 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 colorectal cancer (e.g., colorectal 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 colorectal cancer (e.g., colorectal adenocarcinoma) tumors, and in some embodiments, the extracellular vesicles are shed from or derived from non-colorectal cancer (e.g., non-colorectal adenocarcinoma) tumors. 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 benign colorectal tumors. In some embodiments, the extracellular vesicles are shed from or derived from tissue of a subject with symptoms (e.g., non-specific symptoms) associated with colorectal cancer (e.g., colorectal adenocarcinoma).

[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., colon tissue-specific or rectal tissue-specific). In some embodiments, such a polypeptide may be non-specific, for example, 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 activities, experiences, medical history, and / or exposures of an individual that may directly or indirectly increase the individual's risk for a condition, e.g., cancer, such as colorectal adenocarcinoma, compared with 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., colorectal 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 who does not have non-benign colorectal cancer, more specifically, colorectal 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 who is 55 years of age or older. In some embodiments, the non-cancer subject is a subject who has a non-colon-related health disease, disorder, or condition. In some embodiments, the non-cancer subject is a subject who has a benign tumor in the colorectal cavity and surrounding area.

[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., colorectal 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 called carcinoma in situ 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 assigned 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).

[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 or female subject. In some embodiments, the subject is afflicted with colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject is predisposed to colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject exhibits one or more symptoms or characteristics of colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject exhibits one or more non-specific symptoms of colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject does not exhibit any symptoms or characteristics of colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject is a subject with one or more features characteristic of susceptibility to or risk of colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is administered. In some embodiments, the subject is an asymptomatic subject. Such an asymptomatic subject may be at average population risk or at genetic risk. For example, such an asymptomatic subject may be a subject with a family history of cancer, a subject previously treated for cancer, a subject at risk of cancer recurrence after cancer treatment, a subject in remission after cancer treatment, and / or a subject who has previously or periodically been screened for the presence of at least one cancer biomarker. Alternatively, in some embodiments, an asymptomatic subject may be a subject who has not previously been screened for cancer, a subject who has not been diagnosed with cancer, and / or a subject who has not previously received cancer therapy.In some embodiments, subjects suitable for the provided technologies 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 colorectal 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 stool-derived sample, etc.) of a subject suffering from or susceptible to colorectal 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 colorectal 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 its stage. In some embodiments, the target biomarker signature may be correlated with colorectal cancer (e.g., colorectal 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 colorectal cancer (e.g., colorectal adenocarcinoma), or a subtype and / or disease state thereof, although one or more biomarkers in such a combination may be directed to targets (e.g., surface biomarkers, intravesicular biomarkers, and / or intravesicular RNA) that are not specific for colorectal cancer (e.g., colorectal adenocarcinoma). For example, in some embodiments, the target biomarker signature may include at least one biomarker specific to colorectal adenocarcinoma, or a stage and / or subtype thereof (i.e., a colorectal adenocarcinoma-specific target), and may further include biomarkers that are not necessarily or completely specific to colorectal 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 those 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 (i.e., sufficiently distinguish) the relevant target biological entity for detection (e.g., colorectal adenocarcinoma cells of interest, or extracellular vesicles secreted by colorectal adenocarcinoma cells) of interest (i.e., sufficiently distinguish the relevant target biological entity for detection (e.g., colorectal adenocarcinoma cells of interest, or extracellular vesicles secreted by colorectal 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 in accordance with 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 about and / or classifying the results of a measurement, e.g., 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-colorectal adenocarcinoma vs. colorectal 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] Colorectal cancer was responsible for an estimated 53,200 deaths and 147,950 new cases in 2020, with a 5-year relative survival rate of 64.6% from 2010 to 2016 (new cases from SEER 13; deaths from US Mortality). The majority of these deaths are attributable to delayed diagnosis. Patients with localized disease at the time of diagnosis had a 5-year survival rate of 90.2%; however, the majority of patients were initially diagnosed when distant metastases had already formed, and these patients had a dismal 5-year survival rate of approximately 14.3%.

[0134] The majority of colorectal cancers are adenocarcinomas, which typically begin in the cells that make the mucus that lubricates the colon and rectum. Colorectal cancers (e.g., colorectal adenocarcinomas) usually develop from growths, or polyps, on the lining of the colon or rectum. Some polyps become cancerous, while others do not; however, progression to cancer can take a long time and depends on the type of polyp. Adenomatous polyps can turn into cancer and are considered precancerous. Three types of adenomas include tubular, villous, and ductal ciliary.

[0135] When cancerous polyps form, they can grow through many layers into the wall of the colon or rectum. This becomes a problem because if cancer cells are present in the wall of the colon or rectum, they can then grow into the blood or lymphatic vessels and travel to other parts of the body.

[0136] Current methodologies for colorectal cancer screening include colonoscopy, which allows doctors to sedate patients and then physically examine them for polyps using a lighted tube (e.g., a colonoscope). Other screening methods include stool tests and CT scans. However, there are currently no inexpensive or widely available screening methods for detecting colorectal cancer that use blood samples and / or obtain information on pre-polyp status. The ability to avoid invasive screening methods such as colonoscopy would save patients time, money, and the psychological trauma of being sedated and / or undergoing lengthy testing processes. In addition, asymptomatic screening is not readily available.

[0137] The present disclosure identifies, among other things, the origins of problems with certain previous technologies, including, for example, certain conventional approaches to the detection and diagnosis of colorectal cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as colonoscopy, stool tests, 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 solve such problems by, among other things, identifying combinations of biomarkers that are predicted to exhibit high sensitivity and specificity for colorectal cancer based on bioinformatics analysis. In some embodiments, the present disclosure provides techniques (including systems, compositions, and methods) that solve such problems by detecting the co-localization of target biomarker signatures for colorectal 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 in extracellular vesicles associated with colorectal 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 colorectal 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 having 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 colorectal cancer in individual nanoparticles having 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 colorectal cancer screening, for example, for the early detection of colorectal cancer, including, but not limited to, colorectal adenocarcinoma, among others. In some embodiments, the present disclosure provides techniques for the early detection of colorectal cancer in subjects who may be experiencing another symptom associated with colorectal cancer. In some embodiments, the present disclosure provides techniques for the early detection of colorectal cancer in subjects who are at genetic risk for colorectal cancer. In some embodiments, the present disclosure provides techniques for the early detection of colorectal cancer in subjects who may be at genetic risk for colorectal 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 colorectal cancer in subjects who may have life history 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, life history-related risk, or average risk) for early stage colorectal cancer (e.g., colorectal adenocarcinoma). Colon cancer is relatively common compared to other cancer types, with 22% of cases detected at an advanced metastatic stage (SEER 18 2010-2016 by SEER Summary Stage 200, All Races, Both Sexes; see Figure 7). In some embodiments, provided techniques are effective for detecting early-stage colorectal cancer (e.g., colorectal adenocarcinoma). In some embodiments, provided techniques are effective when applied to populations that include or consist of individuals with one or more symptoms that may be associated with colorectal cancer. In some embodiments, provided techniques are effective even when applied to populations that include or consist of asymptomatic or symptomatic individuals (e.g., due to results of sufficiently high sensitivity and / or low false positive and / or false negative rates). In some embodiments, provided techniques are effective when applied to populations that include or consist of individuals without a genetic and / or lifestyle-related risk of developing colorectal 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 with a genetic risk of developing colorectal 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 colorectal cancer (e.g., individuals with a 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 will 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 characteristics of colorectal 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, stool tests, diabetes (type 2) screening, colonoscopy, blood pressure screening, thyroid function tests, colorectal cancer screening, mammograms, HPV / Pap smears, 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 colorectal cancer. In some embodiments, the present disclosure provides colorectal cancer screening systems that can be implemented to detect early stage cancers, including early stage cancers in asymptomatic individuals (e.g., without genetic and / or life history-related risk for colorectal cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals (e.g., with or without genetic risk for colorectal cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of symptomatic individuals (e.g., with or without genetic and / or life history-related risk for colorectal 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 for providing and / or using the same. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them. I. Colorectal Cancer Detection

[0142] Today, there are no colorectal cancer blood screening tests recommended by the CDC or the United States Defense Medical Task Force (USPSTF) for screening average-risk asymptomatic individuals. However, in the United States, the age-adjusted incidence rate of colorectal cancer was 42.4 per 100,000 men and 32.9 per 100,000 women per year in 2017. Colorectal cancer is one of the most common cancer types, and although it is less lethal than some other cancer types, it remains lethal even in early-stage (localized) disease, with a 5-year survival rate of 90.2% (Figure 6). In 2020 alone, there were an estimated 147,950 new cases and 53,200 deaths from colorectal cancer. While total deaths and new cases have declined slightly over the past few years, however, colorectal cancer remains a significant problem. (New cases from SEER 13. Deaths from US Mortality; https: / / seer.cancer.gov / statfacts / html / pancreas.html are incorporated herein by reference for purposes described herein.) The 5-year relative survival rate for localized stage is 90.2%, regional stage is 71.8%, and distant metastatic stage is 14.3%. Thus, even with early screening, approximately 1 in 10 patients will die within the first 5 years (Figure 6). Currently, the detection range is rather dismal, with 38% of colorectal cancer cases detected while in the localized stage, 35% of cases detected in the regional stage, and 22% of cases detected in the distant metastatic stage (Figure 7).

[0143] Surveillance, Epidemiology, and End Results (SEER) data from 2000 to 2017 provide extensive information on the prevalence and epidemiology of colorectal cancer in the United States. SEER reported that in 2017, there were 163 cases per 100,000 individuals for colorectal cancer in those aged 65 years or older in the United States, 70.1 cases per 100,000 individuals for those aged 50 to 64 years, and only 8.5 cases per 100,000 individuals for those under 50 years. Rates in men were, on average, higher than rates in women. In 2017, there were 42.4 cases per 100,000 individuals for men and 32.9 cases per 100,000 individuals for women. This difference may be genetic, diet-related, or associated with unknown causes. In all cases, outcomes over a 5-year period were unpredictable. The techniques disclosed herein are designed to address current shortcomings in screening technology.

[0144] According to the American Cancer Society, controllable risk factors for colorectal cancer include, for example, weight, diet, and exercise, which have a more significant impact than other cancer types.A diet that includes more grains, fruits, and vegetables, as well as having sufficient vitamin D, can reduce the rate of colorectal cancer.Alcohol and tobacco use also increase an individual's risk for developing colorectal cancer.Certain high risk factors include, but are not limited to, a personal history of chronic inflammation and / or inflammatory bowel disease (IBD).

[0145] 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), which are 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 colorectal cancer (e.g., colorectal adenocarcinoma) 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 activation of the K-ras oncogene, which has been detected in human colorectal cancer.

[0146] In some embodiments, the present disclosure provides techniques for the effective screening of colorectal cancer in individuals at genetic risk or individuals with lifestyle-related risk. In some embodiments, the present disclosure provides techniques for the effective screening of colorectal cancer in average-risk individuals. In some embodiments, the present disclosure provides techniques for the effective screening of colorectal cancer in individuals with one or more symptoms associated with colorectal cancer. In some embodiments, the present disclosure provides techniques for the effective screening of colorectal cancer in asymptomatic individuals. Despite its relative prevalence in both men and women, there are currently no recommended non-invasive colorectal cancer screening tools based on a subject's blood sample that are intended for screening asymptomatic and / or average-risk individuals (e.g., individuals under 55 years of age or over 55 years of age). This is due, in part, to the cost, limited availability, potential side effects, and / or poor performance (e.g., high false positive rates or ineffectiveness) of existing colorectal cancer and colorectal cancer screening technologies. Given the incidence of colorectal cancer in average-risk individuals, inadequate test specificity (<99.5%) can result in false-positive results that exceed the number of true-positives by more than an order of magnitude. This places a significant burden on the healthcare system and on the false-positive results and screened individuals, resulting in additional testing, unnecessary surgery, and emotional / physical distress (Wu et al., 2016). In some embodiments, the present disclosure provides insight that a particularly useful colorectal cancer screening test would be characterized by (1) ultra-high specificity (>99.5%) that minimizes the number of false-positives, and (2) high sensitivity (>40%) for stage I and II colorectal cancer (i.e., when the prognosis is most favorable).

[0147] In some embodiments, the present disclosure provides the insight that a particularly useful colorectal 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 colorectal cancer (i.e., when the prognosis is most favorable). For example, in some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of >98% and a sensitivity of >50%, for example, for stage I and II colorectal cancer. In some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of >98% and a sensitivity of >60%, for example, for stage I and II colorectal cancer. In some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of >98% and a sensitivity of >70%, for example, for stage I and II colorectal cancer. In some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >65%, for example, for stage I and II colorectal cancer. In some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >60%, for example, for stage I and II colorectal cancer. In some embodiments, a particularly useful colorectal cancer screening test may be characterized by a specificity of 99% or higher and a sensitivity of >10% or higher (e.g., >15%, >20%, >25%). In some embodiments, a particularly useful colorectal 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 colorectal 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 a colorectal cancer screening test 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 an assay compared to that achieved with a single set of biomarker combinations. For example, in some embodiments, a colorectal cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve a specificity of at least 98% and a sensitivity of at least 50%. In some embodiments, a colorectal cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve a specificity of at least 98% and a sensitivity of at least 60%. In some embodiments, a colorectal cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve a specificity of 99% and a sensitivity of 50% or higher.

[0149] In some embodiments, the present disclosure provides insight that particularly useful colorectal cancer screening tests 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 colorectal 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 colorectal 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% or higher, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for a subject at genetic risk for colorectal cancer, or a specificity cutoff of at least 99.5% for a subject experiencing one or more symptoms associated with colorectal cancer).

[0150] In some embodiments, the assays described herein are particularly useful as primary screening tests for detecting early colorectal cancer. In some embodiments, subjects who receive a positive test result from the assays described herein are recommended to undergo follow-up testing, such as colonoscopy. In some such embodiments, the assays described herein can be useful for detecting early colorectal cancer, achieving a PPV of more than 2% or higher, including, for example, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, more than 20%, or more than 25%, or higher. In some embodiments, the assays 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 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 a subject at genetic risk for colorectal cancer, or a specificity cutoff of at least 99.5% for a subject experiencing one or more symptoms associated with colorectal cancer).

[0151] Several different biomarker classes, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), bulk proteins, and extracellular vesicles (EVs), have been investigated for colorectal 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 research has focused on bulk EV measurements or low-throughput single-EV analysis. II. PROVIDED BIOMARKERS AND / OR TARGET BIOMARKER SIGNATURES FOR THE DETECTION OF COLORECTAL CANCER

[0152] The present disclosure provides, among other things, various target biomarkers or combinations thereof (e.g., target biomarker signatures) for colorectal cancer. Such target biomarker signatures that are predicted to exhibit high sensitivity and specificity for colorectal cancer are discovered through a wide range of bioinformatics analysis and biological approaches, including, for example, in some embodiments, machine learning and / or 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.

[0153] In some embodiments, a target biomarker signature for colorectal cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., in some embodiments, surface polypeptides present on extracellular vesicles associated with colorectal 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 target biomarker signature for colorectal 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 colorectal cancer, which have the most favorable prognosis.

[0154] In some embodiments, the present disclosure recognizes that, in certain embodiments, the sensitivity and specificity ratios for subjects with different colorectal 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 colorectal cancer (e.g., patients with lifestyle-related risk factors, symptomatic patients, or patients with a family history of colorectal cancer) compared to those for patients at lower risk for colorectal cancer. For example, in some embodiments, the combination of biomarkers described herein useful for detecting colorectal 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 colorectal 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.

[0155] In certain embodiments, subjects at risk for colorectal 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 colorectal 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, assays described herein for detecting colorectal 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, assays described herein for detecting colorectal 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, techniques and / or assays described herein for detecting colorectal cancer in symptomatic subjects may have lower sensitivity and / or specificity requirements than those for detecting colorectal cancer in asymptomatic subjects. In some embodiments, assays described herein for detecting colorectal 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, assays described herein for detecting colorectal 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%.

[0156] In some embodiments, the present disclosure recognizes that, among other things, a biomarker signature for colorectal cancer that provides a positive predictive value (PPV) of 2% or higher may be useful for screening at-risk individuals for colorectal cancer. In some embodiments, a target biomarker signature for colorectal cancer comprises at least one surface biomarker (e.g., a surface biomarker present on the surface of an extracellular vesicle associated with colorectal 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 represents a target biomarker signature for colorectal cancer that provides a positive predictive value (PPV) of at least 2% or higher in a high-risk population, including, for example, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% or higher, at least 15% or higher, at least 20% or higher, at least 25% or higher, and / or at least 30% or higher.

[0157] 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.

[0158] 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.

[0159] In some embodiments, the target biomarkers included in the target biomarker signature for colorectal cancer are long-chain fatty acid-CoA ligase 5 (ACSL5) polypeptide, activin receptor type 2B (ACVR2B) polypeptide, delta-1-pyrroline-5-carboxylic acid synthase (ALDH18A1) polypeptide, dolichyl-phosphate beta-glucosyltransferase (ALG5) polypeptide, AP-1 complex subunit mu-2 (AP1M2) polypeptide, sodium / potassium transporting ATPase subunit beta-1 (ATP1B1) polypeptide, N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) polypeptide, B-cell receptor-associated protein 31 (BCAP31) polypeptide, peripheral cell membrane protein CASK (CAS K) polypeptide, prominin-1 (CD133) polypeptide, cadherin-1 (CDH1) polypeptide, cadherin-17 (CDH17) polypeptide, cadherin-3 (CDH3) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) polypeptide, complement factor B (CFB) polypeptide, cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide, choline dehydrogenase, mitochondrial (CHDH) polypeptide, charged multivesicular body protein 4b (CHMP4B) polypeptide, CDGSH iron-sulfur domain-containing protein 2 (CISD2) polypeptide, chloride intracellular channel protein 1 (CLIC1) polypeptide, coatomer subunit gamma-2 (COPG2) polypeptide, cytochrome P450 2S1 (CYP2S1) polypeptide, dipeptidase 1 (DPEP1) polypeptide, desmoglein-2 (DSG2) polypeptide, tumor necrosis factor receptor superfamily member EDAR (EDAR) polypeptide, epithelial cell adhesion molecule (EPCAM) polypeptide, ephrin type B receptor 2 (EPHB2) polypeptide, ephrin type B receptor 3 (EPHB3) polypeptide, endoplasmic reticulum metallopeptidase 1 (ERMP1) polypeptide, fermitin family homolog 1 (FERMT1) polypeptide,Polypeptides N-acetylgalactosaminyltransferase 3 (GALNT3) polypeptide, glucosamine 6-phosphate N-acetyltransferase (GNPNAT1) polypeptide, Golgi integral membrane protein 4 (GOLIM4) polypeptide, cell surface A33 antigen (GPA33) polypeptide, retinoic acid-inducible protein 3 (GPCR5A) polypeptide, very long chain (3R)-3-hydroxyacyl-CoA dehydratase 3 (HACD3) polypeptide, hephaestin (HEPH) polypeptide, hexokinase HKDC1 ( HKDC1 polypeptide, Indian hedgehog protein (IHH) polypeptide, immunoglobulin-like domain-containing receptor 1 (ILDR1) polypeptide, integrin alpha-2 (ITGA2) polypeptide, potassium voltage-gated channel subfamily KQT member 1 (KCNQ1) polypeptide, Kell blood group glycoprotein (KEL) polypeptide, importin subunit alpha-1 (KPNA2) polypeptide, rhaginin-1 (LAD1) polypeptide, laminin subunit gamma-2 (LAMC2) polypeptide peptide, delta(14)-sterol reductase LBR (LBR) polypeptide, laminin-B1 (LMNB1) polypeptide, laminin-B2 (LMNB2) polypeptide, lipolysis-stimulating lipoprotein receptor (LSR) polypeptide, ensconsin (MAP7) polypeptide, MARCKS-related protein (MARCKSL1) polypeptide, malectin (MLEC) polypeptide, mucin-1 (MUC1) polypeptide, mucin-13 (MUC13) polypeptide, neutral cholesterol ester hydrolase 1 (NCEH1) polypeptide Polypeptide, NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial (NDUFS6) polypeptide, neurolysin, mitochondrial (NLN) polypeptide, NADPH oxidase 1 (NOX1) polypeptide, nuclear pore membrane glycoprotein 210 (NUP210) polypeptide, OCIA domain-containing protein 2 (OCIAD2) polypeptide, serine / threonine-protein phosphatase PGAM5, mitochondrial (PGAM5) polypeptide, polymeric immunoglobulin receptor (PIGR) polypeptide,GPI transamidase component PIG-T (PIGT) polypeptide, inactive tyrosine-protein kinase 7 (PTK7) polypeptide, Ras-related protein Rab-25 (RAB25) polypeptide, Ras-related protein Rap-2a (RAP2A) polypeptide, Ras-related protein Rap-2b (RAP2B) polypeptide, protein RCC2 (RCC2) polypeptide, E3 ubiquitin-protein ligase RNF43 (RNF43) polypeptide, dolityl-diphosphooligosaccharide-protein glycosyltransferase glycosyltransferase subunit 1 (RPN1) polypeptide, dolityl-diphosphooligosaccharide-protein glycosyltransferase subunit 2 (RPN2) polypeptide, 40S ribosomal protein S3 (RPS3) polypeptide, RuvB-like 2 (RUVBL2) polypeptide, protein S100-P (S100P) polypeptide, solute carrier family 12 member 2 (SLC12A2) polypeptide, ADP / ATP translocase 3 (SLC25A6) polypeptide, solute carrier family 2, facilitative glucose transporter member 1 (SLC25A6) polypeptide LC2A1) polypeptide, small integral membrane protein 22 (SMIM22) polypeptide, beta-1-syntrophin (SNTB1) polypeptide, sorbitol dehydrogenase (SORD) polypeptide, translocon-associated protein subunit delta (SSR4) polypeptide, suppressor of tumorigenicity 14 protein (ST14) polypeptide, stomatin-like protein 2, mitochondrial (STOML2) polypeptide, dolityl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3B (STT3B) polypeptide, synapse-associated protein 1 (SYAP1) polypeptide, nine-transmembrane superfamily member 2 (TM9SF2) polypeptide, transmembrane emp24 domain-containing protein 2 (TMED2) polypeptide, lamina-associated polypeptide 2, isoform alpha (TMPO) polypeptide, mitochondrial import receptor subunit TOM22 homolog (TOMM22) polypeptide, mitochondrial import receptor subunit TOM34 (TOMM34) polypeptide, anti-Müllerian hormone type 2 receptor (AMHR2) polypeptide,CanAg (glycoform of MUC1), claudin-1 (CLDN1) polypeptide, delta-like protein 4 (DLL4) polypeptide, epidermal growth factor receptor (EGFR) polypeptide, receptor tyrosine-protein kinase erbB-2 (ERBB2) polypeptide, prolyl endopeptidase FAP (FAP) polypeptide, fibroblast growth factor receptor 4 (FGFR4) polypeptide, folate receptor alpha (FOLR1) polypeptide, heat-stable enterotoxin receptor (GUCY2C) polypeptide, insulin-like growth factor 1 receptor (IGF1R) polypeptide, interleukin-1 alpha (IL1A) polypeptide, integrin alpha-V (ITGAV) polypeptide, keratin, type II cytoskeleton 8 (KRT8) polypeptide, Lewis Y / B antigen, Lewis The surface biomarker is or comprises a surface biomarker selected from the group consisting of B antigen, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) polypeptide, (LPR6) polypeptide, hepatocyte growth factor receptor (MET) polypeptide, macrophage-stimulating protein receptor (MST1R) polypeptide, mucin-5AC (MUC5AC) polypeptide, sialyltetraosyl carbohydrate, tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) polypeptide, vascular endothelial growth factor A (VEGFA) 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, and combinations thereof.

[0160] In some embodiments, the target biomarkers included in the target biomarker signature for colorectal cancer are activin receptor type 2B (ACVR2B) polypeptide, N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) polypeptide, prominin-1 (CD133) polypeptide, cadherin-17 (CDH17) polypeptide, cadherin-3 (CDH3) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) polypeptide, complement factor B (CFB) polypeptide, cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide, cytochrome P450 2S1 (CYP2S1) polypeptide, tumor necrosis factor receptor superfamily member EDAR (EDAR) polypeptide, epithelial cell adhesion molecule (EPCAM) polypeptide, ephrin type B receptor 2 (EPHB2) polypeptide, ephrin type B receptor 3 (EPHB3) polypeptide, retinoic acid-inducible protein 3 (GPCR5A) polypeptide, Indian hedgehog protein (IHH) polypeptide, immunoglobulin-like domain-containing receptor 1 (ILDR1) polypeptide, potassium voltage-gated channel subfamily KQT member 1 (KCNQ1) polypeptide, Kell blood group glycoprotein (KEL) polypeptide, MARCKS-related protein (MARCKSL1) polypeptide The surface biomarker is or comprises a surface biomarker selected from the group consisting of a tyrosine-linked phosphodiesterase (TGP) polypeptide, a mucin-1 (MUC1) polypeptide, a NADPH oxidase 1 (NOX1) polypeptide, an OCIA domain-containing protein 2 (OCIAD2) polypeptide, an E3 ubiquitin-protein ligase RNF43 (RNF43) polypeptide, a small integral membrane protein 22 (SMIM22) polypeptide, a delta-like protein 4 (DLL4) polypeptide, a receptor tyrosine-protein kinase erbB-2 (ERBB2) polypeptide, a prolyl endopeptidase FAP (FAP) polypeptide, an integrin alpha-V (ITGAV) polypeptide, a macrophage-stimulating protein receptor (MST1R) polypeptide, a mucin-5AC (MUC5AC) 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.

[0161] In some embodiments, the target biomarker signature is an ACSL5 polypeptide, an ACVR2B polypeptide, an ALDH18A1 polypeptide, an ALG5 polypeptide, an AP1M2 polypeptide, an ATP1B1 polypeptide, a B3GNT3 polypeptide, a BCAP31 polypeptide, a CASK polypeptide, a CDH1 polypeptide, a CD133 polypeptide, a CDH17 polypeptide, a CDH3 polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, a CFB polypeptide, a CFTR polypeptide, a CHDH polypeptide, a CHMP4B polypeptide, a CISD2 polypeptide, a CLIC1 polypeptide, a COPG2 polypeptide, a CYP2S1 polypeptide, a DPEP1 polypeptide, a DSG2 polypeptide, an EDAR polypeptide, an EPCAM polypeptide, an EPHB2 polypeptide, an EPHB3 polypeptide, an ERMP1 polypeptide, a FERMT1 polypeptide, a GALNT3 polypeptide, a GNPNAT1 polypeptide, a GPCR5A polypeptide, a GOLIM4 polypeptide, a GPA33 polypeptide, a HACD3 polypeptide, a HEPH polypeptide, a HKDC1 polypeptide, a peptide, IHH polypeptide, ILDR1 polypeptide, ITGA2 polypeptide, KCNQ1 polypeptide, KEL polypeptide, KPNA2 polypeptide, LAD1 polypeptide, LAMC2 polypeptide, LBR polypeptide, LMNB1 polypeptide, LMNB2 polypeptide, LSR polypeptide, MAP7 polypeptide, MARCKSL1 polypeptide, MLEC polypeptide, MUC1 polypeptide, MUC13 polypeptide, NCEH1 polypeptide, NDUFS6 polypeptide, NLN polypeptide, NOX1 polypeptide, NUP210 polypeptide, OCIAD2 polypeptide, PGAM5 polypeptide, PIGR polypeptide, PIGT polypeptide, PTK7 polypeptide, RAB25 polypeptide, RAP2A polypeptide, RAP2B polypeptide, RCC2 polypeptide, RNF43 polypeptide, RPN1 polypeptide, RPN2 polypeptide, RPS3 polypeptide, RUVBL2 polypeptide, S100P polypeptide, SLC12A2 polypeptide, SLC25A6 polypeptide, SLC2A1 polypeptide, SMIM22 polypeptide, SNTB1 polypeptide,SORD polypeptide, SSR4 polypeptide, ST14 polypeptide, STOML2 polypeptide, STT3B polypeptide, SYAP1 polypeptide, TM9SF2 polypeptide, TMED2 polypeptide, TMPO polypeptide, TOMM22 polypeptide, TOMM34 polypeptide, AMHR2 polypeptide, CanAg (glycoform of MUC1), CLDN1 polypeptide, DLL4 polypeptide, EGFR polypeptide, ERBB2 polypeptide, FAP polypeptide, FGFR4 polypeptide, FOLR1 polypeptide, GUCY2C polypeptide, IGF1R polypeptide, IL1A polypeptide, ITGAV polypeptide, KRT8 polypeptide, Lewis Y / B antigen, Lewis B antigen, LGR5 polypeptide, LPR6 polypeptide, MET polypeptide, MST1R polypeptide, MUC5AC polypeptide, sialyltetraosyl carbohydrate, TNFRSF10B polypeptide, VEGFA 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), NeuGcGM3, and combinations thereof.

[0162] In some embodiments, the target biomarker signature is selected from the group consisting of an ACVR2B polypeptide, a B3GNT3 polypeptide, a CD133 polypeptide, a CDH17 polypeptide, a CDH3 polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, a CFB polypeptide, a CFTR polypeptide, a CYP2S1 polypeptide, an EDAR polypeptide, an EPCAM polypeptide, an EPHB2 polypeptide, an EPHB3 polypeptide, a GPCR5A polypeptide, an IHH polypeptide, an ILDR1 polypeptide, a KCNQ1 polypeptide, a KEL polypeptide, a MARCKSL1 polypeptide, a MUC1 polypeptide, a NOX1 polypeptide, a RNF43 polypeptide, a SMIM22 polypeptide, a DLL4 polypeptide, an ERBB2 polypeptide, a FAP polypeptide, an ITGAV polypeptide, a MST1R polypeptide, a MUC5AC 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 X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, and combinations thereof.

[0163] 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 PIGT polypeptide, a FERMT1 polypeptide, an EPCAM polypeptide, a CYP2S1 polypeptide, an EPHB2 polypeptide, a CEACAM6 polypeptide, a CEACAM5 polypeptide, a CDH17 polypeptide, a MARCKSL1 polypeptide, a TOMM34 polypeptide, an S100P polypeptide, an AP1M2 polypeptide, an EPHB3 polypeptide, a CDH1 polypeptide, an LSR polypeptide, a MAP7 polypeptide, a HEPH polypeptide, a MUC13 polypeptide, a SLC12A2 polypeptide, a RAB25 polypeptide, a GALNT3 polypeptide, a LAMC2 polypeptide, a PGAM5 polypeptide, a RPN2 polypeptide, a DSG2 polypeptide, a CASK polypeptide, an ALG5 polypeptide, a LAD1 polypeptide, a HACD3 polypeptide, a LMNB2 polypeptide, 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 PIGT polypeptide, a FERMT1 polypeptide, an EPCAM polypeptide, a CYP2S1 polypeptide, an EPHB2 polypeptide, a CEACAM6 polypeptide, a CEACAM5 polypeptide, a CDH17 polypeptide, a MARCKSL1 polypeptide, a TOMM34 polypeptide, an S100P polypeptide, an AP1M2 polypeptide, an EPHB3 polypeptide, a CDH1 polypeptide, an LSR polypeptide, and combinations thereof.

[0165] In some embodiments, the target biomarker signature is selected from the group consisting of an ACVR2B polypeptide, a B3GNT3 polypeptide, a CD133 polypeptide, a CDH17 polypeptide, a CDH3 polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, a CFB polypeptide, a CFTR polypeptide, a CYP2S1 polypeptide, an EDAR polypeptide, an EPCAM polypeptide, an EPHB2 polypeptide, an EPHB3 polypeptide, a GPCR5A polypeptide, an IHH polypeptide, an ILDR1 polypeptide, a KCNQ1 polypeptide, a KEL polypeptide, a MARCKSL1 polypeptide, a MUC1 polypeptide, a NOX1 polypeptide, a RNF43 polypeptide, a SMIM22 polypeptide, a DLL4 polypeptide, an ERBB2 polypeptide, a FAP polypeptide, an ITGAV polypeptide, a MST1R polypeptide, a MUC5AC 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 X (sLex) antigen (also known as sialyl SSEA-1 (SLX)), T antigen, Tn antigen, and combinations thereof.

[0166] 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 FERMT1 polypeptide, an EPCAM polypeptide, an EPHB2 polypeptide, a CEACAM6 polypeptide, a CEACAM5 polypeptide, a CDH17 polypeptide, a MARCKSL1 polypeptide, a TOMM34 polypeptide, an S100P polypeptide, an EPHB3 polypeptide, a CDH1 polypeptide, a MUC13 polypeptide, a SLC12A2 polypeptide, a RAB25 polypeptide, a LAMC2 polypeptide, and combinations thereof.

[0167] In some embodiments, the target biomarkers in the target biomarker signature for colorectal cancer are an AGMAT polypeptide, an AGR2 polypeptide, an AGR3 polypeptide, an ANKS4B polypeptide, an AP1M2 polypeptide, an ARSE polypeptide, an ASCL2 polypeptide, a BSPRY polypeptide, a C10orf99 polypeptide, a C15orf48 polypeptide, a C1orf106 polypeptide, a C9orf152 polypeptide, a CBLC polypeptide, a CCL24 polypeptide, a CDCA7 polypeptide, a CDX1 polypeptide, a CDX2 polypeptide, a DDC polypeptide, a DSG2 polypeptide, an EHF polypeptide, an ELF3 polypeptide, an EPS8L3 polypeptide, an ESRP1 polypeptide, an ESRP2 polypeptide, an ETV4 polypeptide, an EVPL polypeptide, a FABP1 polypeptide, a FAM3D polypeptide, a FAM83E polypeptide, a FAM84A polypeptide, a FERMT1 polypeptide, a FOXA2 polypeptide, a FOXA3 polypeptide, a FOXQ1 polypeptide, a GPX2 polypeptide In some embodiments, the intravesicular biomarker is or comprises an intravesicular biomarker selected from the group consisting of a GRB7 polypeptide, a HKDC1 polypeptide, a HMGCS2 polypeptide, a HNF4A polypeptide, a HOXB9 polypeptide, a KCNN4 polypeptide, a KLK1 polypeptide, a KRT20 polypeptide, a KRT23 polypeptide, a KRT8 polypeptide, an LGALS4 polypeptide, a METTL7B polypeptide, a MISP polypeptide, a MUC2 polypeptide, a MYB polypeptide, a MYBL2 polypeptide, a MYO1A polypeptide, a PHGR1 polypeptide, a PITX1 polypeptide, a PKP3 polypeptide, a PLAC8 polypeptide, a PLEK2 polypeptide, a PLS1 polypeptide, a PPP1R14D polypeptide, a PRR15 polypeptide, a PTK6 polypeptide, an S100A14 polypeptide, an S100P polypeptide, a SAPCD2 polypeptide, a SERPINB5 polypeptide, a SPDEF polypeptide, a TRIM15 polypeptide, a TRIM31 polypeptide, a USH1C polypeptide, a VIL1 polypeptide, and a combination thereof. In some embodiments, the intravesicular biomarker described herein may comprise at least one post-translational modification.

[0168] AGMAT RNA, AGR2 RNA, AGR2 RNA, AGR2 RNA, 4B RNA、ANO9 RNA、AP1M2 RNA、ARSE RNA、ASCL2 RNA、ATP10B RNA、B3GNT3 RNA、BIK RNA、BSPRY RNA、C10orf99 RNA、C15orf48 RNA、C10orf99 RNA、C15orf48 RNA、C10610f210fRNA RNA、C9orf152 RNA、C12 RNA、CBLC RNA、CCL24 RNA、CD24 RNA、CDCA7 RNA、CDH1 RNA、CDH17 RNA、CDH3 RNA、CDHR1 RNA、CDHR5 RNA CDHR5 RNA1XXXXXX RNA、CEACAM6 RNA、CEACAM7 RNA、CFTR RNA、CLDN2 RNA、CLDN3 RNA、CLDN4 RNA、CLDN7 RNA、CLRN3 RNA、COL17A1 RNA、CRB3 RNA、CYP2S1 RNA、DDP1 RNA RNA, DSG2 RNA, EHF RNA, ELF3 RNA, EPCAM RNA, EPHB3 RNA, EPS8L3 RNA, ERN2 RNA, ESRP1 RNA, ESRP2 RNA, ETV4 RNA, EVPL RNA, FA2HF RNA, and FA2 RNA、FAM83E RNA、FAM84A RNA、FAT1 RNA、FERMT1 RNA、FOXA2 RNA、FOXA3 RNA、FOXQ1 RNA、FUT2 RNA、FUT3 RNA、FXYD3 RNA、NT3GRNAGGG16GRNA RNA、GJB3 RNA、GPA33 RNA、GPR160 RNA、GPR35 RNA、GPX2 RNA、GRB7 RNA、GUCY2C RNA、HKDC1 RNA、HMGCS2 RNA、HNF4A RNA9 HMGCS2 RNA、HNF4A RNA9 RNA、KCNN4 RNA、KIAA1324 RNA、KLK1 RNA、KRT20 RNA、KRT23 RNA、KRT8 RNA、LGALS4 RNA、LGR5 RNA、LY6G6D RNA、MEP1A RNAET7MRNA、MRNA 13. RNA、MUC2RNA, MYB RNA, MYBL2 RNA, MYO1A RNA, NOX1 RNA, PDZK1IP1 RNA, PHGR1 RNA, PIGR RNA, PITX1 RNA, PKP3 RNA, PLAC8 RNA, PLEK2 RNA, PLS1 RNA, POF1B RNA, PPP1R14D RNA, PROM1 RNA, PRR15 RNA, PRSS8 RNA, PTK6 RNA, RAB25 RNA, RNF128 RNA, RNF186 RNA, RNF43 RNA, S100A14 RNA, S100P RNA, SAPCD2 RNA, SERPINB5 RNA, SLC26A3 RNA, SLC39A5 RNA, SLC44A4 RNA, SLC5A1 RNA, SMIM22 RNA, SPDEF RNA, ST6GALNAC1 RNA, TJP3 RNA, TM4SF5 RNA, TMC5 RNA, TMEM45B RNA, TMPRSS2 RNA, TMPRSS4 RNA, TNS4 The vesicle comprises one or more intravesicular RNA biomarkers selected from the list consisting of RNA, TRABD2A RNA, TRIM15 RNA, TRIM31 RNA, TSPAN1 RNA, TSPAN8 RNA, UGT2B17 RNA, UGT8 RNA, USH1C RNA, VIL1 RNA, CLDN6 RNA, CRABP2 RNA, KLK7 RNA, MIF RNA, S100A1 RNA, PRAME RNA, and combinations thereof.

[0169] In some embodiments, the target biomarker signature for colorectal 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, e.g., 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.

[0170] In some embodiments, the target biomarker signature for colorectal 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, the at least one extracellular vesicle-associated surface biomarker and the at least one surface biomarker are the same.

[0171] In some embodiments, at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker of the target biomarker signature for colorectal cancer are distinct. For example, in some embodiments, the target biomarker signature for colorectal cancer comprises at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker.

[0172] In some embodiments, a target biomarker signature for colorectal 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.

[0173] In some embodiments, the target biomarker signature for colorectal 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.

[0174] In some embodiments, a target biomarker signature for colorectal 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, for example, 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, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers may be encoded by different genes.

[0175] In some embodiments, the target biomarker signature for colorectal 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.

[0176] 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.

[0177] 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).

[0178] 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.).

[0179] 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-.

[0180] 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).

[0181] In some embodiments, the target biomarker signature comprises a combination of at least two biomarkers, the combination being one of: a CYP2S1 polypeptide and a FERMT1 polypeptide; or a HKDC1 polypeptide and a TOMM34 polypeptide; or a CYP2S1 polypeptide and an S100P polypeptide; or a CEACAM6 polypeptide and an HKDC1 polypeptide; or a CYP2S1 polypeptide and an NLN polypeptide; or a CEACAM6 polypeptide and an HACD3 polypeptide; or a FERMT1 polypeptide and an S100P polypeptide; or a CASK polypeptide and an S100P polypeptide; or a CYP2S1 polypeptide and an LBR polypeptide; or a CYP2S1 polypeptide and an LMNB1 polypeptide; or a CEACAM6 polypeptide and an NLN polypeptide; or a CEACAM6 polypeptide and a CHMP4B polypeptide; or an ALG5 polypeptide and a CYP2S1 polypeptide; or a CEACAM6 polypeptide and a PGAM5 polypeptide; or a CE an ACAM6 polypeptide and an RPS3 polypeptide; or a BCAP31 polypeptide and a CEACAM6 polypeptide; or a FERMT1 polypeptide and a TOMM22 polypeptide; or a CYP2S1 polypeptide and a PGAM5 polypeptide; or a CEACAM6 polypeptide and an ITGA2 polypeptide; or an HKDC1 polypeptide and an S100P polypeptide; or a CYP2S1 polypeptide and a RAP2A polypeptide; or a CYP2S1 polypeptide and an SLC25A6 polypeptide; or a HEPH polypeptide and a TOMM34 polypeptide; or a DSG2 polypeptide and a TOMM34 polypeptide; or an EPHB3 polypeptide and an HKDC1 polypeptide; or a CEACAM5 polypeptide and a DPEP1 polypeptide; or a CEACAM6 polypeptide and a FERMT1 polypeptide; or a CHDH polypeptide and an EPHB2 polypeptide; or a CHMP4B polypeptide and a CYP2S1 polypeptide; or a CEACAM6 polypeptide and an LAD1 polypeptide;or a MARCKSL1 polypeptide and an S100P polypeptide; or a CDH1 polypeptide and a FERMT1 polypeptide; or an EPHB2 polypeptide and an HACD3 polypeptide; or a FERMT1 polypeptide and a TOMM34 polypeptide; or an EPHB2 polypeptide and an LSR polypeptide; or an EPHB3 polypeptide and a FERMT1 polypeptide; or an EPHB2 polypeptide and a MARCKSL1 polypeptide; or an EPHB2 polypeptide and a LAMC2 polypeptide; or an EPHB2 polypeptide and a SORD polypeptide; or an HKDC1 polypeptide and a LAMC2 polypeptide; or an EPHB3 polypeptide and an S100P polypeptide; or an ACSL5 polypeptide and a LAMC2 polypeptide; or an EPCAM polypeptide and a PGAM5 polypeptide; or an HKDC1 polypeptide and an SNTB1 polypeptide; or a MAP7 polypeptide and an S100P polypeptide; or a DPEP1 polypeptide and an SNTB1 polypeptide; or C an HMP4B polypeptide and an EPHB2 polypeptide; or a FERMT1 polypeptide and an SNTB1 polypeptide; or a BCAP31 polypeptide and an EPCAM polypeptide; or a FERMT1 polypeptide and a LAMC2 polypeptide; or a DPEP1 polypeptide and a TOMM34 polypeptide; or a CEACAM5 polypeptide and an ITGA2 polypeptide; or a FERMT1 polypeptide and a RAP2A polypeptide; or a LAMC2 polypeptide and an S100P polypeptide; or a GALNT3 polypeptide and a TOMM34 polypeptide; or a DPEP1 polypeptide and a MARCKSL1 polypeptide; or an ACSL5 polypeptide and a TOMM34 polypeptide; or a DSG2 polypeptide and a MARCKSL1 polypeptide; or an AP1M2 polypeptide and an S100P polypeptide; or an EPCAM polypeptide and a LAMC2 polypeptide; or a BCAP31 polypeptide and an EPHB2 polypeptide; or a CASK polypeptide and an EPHB2 polypeptide;or an ATP1B1 polypeptide and an S100P polypeptide; or an EPCAM polypeptide and an RPN2 polypeptide; or a CDH17 polypeptide and a SORD polypeptide; or an LSR polypeptide and a MARCKSL1 polypeptide; or a CEACAM5 polypeptide and an HACD3 polypeptide; or an EPCAM polypeptide and an SNTB1 polypeptide; or an EPCAM polypeptide and a TOMM34 polypeptide; or a CEACAM5 polypeptide and an SNTB1 polypeptide; or a CHDH polypeptide and a TOMM34 polypeptide; or an ACSL5 polypeptide and an EPHB3 polypeptide; or an ALG5 polypeptide and an EPCAM polypeptide; or a CLIC1 polypeptide and an EPCAM polypeptide; or an ACSL5 polypeptide and a MARCKSL1 polypeptide; or an EPCAM polypeptide and an RPS3 polypeptide; or a CEACAM5 polypeptide and a MARCKSL1 polypeptide; or a CEACAM5 polypeptide and a RAP2A polypeptide; or a CEACAM5 polypeptide and an STT3B polypeptide; or a CDH17 polypeptide and an EPHB3 polypeptide; or a MARCKSL1 polypeptide and a TOMM34 polypeptide; or a CEACAM5 polypeptide and a PTK7 polypeptide; or a CEACAM5 polypeptide and an SLC12A2 polypeptide; or an EPHB3 polypeptide and an LSR polypeptide; or a CEACAM5 polypeptide and an RCC2 polypeptide; or a DSG2 polypeptide and a LAMC2 polypeptide; or a CHDH polypeptide and a MARCKSL1 polypeptide; or a CDH17 polypeptide and a PTK7 polypeptide; or a CLIC1 polypeptide and a MARCKSL1 polypeptide; or a DSG2 polypeptide and a PGAM5 polypeptide; or a DPEP1 polypeptide and an EPHB3 polypeptide; or an EPHB3 polypeptide and a LAMC2 polypeptide; or a SLC2A1 polypeptide and SNTB1 polypeptide; or a DPEP1 polypeptide and a PGAM5 polypeptide;or a CDH17 polypeptide and an RPN2 polypeptide; or a CDH17 polypeptide and an SNTB1 polypeptide; or an HKDC1 polypeptide and an SLC2A1 polypeptide; or a CDH17 polypeptide and an ITGA2 polypeptide; or a DPEP1 polypeptide and a SORD polypeptide; or a CDH17 polypeptide and an LAMC2 polypeptide; or a CEACAM6 polypeptide and a RAP2B polypeptide; or a CEACAM6 polypeptide and a CLIC1 polypeptide; or a CEACAM6 polypeptide and a SYAP1 polypeptide; or a CDH1 polypeptide and a CEACAM6 polypeptide; or an EPHB2 polypeptide and an SLC12A2 polypeptide; or a FERMT1 polypeptide and a RAB25 polypeptide; or a FERMT1 polypeptide and an HKDC1 polypeptide; or a DPEP1 polypeptide and an S100P polypeptide; or an EPHB2 polypeptide and an S100P polypeptide; or an EPHB2 polypeptide and a TOMM34 polypeptide tide; or an RCC2 polypeptide and an S100P polypeptide; or an EPCAM polypeptide and a SORD polypeptide; or an EPCAM polypeptide and an ITGA2 polypeptide; or an LSR polypeptide and a TOMM34 polypeptide; or a HEPH polypeptide and a MARCKSL1 polypeptide; or a CEACAM5 polypeptide and a PGAM5 polypeptide; or a MARCKSL1 polypeptide and a SYAP1 polypeptide; or a DPEP1 polypeptide and an LMNB1 polypeptide; or a CDH17 polypeptide and a SLC12A2 polypeptide; or a HEPH polypeptide and a LAMC2 polypeptide; or an ACSL5 polypeptide and an SNTB1 polypeptide; or a DSG2 polypeptide and an RPN2 polypeptide; or a DPEP1 polypeptide and a RUVBL2 polypeptide; or a HACD3 polypeptide and a HEPH polypeptide; or an EPHB3 polypeptide and a SNTB1 polypeptide; or a CEACAM6 polypeptide and a RAB25 polypeptide;or a CEACAM6 polypeptide and an S100P polypeptide; or an EPHB2 polypeptide and a FERMT1 polypeptide; or a FERMT1 polypeptide and an ITGA2 polypeptide; or a BCAP31 polypeptide and a FERMT1 polypeptide; or an S100P polypeptide and an SLC12A2 polypeptide; or a FERMT1 polypeptide and an SLC12A2 polypeptide; or a CDH17 polypeptide and an S100P polypeptide; or an S100P polypeptide and a SORD polypeptide; or an EPHB2 polypeptide and an LMNB2 polypeptide; or an LMNB2 polypeptide and an S100P polypeptide; or an EPHB2 polypeptide and a RAP2B polypeptide; or a CDH17 polypeptide and a TOMM34 polypeptide; or a CEACAM5 polypeptide and an EPHB3 polypeptide; or a CEACAM5 polypeptide and a TOMM34 polypeptide; or an ST14 polypeptide and a TOMM34 polypeptide; or a CDH1 polypeptide and a TOMM34 polypeptide polypeptide; or an EPCAM polypeptide and an NLN polypeptide; or an EPCAM polypeptide and an EPHB3 polypeptide; or a CASK polypeptide and a CEACAM5 polypeptide; or a CEACAM5 polypeptide and a SORD polypeptide; or a CEACAM5 polypeptide and an RPS3 polypeptide; or a CDH17 polypeptide and an SLC2A1 polypeptide; or an EPHB3 polypeptide and a TOMM34 polypeptide; or a BCAP31 polypeptide and a CEACAM5 polypeptide; or a BCAP31 polypeptide and a CDH17 polypeptide; or a CDH17 polypeptide and an RPS3 polypeptide; or an EPHB3 polypeptide and an SLC2A1 polypeptide; or a CLIC1 polypeptide and a DSG2 polypeptide; or a DSG2 polypeptide and an LMNB2 polypeptide; or an EPHB3 polypeptide and a GPA33 polypeptide; or an ATP1B1 polypeptide and an EPHB3 polypeptide; or a CDH1 polypeptide and an EPHB3 polypeptide;Or a CASK polypeptide and a DSG2 polypeptide; or a GPA33 polypeptide and a SLC2A1 polypeptide; or a DSG2 polypeptide and a RUVBL2 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.

[0182] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CYP2S1 polypeptide and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a HKDC1 polypeptide and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CYP2S1 polypeptide and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an HKDC1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CYP2S1 polypeptide and an NLN polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an HACD3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FERMT1 polypeptide and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CASK polypeptide and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CYP2S1 polypeptide and an LBR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CYP2S1 polypeptide and an LMNB1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an NLN polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a CHMP4B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an ALG5 polypeptide and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a PGAM5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an RPS3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a BCAP31 polypeptide and a CEACAM6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a RAP2B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a LAD1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a CLIC1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a SYAP1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an EPCAM polypeptide and a SNTB1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CDH1 polypeptide and a CEACAM6 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an HKDC1 polypeptide and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an EPHB3 polypeptide and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a FERMT1 polypeptide and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an ITGA2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a RAB25 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a DSG2 polypeptide and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a CEACAM6 polypeptide and a FERMT1 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.

[0183] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ACVR2B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a B3GNT3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CD133 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CDH17 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CDH3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CEACAM5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CEACAM6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CFB polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CFTR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a DLL4 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an EDAR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an EPCAM polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an EPHB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an EPHB3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ERBB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a FAP polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a GPCR5A polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an IHH polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ILDR1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an ITGAV polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a KCNQ1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a KEL 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 an MST1R polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a MUC5AC polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and a NOX1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an OCIAD2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an RNF43 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise a MUC1 polypeptide and an SMIM22 polypeptide. In some embodiments, the target biomarkers in the foregoing combinations may be used as targets for capture probes and / or targets for detection probes in the assays described herein.

[0184] In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ACVR2B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a B3GNT3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CD133 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CDH17 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CDH3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CEACAM5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CEACAM6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CFB polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CFTR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a DLL4 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an EDAR polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an EPCAM polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an EPHB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an EPHB3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ERBB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a FAP polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a GPCR5A polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an IHH polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ILDR1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an ITGAV polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a KCNQ1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a KEL 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 an MST1R polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a MUC5AC polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a NOX1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an OCIAD2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and an RNF43 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sTn antigen and a SMIM22 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 sLex antigen and an ACVR2B polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a B3GNT3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CD133 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CDH17 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CDH3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CEACAM5 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CEACAM6 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CFB polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CFTR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a DLL4 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an EDAR polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an EPCAM polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an EPHB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an EPHB3 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ERBB2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a FAP polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a GPCR5A polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an IHH polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ILDR1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an ITGAV polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a KCNQ1 polypeptide.In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a KEL 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 an MST1R polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a MUC5AC polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and a NOX1 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an OCIAD2 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an RNF43 polypeptide. In some embodiments, the target biomarker signature comprises at least two biomarkers, which are or comprise an sLex antigen and an SMIM22 polypeptide. 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.

[0186] In some embodiments, the target biomarker signature comprises a combination of at least three biomarkers, the combination being: a CYP2S1 polypeptide, an EPHB2 polypeptide, and an S100P polypeptide; or an EPHB2 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide; or a CYP2S1 polypeptide, an EPHB2 polypeptide, and a FERMT1 polypeptide; or a CYP2S1 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide; or a CYP2S1 polypeptide, a FERMT1 polypeptide, and a MARCKSL1 polypeptide; or a CEACAM6 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide; or a CEACAM6 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide; or a CEACAM5 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide; or a CYP2S1 polypeptide, a DSG2 polypeptide, and a FERMT1 polypeptide; or a DPEP1 polypeptide. or a CEACAM6 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide; or a CEACAM6 polypeptide, a FERMT1 polypeptide, and an LAMC2 polypeptide; or a CEACAM6 polypeptide, a CYP2S1 polypeptide, and a MARCKSL1 polypeptide; or a CYP2S1 polypeptide, an EPHB2 polypeptide, and a TOMM34 polypeptide; or a CEACAM6 polypeptide, a CYP2S1 polypeptide, and an EPHB2 polypeptide; or an EPHB2 polypeptide, an EPHB3 polypeptide, and an S100P polypeptide; or an EPHB2 polypeptide, a MARCKSL1 polypeptide, and an S100P polypeptide; or a CEACAM6 polypeptide, a DPEP1 polypeptide, and an S100P polypeptide; or a CEACAM6 polypeptide, an EPHB2 polypeptide, and an S100P polypeptide; or an EPHB2 polypeptide, a LAMC2 polypeptide, and an S100P polypeptide; or a CDH17 polypeptide, an EPHB3 polypeptide, and an S100P polypeptide;or a CEACAM6 polypeptide, a MARCKSL1 polypeptide, and a TOMM34 polypeptide; or a CDH17 polypeptide, an EPHB3 polypeptide, and a SLC2A1 polypeptide; or a CEACAM5 polypeptide, an EPHB3 polypeptide, and a TOMM34 polypeptide; or a CDH17 polypeptide, an EPHB3 polypeptide, and a TOMM34 polypeptide; or a CDH17 polypeptide, an SLC2A1 polypeptide, and a SNTB1 polypeptide; or a CDH17 polypeptide, an SLC2A1 polypeptide, and a TOMM34 polypeptide; or a CEACAM5 polypeptide, an SNTB1 polypeptide, and a TOMM34 polypeptide; or an EPCAM polypeptide, an EPHB3 polypeptide, and a TOMM34 polypeptide; or a CEACAM5 polypeptide, an EPHB3 polypeptide, and a LAMC2 polypeptide; or a CEACAM5 polypeptide, an EPHB3 polypeptide, and a MARCKSL1 polypeptide; or an EPCAM polypeptide, an EPHB3 polypeptide, and a MAR a CKSL1 polypeptide; or a CEACAM5 polypeptide, an EPHB3 polypeptide, and an SLC25A6 polypeptide; or a DPEP1 polypeptide, a MARCKSL1 polypeptide, and an SNTB1 polypeptide; or a GPA33 polypeptide, an SLC2A1 polypeptide, and an SNTB1 polypeptide; or a DPEP1 polypeptide, a LAMC2 polypeptide, and an SNTB1 polypeptide; or an LSR polypeptide, an SLC25A6 polypeptide, and an ST14 polypeptide; or a GPA33 polypeptide, an HACD3 polypeptide, and an SLC2A1 polypeptide; or a DSG2 polypeptide, a LAMC2 polypeptide, and an LMNB1 polypeptide; or a DPEP1 polypeptide, a PGAM5 polypeptide, and an SNTB1 polypeptide; or an LAMC2 polypeptide, an LMNB1 polypeptide, and an LSR polypeptide; or a DSG2 polypeptide, a LAMC2 polypeptide, and a SORD polypeptide; or a HACD3 polypeptide, a MUC13 polypeptide, and an SLC2A1 polypeptide;or a LAMC2 polypeptide, a MUC13 polypeptide, and a SNTB1 polypeptide; or a DSG2 polypeptide, a GALNT3 polypeptide, and a LAMC2 polypeptide; or an HKDC1 polypeptide, an LSR polypeptide, and a SLC2A1 polypeptide; or an AP1M2 polypeptide, an LSR polypeptide, and a RUVBL2 polypeptide; or an AP1M2 polypeptide, an LSR polypeptide, and a SLC25A6 polypeptide; or a GALNT3 polypeptide, a LAMC2 polypeptide, and a LMNB1 polypeptide; or a DPEP1 polypeptide, an RPN2 polypeptide, and a SNTB1 polypeptide; or a GPA33 polypeptide, an SLC12A2 polypeptide, and a SLC2A1 polypeptide; or a MUC13 polypeptide, an SLC2A1 polypeptide, and a SNTB1 polypeptide; or a DSG2 polypeptide, a MAP7 polypeptide, and a NUP210 polypeptide; or an AP1M2 polypeptide, an LMNB2 polypeptide, and an LSR polypeptide; or an AP1M2 polypeptide, SLC25A6 or a DSG2 polypeptide, a GALNT3 polypeptide, and a SORD polypeptide; or a GPA33 polypeptide, a PTK7 polypeptide, and a SLC2A1 polypeptide; or a DPEP1 polypeptide, a HACD3 polypeptide, and a SNTB1 polypeptide; or a DPEP1 polypeptide, a RUVBL2 polypeptide, and a SLC25A6 polypeptide; or a DPEP1 polypeptide, a PGAM5 polypeptide, and a RUVBL2 polypeptide; or a DPEP1 polypeptide, a HACD3 polypeptide, and a LMNB2 polypeptide; or a GPA33 polypeptide, a RPS3 polypeptide, and a SLC25A6 polypeptide; or an LSR polypeptide, a RUVBL2 polypeptide, and a ST14 polypeptide; or a DSG2 polypeptide, a GALNT3 polypeptide, and a GNPNAT1 polypeptide; or a DSG2 polypeptide, a LMNB1 polypeptide, and a SLC12A2 polypeptide; or a CHDH polypeptide, a LMNB2 polypeptide, and an LSR polypeptide;or an HKDC1 polypeptide, an LSR polypeptide, and an SLC25A6 polypeptide; or a DSG2 polypeptide, a GALNT3 polypeptide, and an ITGA2 polypeptide; or a GPA33 polypeptide, an HACD3 polypeptide, and an SLC12A2 polypeptide; or a CASK polypeptide, a CDH1 polypeptide, and an RPN2 polypeptide; or a GPA33 polypeptide, a PTK7 polypeptide, and an SLC25A6 polypeptide; or an ATP1B1 polypeptide, an LMNB1 polypeptide, and a MAP7 polypeptide; or a DSG2 polypeptide, a GALNT3 polypeptide, and an NUP210 polypeptide; or a CLIC1 polypeptide, a HEPH polypeptide, and a RAB25 polypeptide; or an HKDC1 polypeptide, an SLC25A6 polypeptide, and an ST14 polypeptide; or a CDH1 polypeptide, a GOLIM4 polypeptide, and a PGAM5 polypeptide; or a CHMP4B polypeptide, an HKDC1 polypeptide, and a RAB25 polypeptide; or a PIGR polypeptide or a CASK polypeptide, a CDH1 polypeptide, and a KPNA2 polypeptide; or a CDH1 polypeptide, an MLEC polypeptide, and an RPN2 polypeptide; or a CDH1 polypeptide, an HKDC1 polypeptide, and a RAP2B polypeptide; or a CHDH polypeptide, a PGAM5 polypeptide, and an ST14 polypeptide; or a CDH1 polypeptide, a GOLIM4 polypeptide, and an RPN2 polypeptide; or a CDH1 polypeptide, an HACD3 polypeptide, and an LMNB1 polypeptide; or a CLIC1 polypeptide, a HEPH polypeptide, and an ST14 polypeptide; or an ACSL5 polypeptide, an ATP1B1 polypeptide, and a KPNA2 polypeptide; or an ALG5 polypeptide, a CDH1 polypeptide, and an RPN2 polypeptide; or a CHDH polypeptide, an RPN2 polypeptide, and ST14 polypeptide; or a CHDH polypeptide, an LMNB1 polypeptide, and an RPS3 polypeptide;or a CASK polypeptide, an ITGA2 polypeptide, and an MLEC polypeptide; or a CASK polypeptide, a CISD2 polypeptide, and an ITGA2 polypeptide; or a CASK polypeptide, an ITGA2 polypeptide, and an STT3B polypeptide; or a CHDH polypeptide, a CLIC1 polypeptide, and a TMPO polypeptide; or an ACSL5 polypeptide, a COPG2 polypeptide, and an RPN1 polypeptide; or an ALDH18A1 polypeptide, a GOLIM4 polypeptide, and an RPN1 polypeptide; or an ACSL5 polypeptide, a CHDH polypeptide, and a PTK7 polypeptide; or an LAD1 polypeptide, a RAP2B polypeptide, and an SSR4 polypeptide; or an ACSL5 polypeptide, an ATP1B1 polypeptide, and an RCC2 polypeptide; or an ACSL5 polypeptide, a CHDH polypeptide, and a CLIC1 polypeptide; or an ACSL5 polypeptide, a GNPNAT1 polypeptide, and an LAD1 polypeptide; or an ALDH18A1 polypeptide, an ATP1B1 polypeptide, and an RCC2 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;

[0187] In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, an EPHB2 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, an EPHB2 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, a FERMT1 polypeptide, and a MARCKSL1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, a DSG2 polypeptide, and a FERMT1 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a DPEP1 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and an LAMC2 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and a MARCKSL1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and a LAMC2 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, an EPHB3 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a MARCKSL1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPCAM polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a DPEP1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, an EPHB3 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, an EPHB2 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, an EPHB3 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, an EPHB3 polypeptide, and a S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a BCAP31 polypeptide, a CEACAM6 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a S100P polypeptide, and a SLC12A2 polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CASK polypeptide, an EPHB2 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, an EPHB2 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, an EPHB3 polypeptide, and an SLC2A1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a PIGT polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FERMT1 polypeptide, a MARCKSL1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPCAM polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CYP2S1 polypeptide, an EPHB2 polypeptide, and a PIGT polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FERMT1 polypeptide, an LSR polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, a CYP2S1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a CYP2S1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an ACSL5 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a DSG2 polypeptide, a FERMT1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FERMT1 polypeptide, a HKDC1 polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a MARCKSL1 polypeptide, and a PIGT polypeptide.In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a FERMT1 polypeptide, a HEPH polypeptide, and a PIGT polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPCAM polypeptide, a PIGT polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, a PIGT polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, a FERMT1 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPHB2 polypeptide, an EPHB3 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise an EPCAM polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, an EPHB3 polypeptide, and a FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM6 polypeptide, an EPHB2 polypeptide, and an S100P polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CDH17 polypeptide, a FERMT1 polypeptide, and a TOMM34 polypeptide. In some embodiments, the target biomarker signature comprises at least three biomarkers, which are or comprise a CEACAM5 polypeptide, an EPHB3 polypeptide, and a FERMT1 polypeptide. In some embodiments, at least one target biomarker in the aforementioned 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 Colorectal Cancer

[0188] 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 stool-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.

[0189] 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.

[0190] 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.

[0191] 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).

[0192] 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. For example, in some embodiments, the nucleotide sequence encoding the biomarker of interest 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).

[0193] 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.

[0194] 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).

[0195] In some embodiments, methods for detecting one or more provided biomarkers in extracellular vesicles can be based on bulk EV sample analysis.

[0196] 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)."

[0197] 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).

[0198] 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 colorectal 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.

[0199] 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.

[0200] 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.

[0201] In some embodiments, nanoparticles having a desired size range containing extracellular vesicles in a sample may be processed before detecting one or more provided biomarkers of a target biomarker signature for colorectal cancer. Different sample processing and / or preparation can be performed, for example, to stabilize the target (e.g., target biomarker) in the nanoparticles having a desired size range containing extracellular vesicles to be detected, and / or to facilitate 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 containing cells or extracellular vesicles), and / or blocking of non-specific binding sites.

[0202] In one aspect, the present disclosure provides a method for detecting the presence or absence of a target biomarker signature for colorectal cancer in a biological sample from a subject in need thereof, which may, in some embodiments, be a biological sample (e.g., but not limited to, a blood-derived sample, a stool-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 colorectal 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 a biological entity of interest (e.g., nanoparticles having a desired size range, including extracellular vesicles) expressing such a target biomarker signature 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 of a predetermined age range, such as under or over 55 years of age), subjects with non-colon-related health diseases, disorders, or conditions (e.g., subjects with non-colorectal cancers, such as lung cancer, ovarian cancer, etc., or subjects with symptoms of inflammatory bowel disease or disorders), subjects with benign colorectal tumors, and combinations thereof.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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 if 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 a colorectal cancer detection assay (e.g., one 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 a colorectal cancer detection assay (e.g., one described herein) can be achieved. In some embodiments, such a reference threshold level (e.g., cutoff value) may be determined by selecting either (i) a certain number of standard deviations away from the mean value obtained from control subjects, or (ii) a certain number of standard deviations away from the maximum assay signal obtained from control subjects, whichever is less restrictive, so that the desired sensitivity and / or specificity of a colorectal cancer detection assay (e.g., those described herein) can be achieved. In some embodiments, control subjects for determining a reference threshold level (e.g., cutoff value) may include, but are not limited to, healthy subjects, subjects with an inflammatory condition (e.g., Crohn's disease, ulcerative colitis, inflammatory bowel disease, etc.), subjects with benign tumors, and combinations thereof. In some embodiments, healthy subjects and subjects with an inflammatory condition (e.g., inflammatory bowel disease, ulcerative colitis, or Crohn's disease) are included in determining the reference threshold level (e.g., cutoff value). In some embodiments, subjects with benign tumors are not included in determining the reference threshold level (e.g., cutoff value).In some embodiments, the reference threshold level (e.g., cutoff value) is at least 1 mil away from (i) the mean value obtained from the control subjects, or (ii) the maximum assay signal obtained from the control subjects, such that a desired specificity (e.g., at least 95% or higher specificity [e.g., including at least 96%, at least 97%, at least 98%, at least 99%, or higher specificity], e.g., in some embodiments, at least 99.8% specificity) of a colorectal cancer detection assay (e.g., those described herein) can be achieved. The reference threshold level (e.g., cutoff value) may be determined by selecting a reference threshold level that is 0.5 or more standard deviations (SD) away (e.g., including at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, or more SDs). In some embodiments, the reference threshold level (e.g., cutoff value) may be determined by selecting (i) the mean value obtained from control subjects or (ii) at least 2.9 SDs (e.g., at least 2.93 SDs) away from the maximum assay signal obtained from control subjects, such that a desired specificity (e.g., at least 99%, or higher) of a colorectal cancer detection assay (e.g., one described herein) can be achieved. In some embodiments, a reference threshold level (e.g., a cutoff value) may be determined by selecting a value at least 2.9 SD (e.g., at least 2.93 SD) away from either (i) the mean value obtained from control subjects, or (ii) the less restrictive of the maximum assay signal obtained from control subjects, so that a desired specificity (e.g., at least 99%, or higher) of a colorectal cancer detection assay (e.g., one described herein) can be achieved.In some embodiments, such a reference threshold level (e.g., cutoff value) may be determined based on the expression level (e.g., transcription level) of the target biomarker in normal healthy tissue versus colorectal cancer samples, such that a desired specificity and / or sensitivity (e.g., as described herein) can be achieved. In some embodiments, the reference threshold level (e.g., cutoff value) may vary depending on, for example, the stage and / or subtype of colorectal cancer, and / or patient characteristics, such as patient age, risk factors for colorectal cancer (e.g., genetic risk versus average risk, lifestyle-related risk factors), symptomatic / asymptomatic status, and combinations thereof.

[0207] In some embodiments, a reference threshold level (e.g., cutoff value) may be determined based on a log-normal distribution across healthy subjects (e.g., subjects of a predetermined age range) and, optionally, subjects with an inflammatory condition (e.g., inflammatory bowel disease, ulcerative colitis, or Crohn's disease), and selection of a level necessary to achieve a desired specificity, e.g., based on the prevalence of colorectal cancer or its subtypes (e.g., including, but not limited to, colorectal adenocarcinoma). In some embodiments, a desired specificity may be at least 70%, including, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or higher.

[0208] The present disclosure also provides, among other things, a technique for determining whether a subject has or is susceptible to colorectal cancer from a sample containing nanoparticles having a desired size range, including extracellular vesicles. For example, in some embodiments, if a body fluid-derived sample (such as, but not limited to, a blood-derived sample, a stool-derived sample, etc.) from a subject in need thereof shows a level of target biomarker signature-expressing extracellular vesicles that is at or above a reference threshold level, e.g., a cutoff value (e.g., as determined according to the present disclosure), the subject is classified as having or susceptible to colorectal cancer. In some such embodiments, the reference threshold level (e.g., a cutoff value) may be determined based on a log-normal distribution across healthy subjects (e.g., subjects within a predetermined age range) and, if necessary, subjects with an inflammatory condition (e.g., inflammatory bowel disease), and the level required to achieve the desired specificity, for example, based on the prevalence of colorectal cancer or its subtypes (e.g., colorectal adenocarcinoma). In some embodiments, the desired specificity may be 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%, at least 99.5%, or higher.

[0209] In some embodiments, a reference threshold level (e.g., cutoff value) may be determined based on the expression levels (e.g., transcription levels) of individual target biomarkers of the target biomarker signature in normal healthy tissue versus colorectal cancer samples, such that a desired specificity and / or sensitivity (e.g., as described herein) can be achieved. In some embodiments, the reference threshold level (e.g., cutoff value) may vary depending on, for example, the stage and / or subtype of colorectal cancer, and / or patient characteristics, such as patient age, risk factors for colorectal cancer (e.g., genetic risk versus average risk, lifestyle-related risk factors), symptomatic / asymptomatic status, and combinations thereof.

[0210] In some embodiments, if a biological sample from a subject in need thereof shows a level of a combination of biomarkers that meets the reference threshold level, the subject is classified as having or susceptible to colorectal cancer. For example, in some embodiments, if a body fluid-derived sample (such as, but not limited to, a blood-derived sample, a stool-derived sample, etc.) from a subject in need thereof shows an elevated level of target biomarker signature-expressing extracellular vesicles compared to the reference threshold level, the subject is classified as having or susceptible to colorectal cancer.

[0211] In some embodiments, a subject in need thereof is classified as having or susceptible to colorectal cancer if the subject's bodily fluid-derived sample (for example, but not limited to, a blood-derived sample, a stool-derived sample, etc.) exhibits a level of target biomarker signature-expressing extracellular vesicles that 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 a reference threshold level. In some embodiments, a subject in need thereof is classified as having or susceptible to colorectal cancer if the subject's bodily fluid-derived sample (for example, but not limited to, a blood-derived sample, a stool-derived sample, etc.) exhibits a level of target biomarker signature-expressing extracellular vesicles that 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 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, or higher, compared to a reference threshold level.

[0212] If a biological sample from a subject in need thereof shows a level comparable to the reference threshold level, the subject is classified as possibly not having colorectal cancer or possibly not susceptible to it. In some such embodiments, the reference threshold level corresponds to the level of extracellular vesicles expressing the target biomarker signature in a comparable sample from a reference subject, for example, a group 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 55 years o...

Claims

A method for obtaining the amount of extracellular vesicles as an indicator for screening colorectal 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 colorectal 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 colorectal cancer biomarker; contacting the captured extracellular vesicles with a second oligonucleotide probe, wherein the second oligonucleotide probe specifically binds to a third colorectal 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 colorectal cancer A method comprising. Claim 2. The second and third biomarkers are selected from the group consisting of surface biomarkers and intra-vesicular biomarkers, The surface biomarker is (i) a polypeptide encoded by the following human genes: ACSL5, ACVR2B, ALDH18A1, ALG5, AP1M2, ATP1B1, B3GNT3, BCAP31, CASK, CD133, CDH1, CDH17, CDH3, CEACAM5, CEACAM6, CFB, CFTR, CHDH, CHMP4B, CISD2, CLIC1, COPG2, CYP2S1, DPEP1, DSG2, EDAR, EPCAM, EPHB2, EPHB3, ERMP1, FERMT1, GALNT3, GNPNAT1, GOLIM4, GPA33, GPCR5A, HACD3, HEPH, HKDC1, IHH, ILDR1, ITGA2, KCNQ1, KEL, KPNNA2, LAD1, LAMC2, LBR, LMNBl, LMNBl, LSR, MAP7, MARCKSL1, MLEC, MUC1, MUC13, NCEH1, NDUFS6, NLN, NOX1, NUP210, OCIAD2, PGAM5, PIGR, PIGT, PTK7, RAB25, RAP2A, RAP2B, RCC2, RNF43, RPN1, RPN2, RPS3, RUVBL2, S100P, SLC12A2, SLC25A6, SLC2A1, SMIM22, SNTB1, SORD, SSR4, ST14, STOML2, STT3B, SYAP1, TM9SF2, TMED2, TMPO, TOMM22, TOMM34, AMHR2, CLDN1, DLL4, EGFR, ERBB2, FAP, FGFR4, FOLR1, GUCY2C, IGF1R, IL1A, ITGAV, KRT8, LGR5, LPR6, MET, MST1R, MUC5AC, TNFRSF10B, VEGFA, and combinations thereof; and / or (ii) a carbohydrate-dependent marker selected from: CanAg (a glycoform of MUC1), Lewis Y / B antigen, Lewis B antigen, sialyltetraosyl carbohydrate, 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)), sialyl Lewis A antigen (also known as CA19-9), SSEA-1 (also known as Lewis X antigen), and NeuGcGM3 (N-glycolyl GM3 ganglioside);and; The method according to claim 1, wherein the intracellular biomarker is selected from polypeptides encoded by the following human genes: AGMAT, AGR2, AGR3, ANKS4B, AP1M2, ARSE, ASCL2, BSPRY, C10orf99, C15orf48, C1orf106, C9orf152, CBL C, CCL24, CDCA7, CDX1, CDX2, DDC, DSG2, EHF, ELF3, EPS8L3, ESRP1, ESRP2, ETV4, EVPL, FABP1, FAM3D, FAM83E, FAM84A, FERMT1, FOXA2, FOXA3, FOXQ1, GPX2, GRB7, HKDC1, HMGCS2, HNF4A, HOXB9, KCNN4, KLK1, KRT20, KRT23, KRT8, LGALS4, METTL7B, MISP, MUC2, MYB, MYBL2, MYO1A, PHGR1, PITX1, PKP3, PLAC8, PLEK2, PLS1, PPP1R14D, PRR15, PTK6, S100A14, S100P, SAPCD2, SERPINB5, SPDEF, TRIM15, TRIM31, USH1C, and VIL1.

3. The method according to claim 1, wherein the first biomarker is a polypeptide encoded by the following human genes: FERMT1, EP-CAM, EphB2, CEACAM6, CEACAM5, CDH17, MARCKSL1, TOMM34, S100P, EphB3, CDH1, MUC13, SLC12A2, RAB25, LAMC2, or a combination thereof, or comprises the same.

4. The method according to claim 1, wherein the capture agent comprises an antibody.

5. The method according to claim 4, wherein the antibody is attached to a solid substrate.

6. The method according to claim 5, wherein the solid substrate is magnetic beads.

7. The method according to claim 1, wherein the sample is subjected to size exclusion chromatography to isolate the extracellular vesicles.

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.

9. The second and third biomarkers are in the following combinations: - at least two distinct surface biomarkers; - at least two distinct intracellular biomarkers; and - Surface biomarkers and intracellular biomarkers; The method according to claim 1, comprising one of them. **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, 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 a benign tumor, subjects having a colon-related disease, and subjects having a non-colon-related disease, disorder, and / or condition. **Claim 12**: The method according to claim 1, wherein when the first and / or second biomarker comprises at least one surface biomarker and / or intracellular 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. **Claim 15**: The method according to claim 14, wherein the oligonucleotides of the first and second oligonucleotide probes are different. **Claim 16**: The method according to claim 1, wherein the method is performed to obtain the amount of extracellular vesicles as an indicator for screening for early colorectal cancer, advanced colorectal cancer, or recurrent colorectal cancer in the subject. **Claim 17**: The subject has the following characteristics: (i) An asymptomatic subject prone to colorectal cancer; (ii) A subject having a family history of colorectal cancer; (iii) A subject who is a smoker or was a smoker; (iv) An obese subject; (v) A subject who consumes an excessive amount of alcohol; (vi) A subject 40 years old or older; (vii) A subject having one or more non-specific symptoms of colorectal cancer, and optionally, at least one of the non-specific symptoms is similar to one or more common 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 colorectal tumor confirmed by imaging; (x) A subject having a benign colon tumor; (xi) A subject previously treated for colorectal 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 intake; (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.

18. The method is the following health evaluation and / or diagnostic assay: (i) A health check-up for the subject once a year; (ii) Imaging examination; (iii) Digital rectal examination; (iv) A genetic assay for screening plasma for gene mutations in circulating tumor DNA and / or cancer-related protein biomarkers; (v) An assay including immunofluorescence staining for identifying cell phenotypes and marker expression, followed by amplification and analysis by next-generation sequencing; (vi) Immunochemical fecal occult blood test (FTI); and (vii) Serum biomarkers The method according to claim 1, used in combination with one or more of the above.

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

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

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