Compositions and methods for detecting ovarian cancer

Abstract

The present disclosure provides, in one aspect, a technique for detecting ovarian cancer, e.g., for early detection of ovarian 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 determined to have or be susceptible to ovarian cancer. In some embodiments, the technique provided herein is useful for developing companion diagnostics, e.g., 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, e.g., by identifying biomarkers in body fluid samples (e.g., blood samples) of female subjects that are associated with treatment response. In some embodiments, the technique provided herein is useful for distinguishing benign adnexal masses from ovarian cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 280603, filed November 17, 2021, U.S. Provisional Application No. 63 / 328250, filed April 6, 2022, and U.S. Provisional Application No. 63 / 417309, filed October 18, 2022, the entire contents of which are hereby incorporated by reference. [Background technology]

[0002] background Early detection of cancer greatly increases the chance of successful treatment. However, many cancers, including ovarian cancer, still lack effective screening recommendations or patient compliance with these recommendations. Typical challenges for cancer screening tests include limited sensitivity and specificity. High false positive rate 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 rate results will miss patients who need therapy, resulting in delayed treatment and consequently reduced chances of success, and therefore fail to meet the purpose of screening tests. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides, among other things, insights and techniques for achieving effective ovarian 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 provided techniques are effective for detecting early-stage ovarian cancer. 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 ovarian cancer (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 ovarian cancer). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals at risk for ovarian cancer (e.g., individuals with genetic and / or lifestyle-related risk factors for ovarian cancer). In some embodiments, the provided technology may be or may include one or more compositions (e.g., molecular entities or complexes, systems, cells, collections, combinations, or kits) and / or methods (e.g., methods of making, using, or assessing), 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 source of certain prior technology challenges, including certain conventional approaches to the detection and diagnosis of ovarian cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as those based on bulk analysis of cell-free nucleic acids, serum biomarkers (e.g., CA-125, which is part of the MUC16 polypeptide), and / or 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 detecting the colocalization of ovarian cancer target biomarker signatures in individual nanoparticles of a desired size range, including extracellular vesicles, which include (i) at least one extracellular vesicle-associated surface biomarker and (ii) at least one target biomarker, including one or more surface biomarkers. In some embodiments, such a target biomarker signature may further include one or more internal biomarkers (e.g., those described herein) and / or one or more RNA biomarkers (e.g., those described herein).

[0005] In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems by detecting, among other things, target biomarker signatures of ovarian cancer using target entity detection approaches developed by applicant and described in US2020 / 0299780 and WO2020180741, which are based on the interaction and / or co-localization of at least two or more target entities (e.g., target biomarker signatures) in individual nanoparticles (e.g., including extracellular vesicles).

[0006] 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 ovarian cancer in individual nanoparticles (hereinafter "nanoparticles" as defined herein) having a desired size range, including extracellular vesicles. In some embodiments, such individual nanoparticles have a size range of about 30 nm to about 1000 nm. 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.

[0007] The present inventors have previously identified certain biomarker combinations and / or biomarker signatures useful for detecting ovarian cancer (see, e.g., WO 2121 / 146659). The present disclosure provides additional biomarker combinations and / or biomarker signatures that have been demonstrated to achieve 90-100% specificity (e.g., as described herein) at certain sensitivities when distinguishing ovarian cancer samples from reference samples (e.g., normal healthy samples, benign tumor samples, and / or off-target cancer samples). In some embodiments, the present disclosure provides biomarker combinations that are particularly useful for detecting early-stage ovarian cancer, e.g., with about 90-100% specificity and / or about 80-95% sensitivity. In some embodiments, the present disclosure provides biomarker combinations that are particularly useful for distinguishing ovarian cancer from benign adnexal masses, e.g., with about 90-100% specificity and / or about 80-100% or about 95%-100% sensitivity. In some embodiments, the present disclosure provides biomarker combinations that are particularly useful for distinguishing between benign adnexal masses and ovarian cancer, e.g., with a positive predictive value of greater than 70% and / or a negative predictive value of greater than 98%.

[0008] 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 ovarian cancer. In some embodiments, the present disclosure provides ovarian cancer screening systems that can be implemented to detect early stage cancers, including, in some embodiments, early stage cancers in asymptomatic individuals (e.g., not at genetic risk for ovarian cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals (e.g., not at genetic risk for ovarian 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., symptomatic or asymptomatic individuals), as well as methods of providing and / or using them. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them.

[0009] 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 ovarian 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) adequate 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 women's regular physical examinations, such as mammograms, HPV and / or Pap smear screening. 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.

[0010] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to ovarian cancer. In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to ovarian cancer. In some embodiments, provided methods or assays include (a) detecting nanoparticles (having a size range of interest, including extracellular vesicles) that express a target biomarker signature for ovarian cancer in a biological sample from a subject in need thereof (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample), wherein the target biomarker signature is expressed by (i) intact or truncated polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2, and combinations thereof; and / or (ii) one of the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis (b) comparing sample information indicating the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to ovarian cancer if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff that references the reference threshold level.

[0011] In some embodiments, the at least one target biomarker comprises one or more surface biomarkers selected from (i) intact or truncated polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0012] 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.

[0013] In some embodiments, the extracellular vesicle-associated surface biomarkers for use in the target biomarker signatures for ovarian cancer used and / or described herein may be or include tumor-specific and / or tissue-specific biomarkers (e.g., ovarian 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 may include, but are not limited to, (i) intact or truncated polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof. In some embodiments, such extracellular vesicle-associated surface biomarkers may include, but are not limited to, (i) intact or truncated polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, and combinations thereof; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0014] In some embodiments, a target biomarker signature for ovarian cancer detection includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes an intact or truncated polypeptide encoded by the human gene SLC34A2; and (ii) one or more target surface biomarkers that include intact or truncated polypeptides encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

[0015] In some embodiments, a target biomarker signature for ovarian cancer detection comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker that comprises a sialyl-Tn (sTn) antigen.

[0016] In some embodiments, a target biomarker signature for ovarian cancer detection includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes an intact or truncated polypeptide encoded by the human gene BST2; and (ii) one or more target surface biomarkers that include an intact or truncated polypeptide encoded by the human gene FOLR1.

[0017] In some embodiments, a target biomarker signature for ovarian cancer detection comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

[0018] In some embodiments, a target biomarker signature for ovarian cancer detection comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC1; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0019] In some embodiments, a target biomarker signature for ovarian cancer detection comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an intact or truncated polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0020] In some embodiments, the target biomarker signature for detecting ovarian cancer comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including a sialyl-Tn (sTn) antigen; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof. In some such embodiments, the surface biomarker encoded by the human gene MUC16 may be an intact MUC16 polypeptide. In some such embodiments, the surface biomarker encoded by the human gene MUC16 may be a truncated MUC16 polypeptide.

[0021] In some embodiments, a target biomarker signature for ovarian cancer detection comprises one or more target surface biomarkers: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker, including the Thomsen-Friedenreich (T,TF) antigen; and (ii) at least one intact or truncated polypeptide encoded by the human gene BST2.

[0022] 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 nanoparticles (having a size range of interest, including extracellular vesicles) observed in comparable samples from a population of non-ovarian cancer subjects.

[0023] In some embodiments, extracellular vesicle-associated surface biomarkers included in the target biomarker signature may be detected using affinity agents (for example, but not limited to, antibody-based agents). In some embodiments, extracellular vesicle-associated surface biomarkers may be detected using a capture assay comprising an antibody-based agent. For example, in some embodiments, a capture assay for detecting the presence of extracellular vesicle-associated surface biomarkers in extracellular vesicles may include contacting a biological sample (e.g., in some embodiments, a body fluid-derived sample, such as, but not limited to, a blood-derived sample) containing nanoparticles 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., antibody agent) that targets the extracellular vesicle-associated surface biomarker.

[0024] 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 biomarkers, such as mRNA, small nuclear RNA (snRNA), microRNA (miRNA), small interfering RNA (siRNA), orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA, may be detected using nucleic acid-based agents, e.g., using quantitative reverse transcription PCR.

[0025] For example, in some embodiments in which the target biomarker is or comprises a surface biomarker and / or an intravesicular biomarker, such a target biomarker may be detected, for example, by a proximity ligation assay following a capture assay (e.g., as described herein) for capturing nanoparticles 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 biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample) containing nanoparticles with a set of detection probes each targeting a target biomarker, the set including at least two distinct detection probes, such that a combination comprising the nanoparticles and the set of detection probes is created, the two detection probes each including: (i) a binding moiety directed to the surface biomarker and / or the intravesicular biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain including a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain. The single-stranded overhang portion of the detection probe is characterized in that when the detection probes are bound to the same extracellular vesicle, they can hybridize with each other.The combination of nanoparticles 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 nanoparticles, so that the detection probes can bind to the same extracellular vesicle and form double-stranded complexes.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.The presence of this ligated template indicates the presence of nanoparticles that are positive for the target biomarker signature of ovarian cancer.Such proximity ligation assays may perform better, e.g., with greater specificity and / or sensitivity, than other existing proximity ligation assays, although those of skill in the art reading this disclosure will recognize that other forms of proximity ligation assays known in the art may alternatively be used.

[0026] In some embodiments, where the target biomarker is or includes an intravesicular RNA (e.g., mRNA, snRNA, miRNA, siRNA, orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA) marker, such target biomarker may be detected using a nucleic acid detection assay. In some embodiments, an exemplary nucleic acid detection assay may be or include reverse transcription PCR.

[0027] In some embodiments in which the target biomarker is or comprises an intravesicular biomarker and / or an intravesicular RNA (e.g., mRNA, snRNA, miRNA, siRNA, orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA) biomarker, such target biomarker may be detected by treating the sample (e.g., fixation and / or permeabilization) prior to the detection assay (e.g., a proximity ligation assay described herein) to expose such biomarker within the nanoparticles for subsequent detection.

[0028] The present disclosure recognizes, among other things, that detection of a single ovarian cancer-associated serum protein or multiple ovarian 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 ovarian cancer. The present disclosure provides technologies, including systems, compositions, and / or methods, that solve such problems, including, among other things, by specifically requiring that nanoparticles having a size range of interest, including individual extracellular vesicles, 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, including one or more surface biomarkers (e.g., as described herein). In certain embodiments, the present disclosure teaches techniques that require such individual nanoparticles characterized by the presence (e.g., by expression) of such a target biomarker signature for ovarian cancer, while nanoparticles that do not include the target biomarker signature do not produce a detectable signal (e.g., a level above a reference level, e.g., at least 10% or more above, where in some embodiments the reference level may be the level observed in a negative control sample, such as a sample lacking the individual nanoparticles comprising such a target biomarker signature).

[0029] Thus, in some embodiments, the techniques provided herein may be useful for detecting the occurrence or recurrence of ovarian 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 ovarian cancer. In some embodiments, a target biomarker signature may be selected for the detection of a particular category of ovarian cancer, including, for example, but not limited to, high-grade serous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, low-grade serous ovarian cancer, and / or mucinous ovarian cancer. 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 ovarian cancer or the recurrence of ovarian cancer.

[0030] In some embodiments, subjects suitable for the techniques provided herein for detecting the onset or recurrence of ovarian cancer may be asymptomatic human subjects and / or the asymptomatic population as a whole. Such asymptomatic subjects may include subjects with a family history of ovarian cancer, subjects with a lifestyle history that places them at increased risk for ovarian cancer, postmenopausal subjects, subjects previously treated for ovarian cancer, subjects at risk for ovarian cancer recurrence after cancer treatment, subjects in remission after ovarian cancer treatment, and / or subjects previously or periodically screened for the presence of at least one ovarian cancer biomarker, such as, but not limited to, CA-125 plasma protein. In some embodiments, such asymptomatic subjects may be subjects whose plasma CA-125 levels have been determined to be normal (e.g., plasma CA-125 levels less than 35 U / mL). In some embodiments, such asymptomatic subjects may be subjects whose plasma CA-125 levels have been determined to be equal to or higher than normal plasma CA-125 levels. Alternatively, in some embodiments, an asymptomatic subject may be a subject who has not previously been screened for ovarian cancer, a subject who has not been diagnosed with ovarian cancer, and / or a subject who has not previously received ovarian cancer therapy.

[0031] 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, use of talc in the perineal area, hormone replacement therapy (HRT), exposure to infectious agents such as viruses, and / or occupational hazards).

[0032] In some embodiments, the techniques provided herein may be useful for selecting a surgery or therapy for a subject suffering from or susceptible to ovarian cancer, hi some embodiments, ovarian cancer surgery, therapy, and / or adjuvant therapy can be selected in light of findings based on the techniques provided herein.

[0033] 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., an ovarian cancer subject).

[0034] In some embodiments, the present disclosure provides techniques for managing patient care, for example, for one or more individual subjects and / or an entire population of subjects. To give some examples, in some embodiments, the present disclosure provides techniques that can be used in screening (e.g., temporally or incidentally motivated screening and / or non-temporally or incidentally motivated screening, for example, periodic screening, once a year, twice a year, once every two years, or some other frequency). For example, in some embodiments, the provided techniques for use in temporally motivated screening can be useful for screening one or more individual subjects or an entire population of subjects (e.g., asymptomatic subjects) who are older than a certain age (e.g., over 40, 45, 50, 55, 60, 65, 70 years old, or older). In some embodiments, the provided techniques for use in incidentally motivated screening can be useful for screening individual subjects who may have experienced an event or occurrence that motivates screening for ovarian 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 ovarian cancer), the identification of one or more risk factors associated with ovarian 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 imaging diagnostic testing (e.g., ultrasound, computed tomography (CT) and / or magnetic resonance imaging (MRI) scan), the occurrence of one or more signs or symptoms characteristic of ovarian cancer (e.g., abnormal bleeding during the menstrual cycle potentially indicative of ovarian cancer, etc.).

[0035] In some embodiments, the provided technology for managing patient care can inform treatment and / or payment (e.g., reimbursement for treatment) decisions and / or actions. For example, in some embodiments, the provided technology can provide a determination of whether an individual subject has one or more indicators of the development or recurrence of ovarian 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., ovarian cancer response biomarkers). In some embodiments, the provided technology can provide 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 ovarian 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.

[0036] 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.

[0037] In some embodiments, the provided techniques can aid in the diagnosis of ovarian cancer in symptomatic individuals with an adnexal mass confirmed by imaging. In some such embodiments, a positive test result is interpreted in conjunction with other clinical findings to diagnose cancer. In some embodiments, such clinical findings for diagnosing cancer can include, for example, pelvic or abdominal pain, inability to eat or feeling "full," and / or increased abdominal size or bloating, as well as other clinical findings described, for example, in Goff et al., Development of an ovarian cancer symptom index. Cancer. 2007;109:221-227, the entire contents of which are incorporated herein by reference for purposes set forth herein.

[0038] Some aspects provided herein relate to systems and kits for use in the provided technology. In some embodiments, the system or kit may include a detection agent (for example, as described herein) for the tumor biomarker signature of ovarian cancer. In some embodiments, such a system or kit may include a capture agent (for example, as described herein) for the extracellular vesicle-associated surface biomarker present in nanoparticles associated with ovarian cancer; and (b) at least one or more detection agents targeting one or more target biomarkers of the target biomarker signature of ovarian cancer, which may be or include additional surface biomarkers (for example, as described herein). In some embodiments, such systems or kits may further include one or more detection agents directed to intravesicular biomarkers (e.g., those used and / or described herein) and / or intravesicular RNA (e.g., mRNA, snRNA, miRNA, siRNA, orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA) biomarkers (e.g., those used and / or described herein) that have been determined to be useful for the detection of ovarian cancer.

[0039] 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.

[0040] 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.

[0041] 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 ovarian cancer. For example, in some embodiments, the provided systems and kits may include at least one set of detection probes for the detection of ovarian 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 ovarian cancer, such as high-grade serous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, low-grade serous ovarian cancer, or mucinous ovarian cancer. In some embodiments, two or more sets may be directed to the detection of different stages of ovarian cancer, hi some embodiments, two or more sets may be directed to the detection of the same stage of ovarian cancer.

[0042] 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.

[0043] In some embodiments, in which the target biomarker comprises an intravesicular RNA (e.g., mRNA, snRNA, miRNA, siRNA, orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA) biomarker, such systems and / or kits may include detection agents for performing a nucleic acid detection assay. 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 an intravesicular RNA (e.g., mRNA, snRNA, miRNA, siRNA, orphan non-coding RNA, long non-coding RNA, or piwi-interacting RNA) target.

[0044] In some embodiments, the provided systems and / or kits may include at least one chemical reagent therein, such as a chemical reagent for processing a sample and / or nanoparticles. In some embodiments, the provided systems and / or kits may include at least one chemical reagent for processing nanoparticles in a sample, including, 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 may be designed to increase the specificity of qPCR in some embodiments. 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).

[0045] In some embodiments, the provided systems and / or kits can be used for screening (e.g., routine screening) and / or other assessment of individuals (e.g., asymptomatic or symptomatic subjects) for detection (e.g., early detection) of ovarian cancer. In some embodiments, the provided systems and / or kits can be used for screening and / or other assessment of individuals predisposed to ovarian 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 ovarian 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 ovarian 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 ovarian cancer. In some embodiments, the provided systems and / or kits can be used to select a therapy for a subject suffering from ovarian cancer. In some embodiments, the provided systems and / or kits can be used to make therapy decisions and / or select a therapy for a subject with one or more symptoms (e.g., non-specific symptoms) associated with ovarian cancer.

[0046] Complexes formed by carrying out the methods described herein and / or using the systems and / or kits described herein are also within the scope of the present disclosure.For example, in some embodiments, the complex comprises extracellular vesicles expressing a target biomarker signature, which comprises at least one extracellular vesicle-associated surface biomarker and at least one target biomarker, including one or more surface biomarkers (e.g., as described herein), and the extracellular vesicles are immobilized on a solid substrate comprising a binding moiety targeting the extracellular vesicle-associated surface biomarker.In some embodiments, such complexes further comprise at least two detection probes targeting at least one target biomarker of the target biomarker signature present in the extracellular vesicles, each detection probe binding to a respective target biomarker and comprising (i) a binding moiety targeting 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 with each other.

[0047] In some embodiments, the extracellular vesicle-associated surface biomarkers present on the complexed extracellular vesicles may include one or more of the following: polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2, and combinations thereof; and / or (ii) one or more of the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0048] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes an intact or truncated polypeptide encoded by the human gene SLC34A2; and (ii) one or more target surface biomarkers that include intact or truncated polypeptides encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

[0049] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes an intact or truncated polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that include at least one intact or truncated polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker that includes a sialyl-Tn (sTn) antigen.

[0050] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes an intact or truncated polypeptide encoded by the human gene BST2; and (ii) one or more target surface biomarkers that include an intact or truncated polypeptide encoded by the human gene FOLR1.

[0051] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles includes (i) at least one extracellular vesicle-associated surface biomarker that is or includes a carbohydrate-dependent marker, including sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more target surface biomarkers that include at least one intact or truncated polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

[0052] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an intact or truncated polypeptide encoded by the human gene MUC1; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0053] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an intact or truncated polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0054] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including a sialyl-Tn (sTn) antigen; and (ii) one or more target surface biomarkers that comprise at least one intact or truncated polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof. In some such embodiments, the surface biomarker encoded by the human gene MUC16 may be an intact MUC16 polypeptide. In some such embodiments, the surface biomarker encoded by the human gene MUC16 may be a truncated MUC16 polypeptide.

[0055] In some embodiments, the target biomarker signature expressed by the ovarian cancer-associated nanoparticles includes one or more target surface biomarkers that are (i) at least one extracellular vesicle-associated surface biomarker that is or includes a carbohydrate-dependent marker, including the Thomsen-Friedenreich (T,TF) antigen; and (ii) at least one intact or truncated polypeptide encoded by the human gene BST2.

[0056] The disclosure herein provides: a) providing or obtaining a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample) from a subject; b) detecting in the biological sample nanoparticles expressing a first target biomarker signature ("first target biomarker signature-expressing nanoparticles"), wherein the first target biomarker signature is i) at least one extracellular vesicle-associated surface biomarker, and ii) at least one target biomarker selected from surface biomarkers Including, iii) the surface biomarkers are selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof; c) comparing the sample information indicating the level of the first target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a first reference threshold level; d) classifying the subject as having or susceptible to ovarian cancer if the biological sample exhibits an elevated level of the first target biomarker signature-expressing nanoparticles compared to a classification cutoff referenced to a first reference threshold level. The method includes:

[0057] In some embodiments of the disclosed methods, when the surface biomarker is a polypeptide encoded by the human gene MUC16, the polypeptide is an intact MUC16 polypeptide.

[0058] In some embodiments of the disclosed methods, when the surface biomarker is a polypeptide encoded by the human gene MUC16, the polypeptide is a truncated MUC16 polypeptide.

[0059] In some embodiments of the disclosed methods, the first target biomarker signature further comprises an intravesicle biomarker and / or an intravesicle RNA biomarker.

[0060] In some embodiments of the disclosed methods, when at least one target biomarker is selected from one or more of the surface biomarkers, the selected surface biomarker and the at least one extracellular vesicle-associated surface biomarker are different.

[0061] In some embodiments of the disclosed methods, steps (b) and (c) are repeated for at least a second target biomarker signature, and the classification cutoff refers to the first reference threshold level and at least a second reference threshold level corresponding to the at least second target biomarker signature.

[0062] In some embodiments of the disclosed methods, the extracellular vesicle-associated surface biomarker is or comprises: (i) a polypeptide encoded by the following human genes: BST2, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0063] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises at least one extracellular vesicle-associated surface biomarker and at least two biomarkers selected from the group consisting of a surface biomarker, an intravesicle biomarker, and an intravesicle RNA biomarker.

[0064] In some embodiments of the disclosed methods, the at least two biomarkers are selected from the following combinations: a) at least two distinct surface biomarkers; b) at least two distinct intravesicular biomarkers; c) at least two distinct intravesicular RNA biomarkers; d) surface and intravesicular biomarkers; e) surface biomarkers and intravesicular RNA biomarkers; and f) Intravesicular biomarkers and intravesicular RNA biomarkers Contains one of the following:

[0065] In some embodiments of the disclosed methods, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene SLC34A2; and (ii) one or more target surface biomarkers that include polypeptides encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

[0066] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker that comprises a sialyl-Tn (sTn) antigen.

[0067] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene BST2; and (ii) one or more target surface biomarkers that comprise a polypeptide encoded by the human gene FOLR1.

[0068] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

[0069] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC1; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0070] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0071] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including a sialyl-Tn (sTn) antigen; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof.

[0072] In some embodiments of the disclosed methods, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is an intact MUC16 polypeptide.

[0073] In some embodiments of the disclosed methods, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is a truncated MUC16 polypeptide.

[0074] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including the Thomsen-Friedenreich (T,TF) antigen; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the human gene BST2.

[0075] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker BST2.

[0076] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BST2.

[0077] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least two target biomarkers that are BST2 and FOLR1.

[0078] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least one target biomarker sTn antigen.

[0079] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are BST2 and MUC1.

[0080] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are FOLR1 and MUC1.

[0081] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and MSLN.

[0082] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are FOLR1 and MUC16.

[0083] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are MUC1 and MUC16.

[0084] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker SLC34A2.

[0085] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a T antigen, and (ii) at least one target biomarker BST2.

[0086] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and cleaved MUC16.

[0087] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least two target biomarkers that are BST2 and MUC16.

[0088] In some embodiments of the disclosed methods, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BCAM.

[0089] In some embodiments of the disclosed methods, the first or second reference threshold level is determined by the level of target biomarker signature-expressing nanoparticles observed in comparable samples from a population of non-cancer subjects.

[0090] In some embodiments of the disclosed methods, the population of non-cancer subjects includes one or more of the following subject populations: healthy subjects, subjects diagnosed with benign tumors, and subjects with non-ovarian-related diseases, disorders, and / or conditions.

[0091] In some embodiments of the disclosed methods, the biological sample is subjected to purification (e.g., size exclusion chromatography) to isolate nanoparticles having a desired size range, including nanoparticles (e.g., directly from the biological sample).

[0092] In some embodiments of the disclosed methods, the detecting step comprises a capture assay.

[0093] In some embodiments of the disclosed methods, the capture assay includes contacting the biological sample with a capture agent comprising a target capture moiety that binds to at least one extracellular vesicle-associated surface biomarker.

[0094] In some embodiments of the disclosed methods, the capture agent is or comprises a solid substrate comprising a target capture moiety conjugated thereto, hi some embodiments, the solid substrate comprises a magnetic bead.

[0095] In some embodiments of the disclosed methods, the target capture moiety is or includes an antibody agent.

[0096] In some embodiments of the disclosed methods, the detecting step comprises a detection assay.

[0097] In some embodiments of the disclosed methods, the detecting step comprises a capture assay and a detection assay, wherein the capture assay is performed before the detection assay.

[0098] In some embodiments of the disclosed methods, when the first and / or second target biomarker signature comprises at least one intravesicle RNA biomarker, the detection assay comprises reverse transcription-qPCR.

[0099] In some embodiments of the disclosed methods, when the first and / or second target biomarker signatures include at least one intravesicular biomarker, the target biomarker signature-expressing nanoparticles are treated by fixation and / or permeabilization prior to the detection assay.

[0100] In some embodiments of the disclosed methods, when the first and / or second target biomarker signatures include at least one surface biomarker and / or intravesicular biomarker, the detection assay comprises an immunoassay (e.g., including immuno-PCR and / or proximity ligation assay).

[0101] In some embodiments of the disclosed methods, the detection assay comprises a proximity ligation assay. In some embodiments, the proximity ligation assay comprises: a) contacting target biomarker signature-expressing nanoparticles expressing at least one extracellular vesicle-associated surface biomarker ("extracellular vesicle-associated surface biomarker-expressing nanoparticles") with a set of detection probes each directed to a target biomarker of the target biomarker signature, the set comprising at least two detection probes, such that a combination comprising the set of nanoparticles and detection probes is created; The detection probe is i) a target binding moiety directed to a target biomarker of the target biomarker signature; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain in which the single-stranded overhang portions of the detection probes are capable of hybridizing with each other when the detection probes are bound to the same extracellular vesicle; Each step includes b) maintaining the combination under conditions that allow binding of the set of detection probes to their respective targets on the nanoparticles, such that at least two detection probes can bind to the same extracellular vesicle expressing the target biomarker signature to form a double-stranded complex; c) contacting the double-stranded complex with a nucleic acid ligase to produce a ligated template; and d) detecting the ligated template, wherein the presence of the ligated template indicates the presence of target biomarker signature-expressing nanoparticles in the biological sample; and e) Optionally, repeating steps a through d at least one additional time using an orthogonal target biomarker signature. Includes.

[0102] In some embodiments, the target binding moieties of at least two detection probes are directed to the same target biomarker. In some embodiments, the oligonucleotide domains of at least two detection probes are different.

[0103] In some embodiments of the disclosed methods, the target capture moiety of the capture assay is or includes at least one antibody agent directed against at least one extracellular vesicle-associated surface biomarker.

[0104] In some embodiments, the disclosed methods are performed to screen for early stage ovarian cancer, late stage ovarian cancer, or recurrent ovarian cancer in a subject.

[0105] In some embodiments of the disclosed methods, the subject has been determined to have a normal plasma CA-125 level.

[0106] In some embodiments of the disclosed methods, the subject has one or more of the following characteristics: a) asymptomatic females (e.g., women) who are susceptible to ovarian cancer (e.g., at average population risk (i.e., no genetic risk) or who have a genetic risk for ovarian cancer); b) postmenopausal women; c) females (e.g., women) with a family history of breast and / or ovarian cancer (e.g., females (e.g., women) with one or more first-degree relatives with a history of breast and / or ovarian cancer); d) females (e.g., women) determined to have one or more germline mutations in ATM, BRCA1, BRCA2, CDKN2A, MSH2, MLH1, MSH2, EPCAM, PALB2, STK11, TP53, BARD, CHEK2, MRE11A, RAD50, RAD51C, RAD51D, and combinations thereof; e) a female (e.g., woman) with breast cancer who has been determined to have a germline mutation in BRCA1, BRCA2, and / or PALB2; f) older age, e.g., women aged 65 or over; g) a female (e.g., a woman) having one or more non-specific symptoms of ovarian cancer, optionally wherein at least one of the non-specific symptoms resembles one or more symptoms of irritable bowel syndrome; and h) females for whom periodic screening with CA-125 / transvaginal ultrasound (TVUS) is recommended (e.g., women); i) females diagnosed with an adnexal mass confirmed by imaging (e.g., females); j) genetically at-risk females (e.g., women) prior to undergoing risk-reducing bilateral salpingo-oophorectomy; k) females (e.g., women) with benign gynecological tumors; l) females (e.g., women) previously treated for ovarian cancer; and m) Females (e.g., women) with a lifestyle-related risk for ovarian cancer The present invention has at least one or more of the following:

[0107] In some embodiments of the disclosed methods, the method comprises the following diagnostic assays: a) Subject's annual health check (e.g., including HPV and / or Pap smear screening for cervical cancer and mammogram screening for breast cancer); b) plasma CA-125 and / or TVUS screening tests; c) genetic assays to screen circulating tumor DNA and / or plasma for genetic mutations in protein biomarkers associated with cancer; d) assays involving immunofluorescence staining to identify cell phenotype and marker expression, followed by amplification and analysis by next-generation sequencing; and e) BRCA1 and / or BRCA2 germline and somatic mutation assays or assays involving cell-free tumor DNA, liquid biopsy, serum protein and cell-free DNA, OVA1 and OVERA testing, and / or circulating tumor cells used in combination with one or more of:

[0108] In some embodiments of the disclosed methods, the ovarian cancer is high-grade serous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, low-grade serous ovarian cancer, or mucinous ovarian cancer.

[0109] In some embodiments of the disclosed methods, the ovarian cancer is high-grade serous ovarian cancer. In some embodiments, the high-grade serous ovarian cancer is in an early stage.

[0110] In some embodiments, the disclosed methods are performed to monitor ovarian cancer patients for response to treatment with an anti-ovarian cancer therapy (e.g., olaparib, cisplatin, rucaparib, niraparib, talazoparib) and / or for cancer recurrence / metastasis.

[0111] In some embodiments, the disclosed methods include detecting co-localization of at least two biomarkers, the combined expression levels of which have been determined to be associated with cancer, on the surface of intact nanoparticles derived from a human blood sample; comparing the detected co-localization level with a determined level; and detecting cancer if the detected co-localization level is at or above the determined level.

[0112] In some embodiments, the disclosed methods include contacting a sample containing exosomes with a set of detection probes that specifically bind to surface biomarkers on exosomes to detect cancer-associated exosomes in the sample with a specificity within the range of 95% to 100% and a sensitivity within the range of 30% to 100%.

[0113] In some embodiments, the disclosed methods include capturing exosomes from a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample) using a capture agent that selectively interacts with a cancer-specific surface biomarker on the exosome; and contacting the captured exosomes with at least one set of at least two detection probes, each of which selectively interacts with a surface biomarker on the exosome; and detecting a product formed when at least two detection probes of the set are in sufficient proximity, wherein such detection indicates co-localization of the surface biomarkers.

[0114] In some embodiments, the disclosed methods comprise contacting a sample comprising exosomes with a set of probes that specifically bind to surface biomarkers on exosomes to detect cancer-associated exosomes in the sample, wherein (i) each probe in the set comprises a target binding moiety directed to a surface biomarker on the exosome; and (ii) the set comprises at least one capture probe and at least two detection probes, each detection probe further comprising a detection moiety.

[0115] In some embodiments, the disclosed methods comprise performing a proximity assay to detect a surface biomarker signature on exosomes from the human subject, wherein the performing is performed a period of time after performing a previous assay to detect a surface biomarker signature on exosomes from the human subject; and comparing the results of the performed assay with the results of the previous assay.

[0116] In some embodiments, the disclosed methods comprise contacting the exosome with at least two detection probes, each detection probe comprising: (i) a binding moiety; and (ii) an oligonucleotide entity, wherein the binding moieties are the same and the oligonucleotide entities are complementary to each other.

[0117] In some embodiments, the disclosed methods include an improvement comprising detecting marker proximity on the surface of an exosome, wherein the step includes contacting the exosome with at least one pair of binding agents each comprising a binding moiety and a proximity moiety, wherein the binding moieties are the same and the proximity moieties are complementary to each other; and detecting an interaction between the proximity moieties.

[0118] One aspect of the disclosure herein is a kit for detecting ovarian cancer, comprising: a) a capture agent comprising a target capture moiety directed to an extracellular vesicle-associated surface biomarker; and b) at least one set of detection probes, the set comprising at least two detection probes each directed to a target biomarker of a target biomarker signature for ovarian cancer, the detection probes being: i) a target binding moiety for a target biomarker of a target biomarker signature for ovarian cancer; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain characterized in that the single-stranded overhang portions of at least two detection probes are capable of hybridizing to each other when the at least two detection probes are bound to the same extracellular vesicle; Each includes; Targeted biomarker signatures for ovarian cancer at least one extracellular vesicle-associated surface biomarker, and at least one target biomarker selected from a surface biomarker; the surface biomarkers are selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof; At least one set of detection probes The kit includes:

[0119] In some embodiments of the disclosed kits, when the surface biomarker is a polypeptide encoded by the human gene MUC16, the polypeptide is an intact MUC16 polypeptide.

[0120] In some embodiments of the disclosed kits, when the surface biomarker is a polypeptide encoded by the human gene MUC16, the polypeptide is a truncated MUC16 polypeptide.

[0121] In some embodiments of the disclosed kits, the first target biomarker signature further comprises an intravesicle biomarker and / or an intravesicle RNA biomarker.

[0122] In some embodiments of the disclosed kits, when at least one target biomarker is selected from one or more of the surface biomarkers, the selected surface biomarker and the at least one extracellular vesicle-associated surface biomarker are different.

[0123] In some embodiments of the disclosed kits, the extracellular vesicle-associated surface biomarkers are or include (i) polypeptides encoded by the following human genes: BST2, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0124] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene SLC34A2; and (ii) one or more target surface biomarkers that include polypeptides encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

[0125] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that include at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker that includes a sialyl-Tn (sTn) antigen.

[0126] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene BST2; and (ii) one or more target surface biomarkers that include a polypeptide encoded by the human gene FOLR1.

[0127] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a carbohydrate-dependent marker including sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more target surface biomarkers that include at least one polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

[0128] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC1; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0129] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0130] In some embodiments of the disclosed kit, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker comprising a sialyl-Tn (sTn) antigen; and (ii) one or more target surface biomarkers comprising at least one polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof. In some embodiments, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is an intact MUC16 polypeptide. In some embodiments, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is a truncated MUC16 polypeptide.

[0131] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a carbohydrate-dependent marker including the Thomsen-Friedenreich (T,TF) antigen; and (ii) one or more target surface biomarkers that include at least one polypeptide encoded by the human gene BST2.

[0132] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker BST2.

[0133] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BST2.

[0134] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least two target biomarkers that are BST2 and FOLR1.

[0135] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least one target biomarker sTn antigen.

[0136] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are BST2 and MUC1.

[0137] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are FOLR1 and MUC1.

[0138] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and MSLN.

[0139] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are FOLR1 and MUC16.

[0140] In some embodiments of the disclosed kits, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are MUC1 and MUC16.

[0141] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker SLC34A2.

[0142] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a T antigen, and (ii) at least one target biomarker BST2.

[0143] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and cleaved MUC16.

[0144] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least two target biomarkers that are BST2 and MUC16.

[0145] In some embodiments of the disclosed kits, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BCAM.

[0146] In some embodiments of the disclosed kits, the target binding moieties of at least two detection probes each target the same target biomarker of the target biomarker signature.

[0147] In some embodiments of the disclosed kits, the oligonucleotide domains of at least two of the detection probes are different.

[0148] In some embodiments of the disclosed kits, the target binding moieties of at least two detection probes are each directed to a distinct target biomarker in the target biomarker signature.

[0149] In some embodiments, the disclosed kits further comprise at least one additional reagent (eg, a ligase, a fixative, and / or a permeabilization agent).

[0150] In some embodiments, the disclosed kits include at least two sets (e.g., including at least three sets) of detection probes, each set including at least two detection probes each directed to a target biomarker of a distinct target biomarker signature for ovarian cancer.

[0151] In some embodiments, the disclosed kit comprises: a) a first capture agent comprising a target capture moiety; b) a second capture agent comprising a target capture moiety; c) at least two sets of detection probes, the detection probes comprising: i) a target binding moiety for a target surface biomarker; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain characterized in that the single-stranded overhang portions of at least two detection probes are capable of hybridizing to each other when the at least two detection probes are bound to the same extracellular vesicle; At least two sets of detection probes, each comprising Includes.

[0152] In some embodiments, the disclosed kit comprises: a) a first capture agent comprising a target capture moiety; b) a second capture agent comprising a target capture moiety; c) a third capture agent comprising a target capture moiety; d) at least three sets of detection probes, the detection probes comprising: i) a target binding moiety for a target surface biomarker; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain characterized in that the single-stranded overhang portions of at least two detection probes are capable of hybridizing to each other when the at least two detection probes are bound to the same extracellular vesicle; At least three sets of detection probes, each containing Includes.

[0153] One aspect disclosed herein is a method for manufacturing a semiconductor device comprising: a) extracellular vesicles expressing a target biomarker signature for ovarian cancer, wherein the target biomarker signature is: at least one extracellular vesicle-associated surface biomarker, and at least one target biomarker selected from a surface biomarker; the surface biomarkers are selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof; The extracellular vesicles are immobilized on a solid substrate that includes a target capture moiety directed against an extracellular vesicle-associated surface biomarker. extracellular vesicles; b) a first detection probe and a second detection probe each bound to an extracellular vesicle, wherein each detection probe is: i) a target binding moiety directed to one of the target biomarkers of the tumor target biomarker signature; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain in which the single-stranded overhang portions of the first and second detection probes hybridize to each other; a first detection probe and a second detection probe, It is a complex containing

[0154] In some embodiments of the disclosed complexes, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is an intact MUC16 polypeptide.

[0155] In some embodiments of the disclosed complexes, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is a truncated MUC16 polypeptide.

[0156] In some embodiments of the disclosed complexes, the first target biomarker signature further comprises an intravesicular biomarker and / or an intravesicular RNA biomarker.

[0157] In some embodiments of the disclosed complexes, when at least one target biomarker is selected from one or more surface biomarkers, the selected surface biomarker and the at least one extracellular vesicle-associated surface biomarker are different.

[0158] In some embodiments of the disclosed complexes, the extracellular vesicle-associated surface biomarker is or includes (i) a polypeptide encoded by the following human genes: BST2, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0159] In some embodiments of the disclosed complexes, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene SLC34A2; and (ii) one or more target surface biomarkers that include polypeptides encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

[0160] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker that comprises a sialyl-Tn (sTn) antigen.

[0161] In some embodiments of the disclosed complexes, the first and / or second target biomarker signatures include: (i) at least one extracellular vesicle-associated surface biomarker that is or includes a polypeptide encoded by the human gene BST2; and (ii) one or more target surface biomarkers that include a polypeptide encoded by the human gene FOLR1.

[0162] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker including sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

[0163] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC1; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0164] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a polypeptide encoded by the human gene MUC16; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

[0165] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker comprising a sialyl-Tn (sTn) antigen; and (ii) one or more target surface biomarkers comprising at least one polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof. In some embodiments, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is an intact MUC16 polypeptide. In some embodiments, the surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is a truncated MUC16 polypeptide.

[0166] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises: (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a carbohydrate-dependent marker, including a Thomsen-Friedenreich (T,TF) antigen; and (ii) one or more target surface biomarkers that comprise at least one polypeptide encoded by the human gene BST2.

[0167] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker BST2.

[0168] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BST2.

[0169] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least two target biomarkers that are BST2 and FOLR1.

[0170] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least one target biomarker sTn antigen.

[0171] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are BST2 and MUC1.

[0172] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are FOLR1 and MUC1.

[0173] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and MSLN.

[0174] In some embodiments of the disclosed complexes, the first and / or second target biomarker signatures include (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are FOLR1 and MUC16.

[0175] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC16 polypeptide, and (ii) at least two target biomarkers that are MUC1 and MUC16.

[0176] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least one target biomarker SLC34A2.

[0177] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a T antigen, and (ii) at least one target biomarker BST2.

[0178] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises an sTn antigen, and (ii) at least two target biomarkers that are MUC16 and cleaved MUC16.

[0179] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises CA19-9 antigen, and (ii) at least two target biomarkers that are BST2 and MUC16.

[0180] In some embodiments of the disclosed complexes, the first and / or second target biomarker signature comprises (i) at least one extracellular vesicle-associated surface biomarker that is or comprises a MUC1 polypeptide, and (ii) at least one target biomarker BCAM.

[0181] In some embodiments of the disclosed complexes, the target binding moieties of at least two detection probes each target the same target biomarker of the target biomarker signature, hi some embodiments, the oligonucleotide domains of at least two detection probes are different.

[0182] In some embodiments of the disclosed complexes, the target binding moieties of at least two detection probes are each directed to a distinct target biomarker of a target biomarker signature.

[0183] In some embodiments of the disclosed complexes, the solid substrate comprises magnetic beads.

[0184] In some embodiments of the disclosed conjugates, the target capture moiety is or includes an antibody agent.

[0185] In some embodiments, the disclosed complexes comprise: (a) an exosome having at least one target biomarker on its surface; and (b) a first detection probe and a second detection probe, respectively, bound to the exosome, wherein the first detection probe and the second detection probe each comprise: (i) a target-binding moiety directed to a target biomarker expressed by the exosome; 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 first and second detection probes hybridize to one another.

[0186] In some embodiments, the disclosed complexes comprise nanoparticles derived from a human blood sample bound to a set of at least two probes, each of which comprises a biomarker-binding moiety and an oligonucleotide domain, and the two or more bound probes are in close proximity to each other such that their oligonucleotide domains hybridize to each other to form ligatable hybrids.

[0187] In some embodiments, the disclosed complexes comprise: (a) an exosome comprising a cancer-associated target biomarker signature; and (b) at least a first detection probe and a second detection probe, respectively, bound to the exosome, wherein each of the detection probes comprises: (i) a target-binding moiety directed to the target biomarker signature; 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, and the single-stranded overhang portion of the detection probe is at least partially complementary.

[0188] One aspect of the disclosure herein is a set of probes for use in a method, kit, or complex, wherein each of the set of probes comprises: (a) a biomarker-binding moiety that specifically binds to a surface biomarker on a cancer cell-derived nanoparticle; and (b) an oligonucleotide domain, the oligonucleotide domains of the probes in the set being arranged and constructed such that when the probes bind to their target biomarkers, the oligonucleotide domains hybridize to each other to form a hybrid that can be ligated only when the target biomarkers are in close proximity to each other.

[0189] One aspect of the disclosure herein is a) providing or obtaining a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample) from a female subject; b) detecting in the biological sample nanoparticles expressing a first target biomarker signature ("first target biomarker signature-expressing nanoparticles"), wherein the first target biomarker signature is i) at least one extracellular vesicle-associated surface biomarker; and ii) at least one target surface biomarker Including, at least one extracellular vesicle-associated surface biomarker and at least one target surface biomarker, (1) Polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; and (2) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and (3) The combination each independently selected from: c) comparing the sample information indicative of the level of the first target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a first reference threshold level; d) classifying the subject as having or susceptible to ovarian cancer if the biological sample exhibits an elevated level of the first target biomarker signature-expressing nanoparticles compared to a classification cutoff referenced to a first reference threshold level. The method includes:

[0190] In some embodiments of the disclosed methods, at least one extracellular vesicle-associated surface biomarker and at least one target surface biomarker are different.

[0191] In some embodiments of the disclosed methods, steps (b) and (c) are repeated for at least a second target biomarker signature, and the classification cutoff refers to the first reference threshold level and at least a second reference threshold level corresponding to the at least second target biomarker signature.

[0192] In some embodiments of the disclosed methods, steps (b) and (c) are repeated for multiple additional target biomarker signatures, and the classification cutoffs refer to respective reference threshold levels corresponding to each target biomarker signature.

[0193] In some embodiments of the disclosed methods, the first target biomarker signature or at least one of the target biomarker signatures comprises at least one extracellular vesicle-associated surface biomarker and at least one target surface biomarker, the combination of which is as follows: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; and c) Polypeptides encoded by the human gene MUC16 and sialyl-Tn (sTn) antigens is selected from.

[0194] In some embodiments of the disclosed methods, the first target biomarker signature or at least one of the target biomarker signatures comprises at least one extracellular vesicle-associated surface biomarker and at least two target surface biomarkers, the combination of which is as follows: a) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; b) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; c) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and d) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN is selected from.

[0195] In some embodiments of the disclosed methods, the first target biomarker signature and the plurality of additional target biomarker signatures collectively represent a combination of at least one extracellular vesicle-associated surface biomarker and at least one target surface biomarker: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; c) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; d) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; e) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; f) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and g) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN Includes.

[0196] In some embodiments of the disclosed methods, the reference threshold level is determined by the level of corresponding target biomarker signature-expressing nanoparticles observed in comparable samples from a population of non-cancer subjects.

[0197] In some embodiments of the disclosed methods, the population of non-cancer subjects includes one or more of the following subject populations: healthy subjects, subjects diagnosed with benign tumors, and subjects with non-ovarian-related diseases, disorders, and / or conditions.

[0198] In some embodiments of the disclosed methods, the biological sample is subjected to purification (e.g., size exclusion chromatography) to isolate nanoparticles having a desired size range, including nanoparticles (e.g., directly from the biological sample).

[0199] In some embodiments of the disclosed methods, the detecting step comprises a capture assay. In some embodiments, the capture assay comprises contacting the biological sample with a capture probe comprising a target capture moiety that binds to at least one extracellular vesicle-associated surface biomarker. In some embodiments, the target capture moiety of the capture probe is or comprises at least one antibody agent directed against at least one extracellular vesicle-associated surface biomarker. In some embodiments, the at least one extracellular vesicle-associated surface biomarker is or comprises sialyl Lewis A antigen (also known as CA19-9), a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene MUC16, or a sialyl Tn (sTn) antigen. In some embodiments, the capture probe is or comprises a solid substrate comprising a target capture moiety conjugated thereto. In some embodiments, the solid substrate comprises magnetic beads.

[0200] In some embodiments of the disclosed methods, the detecting step comprises a detection assay. In some embodiments, the detecting step comprises a capture assay and a detection assay, wherein the capture assay is performed before the detection assay. In some embodiments, the detection assay comprises an immunoassay (e.g., including immuno-PCR and / or a proximity ligation assay). In some embodiments, the detection assay comprises a proximity ligation assay. In some embodiments, the proximity ligation assay comprises: a) contacting nanoparticles in a biological sample with a set of detection probes each directed to at least one target surface biomarker of a target biomarker signature, the set comprising at least two detection probes, such that a complex comprising the nanoparticles and the set of detection probes is created; The detection probes are i) a target binding moiety directed to one of at least one target surface biomarker of the target biomarker signature; and ii) an oligonucleotide domain coupled to a 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; an oligonucleotide domain in which the single-stranded overhang portions of the detection probes are capable of hybridizing with each other when the detection probes are bound to the same extracellular vesicle; each including the steps; b) maintaining the combination under conditions that allow binding of the set of detection probes to their respective targets on the nanoparticles, such that at least two detection probes can bind to the same extracellular vesicle expressing the target biomarker signature to form a double-stranded complex; c) contacting the double-stranded complex with a nucleic acid ligase to produce a ligated template; d) detecting the ligated template, wherein the presence of the ligated template indicates the presence of target biomarker signature-expressing nanoparticles in the biological sample; and e) optionally repeating steps (a) through (d) at least one additional time using an orthogonal target biomarker signature; Includes.

[0201] In some embodiments, the target binding portions of at least two detection probes are each directed to the same target surface biomarker. In some embodiments, the oligonucleotide domains of at least two detection probes are different. In some embodiments, the same target surface biomarker is or comprises a polypeptide encoded by the human gene BST2. In some embodiments, the target capture portion of a capture probe is or comprises at least one antibody agent directed to the sialyl Lewis A antigen (also known as CA19-9) or to a polypeptide encoded by the human gene MUC1. In some embodiments, the same target surface biomarker is or comprises the sialyl Tn (sTn) antigen. In some embodiments, the target capture portion of a capture probe is or comprises at least one antibody agent directed to a polypeptide encoded by the human gene MUC16. In some embodiments of the disclosed methods, the target binding portions of at least two detection probes are each directed to a distinct target surface biomarker. In some embodiments, the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene BST2, and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene FOLR1. In some embodiments, the target capture portion of the capture probe is or includes at least one antibody agent directed to a polypeptide encoded by the human gene MUC1. In some embodiments, the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene BST2, and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene MUC1. In some embodiments, the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene FOLR1, and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene MUC1.In some embodiments, the target binding moiety of the first detection probe is directed to a polypeptide encoded by the human gene MUC16, and the target binding moiety of the second detection probe is directed to a polypeptide encoded by the human gene MSLN. In some embodiments, the target capture moiety of the capture assay is or includes at least one antibody agent directed to the sialyl-Tn (sTn) antigen.

[0202] In some embodiments of the disclosed methods, the methods are performed to screen for early stage ovarian cancer, late stage ovarian cancer, or recurrent ovarian cancer in a subject.

[0203] In some embodiments of the disclosed methods, the methods are performed to screen for early stage ovarian cancer.

[0204] In some embodiments of the disclosed methods, the subject has one or more of the following characteristics: a) asymptomatic females (e.g., women) who are susceptible to ovarian cancer (e.g., at average population risk (i.e., no genetic risk) or who have a genetic risk for ovarian cancer); b) postmenopausal women; c) females (e.g., women) with a family history of breast and / or ovarian cancer (e.g., females (e.g., women) with one or more first-degree relatives with a history of breast and / or ovarian cancer); d) females (e.g., women) determined to have one or more germline mutations in ATM, BRCA1, BRCA2, CDKN2A, MSH2, MLH1, MSH2, EPCAM, PALB2, STK11, TP53, BARD, CHEK2, MRE11A, RAD50, RAD51C, RAD51D, and combinations thereof; e) a female (e.g., woman) with breast cancer who has been determined to have a germline mutation in BRCA1, BRCA2, and / or PALB2; f) older age, e.g., women aged 65 or over; g) a female (e.g., a woman) having one or more non-specific symptoms of ovarian cancer, optionally wherein at least one of the non-specific symptoms resembles one or more symptoms of irritable bowel syndrome; and h) females for whom periodic screening with plasma CA-125 / transvaginal ultrasound (TVUS) is recommended (e.g., women); i) females diagnosed with an adnexal mass confirmed by imaging (e.g., females); j) females at genetic risk for ovarian cancer (e.g., women) prior to undergoing risk-reducing bilateral salpingo-oophorectomy; k) females (e.g., women) with benign gynecological tumors; l) females (e.g., women) previously treated for ovarian cancer; and m) Females (e.g., women) with a lifestyle-related risk for ovarian cancer The present invention has at least one or more of the following:

[0205] In some embodiments of the disclosed methods, the subject has been determined to have a normal serum CA-125 level (eg, less than or equal to 25 U / mL).

[0206] In some embodiments of the disclosed methods, the female subject has been diagnosed with an adnexal mass confirmed by imaging, hi some embodiments, the female subject has been determined to have an elevated plasma or serum CA-125 level (e.g., greater than 25 U / mL).

[0207] In some embodiments of the disclosed methods, the method comprises the following diagnostic assays: a) Subject's annual health check (e.g., including HPV and / or Pap smear screening for cervical cancer and mammogram screening for breast cancer); b) plasma or serum CA-125 and / or TVUS screening tests; c) genetic assays to screen circulating tumor DNA and / or plasma for genetic mutations in protein biomarkers associated with cancer; d) assays involving immunofluorescence staining to identify cell phenotype and marker expression, followed by amplification and analysis by next-generation sequencing; and e) BRCA1 and / or BRCA2 germline and somatic mutation assays or assays involving cell-free tumor DNA, liquid biopsy, serum protein and cell-free DNA, OVA1 and OVERA testing, and / or circulating tumor cells used in combination with one or more of:

[0208] In some embodiments of the disclosed methods, the ovarian cancer is high-grade serous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, low-grade serous ovarian cancer, or mucinous ovarian cancer.

[0209] In some embodiments of the disclosed methods, the ovarian cancer is high-grade serous ovarian cancer. In some embodiments, the high-grade serous ovarian cancer is in an early stage.

[0210] One embodiment disclosed herein is a method for distinguishing between a benign adnexal mass and ovarian cancer, comprising: a) detecting co-localization of at least one combination of biomarkers on the surface of intact nanoparticles in a biological sample (e.g., in some embodiments, a body fluid-derived sample, such as, but not limited to, a blood-derived sample) from a female subject determined to have an adnexal mass, wherein the combination of biomarkers comprises at least one capture biomarker and at least one detection biomarker, and the at least one capture biomarker and the at least one detection biomarker are: i) polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; and ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and iii) combinations thereof each independently selected from: b) comparing the detected colocalization level with a reference level; and c) identifying the female subject's adnexal mass as potentially benign if the detected colocalization level is at or comparable to the reference level; or identifying the adnexal mass as cancerous if the detected colocalization level is above the reference level. The method includes:

[0211] In some embodiments, the method has a specificity within the range of 90% to 100% and a sensitivity within the range of 65% to 95% for distinguishing between benign adnexal masses and ovarian cancer. In some embodiments, the female subject has been determined to have an elevated serum CA-125 level (e.g., greater than 25 U / mL).

[0212] One embodiment disclosed herein is a method for detecting early stage ovarian cancer, comprising: a) detecting co-localization of at least one combination of biomarkers on the surface of intact nanoparticles in a biological sample (e.g., in some embodiments, a body fluid-derived sample, such as, but not limited to, a blood-derived sample) from a female subject, wherein the combination of biomarkers comprises at least one capture biomarker and at least one detection biomarker, and the at least one capture biomarker and the at least one detection biomarker are: i) Polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and iii) combinations thereof each independently selected from: b) comparing the detected colocalization level with a reference level; and c) identifying the female subject as negative for ovarian cancer if the detected co-localization level is at or comparable to the reference level; or, identifying the female subject as likely to have or be susceptible to ovarian cancer if the detected co-localization level is above the reference level. The method includes:

[0213] In some embodiments, the specificity of the method for detecting early stage ovarian cancer is within the range of 90% to 100% and the sensitivity is within the range of 80% to 95%. In some embodiments, the female subject has been determined to have a normal plasma or serum CA-125 level (e.g., less than or equal to 25 U / mL).

[0214] In some embodiments of the disclosed methods, the detecting step comprises detecting co-localization of at least one combination of biomarkers on the surface of the intact nanoparticle, wherein the at least one combination of biomarkers is one of the following: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; c) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; d) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; e) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; f) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and g) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN is selected from one of the following:

[0215] In some embodiments of the disclosed methods, the detecting step comprises detecting a combination of the following biomarkers on the surface of the intact extracellular vesicles: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; c) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; d) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; e) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; f) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and g) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN and detecting the co-localization of each of the following:

[0216] In some embodiments of the disclosed methods, the detecting step comprises: a) capturing intact nanoparticles from a biological sample using a capture probe that selectively interacts with at least one capture biomarker on the intact nanoparticles; b) contacting the captured nanoparticles with at least one set of at least two detection probes that each selectively interact with at least one detection biomarker on the intact nanoparticles; and c) detecting the product formed when at least two detection probes of the set are in sufficient proximity on each nanoparticle. Includes.

[0217] In some embodiments, the capture probe comprises a target capture moiety that binds to the capture biomarker. In some embodiments, the target capture moiety is or comprises an antibody agent directed against the capture biomarker.

[0218] In some embodiments of the disclosed methods, the capture biomarker is or comprises sialyl Lewis A antigen (also known as CA19-9), a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene MUC16, or a sialyl Tn (sTn) antigen.

[0219] In some embodiments of the disclosed methods, the capture probe is or comprises a solid substrate that comprises a target capture moiety conjugated thereto, and in the disclosed methods, the solid substrate comprises magnetic beads.

[0220] In some embodiments of the disclosed methods, at least two detection probes comprise: a) a target binding moiety directed to one of the at least two detectable biomarkers; and b) an oligonucleotide domain coupled to a 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; an oligonucleotide domain in which the single-stranded overhang portions of the detection probes are capable of hybridizing with each other when the detection probes are bound to the same extracellular vesicle; Each includes:

[0221] In some embodiments of the disclosed methods, a product is formed when at least two detection probes of the set are in sufficient proximity on each nanoparticle such that the single-stranded overhang portions of at least two detection probes of the set hybridize to each other to form a double-stranded complex, hi some embodiments, the product formed comprises a template that is ligated when the double-stranded complex is contacted with a nucleic acid ligase.

[0222] In some embodiments of the disclosed methods, the target binding portions of at least two detection probes each target the same detection biomarker. In some embodiments, the oligonucleotide domains of at least two detection probes are different. In some embodiments, the same detection biomarker is or comprises a polypeptide encoded by the human gene BST2. In some embodiments, the target capture portion of the capture agent is or comprises at least one antibody agent directed to the sialyl Lewis A antigen (also known as CA19-9) or to a polypeptide encoded by the human gene MUC1. In some embodiments, the same detection biomarker is or comprises the sialyl Tn (sTn) antigen. In some embodiments, the target capture portion of the capture agent is or comprises at least one antibody agent directed to a polypeptide encoded by the human gene MUC16.

[0223] In some embodiments of the disclosed method, the target binding portions of at least two detection probes each target a distinct detection biomarker. In some embodiments, the target binding portion of a first detection probe targets a polypeptide encoded by the human gene BST2, and the target binding portion of a second detection probe targets a polypeptide encoded by the human gene FOLR1. In some embodiments, the target capture portion of the capture agent is or includes at least one antibody agent that targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target binding portion of a first detection probe targets a polypeptide encoded by the human gene BST2, and the target binding portion of a second detection probe targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target binding portion of a first detection probe targets a polypeptide encoded by the human gene FOLR1, and the target binding portion of a second detection probe targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target binding portion of a first detection probe targets a polypeptide encoded by the human gene MUC16, and the target binding portion of a second detection probe targets a polypeptide encoded by the human gene MSLN. In some embodiments, the target capture portion of the capture agent is or includes at least one antibody agent directed against a sialyl-Tn (sTn) antigen.

[0224] One aspect of the present disclosure is a kit comprising: a) at least one set of probes for a combination of biomarkers specific for the detection of ovarian cancer, the combination of biomarkers comprising at least one capture biomarker on exosomes and at least one detection biomarker on exosomes, the capture biomarker and the detection biomarker being i) Polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and iii) combinations thereof are each independently selected from At least one set of probes b) a capture probe comprising a target capture moiety directed to a capture biomarker; and c) at least two detection probes, each containing a target binding moiety directed to at least one detection biomarker; The kit includes:

[0225] In some embodiments, the disclosed kits further comprise multiple sets of probes, each set directed to a distinct combination of biomarkers specific for the detection of ovarian cancer. In some embodiments, the combination of biomarkers is selected from the following: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; c) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; d) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; e) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; f) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and g) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN is selected from one of the following:

[0226] In some embodiments, the kit comprises at least seven sets of probes, each set comprising: a) sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; b) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; c) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; d) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; e) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; f) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and g) Sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN for a combination of distinct biomarkers.

[0227] In some embodiments of the disclosed kits, the capture probes and detection probes selectively bind to individual biomarkers on exosomes with a specificity within the range of 90% to 100% and a sensitivity within the range of 65% to 95%.

[0228] In some embodiments of the disclosed kits, the detection probes each further comprise an oligonucleotide domain coupled to a 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, characterized in that the single-stranded overhang portions of at least two detection probes are capable of hybridizing to each other when the at least two detection probes bind to the same exosome.

[0229] In some embodiments of the disclosed kit, the target binding portions of at least two detection probes each target the same detection biomarker on exosomes. In some embodiments, the oligonucleotide domains of at least two detection probes are different. In some embodiments, the same detection biomarker is or comprises a polypeptide encoded by the human gene BST2. In some embodiments, the target capture portion of the capture probe is or comprises at least one antibody agent directed to the sialyl Lewis A antigen (also known as CA19-9) or a polypeptide encoded by the human gene MUC1. In some embodiments, the same detection biomarker is or comprises the sialyl Tn (sTn) antigen. In some embodiments, the target capture portion of the capture probe is or comprises at least one antibody agent directed to a polypeptide encoded by the human gene MUC16.

[0230] In some embodiments of the disclosed kit, the target-binding portions of at least two detection probes each target a distinct detection biomarker on exosomes. In some embodiments, the target-binding portion of a first detection probe targets a polypeptide encoded by the human gene BST2, and the target-binding portion of a second detection probe targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target capture portion of a capture probe is or includes at least one antibody agent targeted to a polypeptide encoded by the human gene MUC1. In some embodiments, the target-binding portion of a first detection probe targets a polypeptide encoded by the human gene BST2, and the target-binding portion of a second detection probe targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target-binding portion of a first detection probe targets a polypeptide encoded by the human gene FOLR1, and the target-binding portion of a second detection probe targets a polypeptide encoded by the human gene MUC1. In some embodiments, the target-binding portion of a first detection probe targets a polypeptide encoded by the human gene MUC16, and the target-binding portion of a second detection probe targets a polypeptide encoded by the human gene MSLN. In some embodiments, the target capture moiety of the capture assay is or comprises at least one antibody agent directed against a sialyl-Tn (sTn) antigen.

[0231] In some embodiments, the disclosed kits further comprise at least one additional reagent (e.g., a ligase, a fixative, and / or a permeabilization agent).

[0232] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims. [Brief explanation of the drawings]

[0233] [Figure 1]Figure 1 is a schematic diagram illustrating an exemplary workflow for profiling individual nanoparticles (EVs). The diagram shows the purification of EVs from plasma using size exclusion chromatography (SEC) and immunoaffinity capture of EVs displaying specific surface biomarkers (Panel A); detection of co-localized target markers (e.g., intravesicular or surface proteins) on the captured EVs using a target entity detection assay according to some embodiments described herein (Panel B).

[0234] [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 specific for the detection of cancer. In some embodiments, a dual system includes a first detection probe for target protein 1 (e.g., cancer marker 1) and a second detection probe for target protein 2 (e.g., cancer marker 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 antibody agent against the target protein) 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 distinct target binding moieties (e.g., antibody agents against target protein 1 and target protein 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 causing ligation of the oligonucleotide domains. A detection signal is generated when the distinct target binding moieties of the first and second detection probes (e.g., antibody agents against target protein 1 and target protein 2, respectively) are localized in the same biological entity (e.g., extracellular vesicles) such that the corresponding single-stranded overhangs hybridize with each other, thus causing 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 protein 1 (e.g., cancer marker 1) and target protein 2 (e.g., cancer marker 2), and as a result, no signal can be detected. However, if a biological entity derived from a cancer sample (e.g., ovarian cancer) expresses target protein 1 and target protein 2, and the target proteins are present within a sufficiently short distance from each other in the same biological entity (e.g., extracellular vesicles), a detection signal will be generated.

[0235] [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 the corresponding single-stranded overhangs of all 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.

[0236] [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.

[0237] [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.

[0238] [Figure 6] Figure 6 is a pie chart showing the prevalence of ovarian cancer by major ovarian cancer subtype. "Other" refers to mixed or transitional cancer that cannot be categorized into a single subtype.See, for example, Gilks ​​et al., 2008, Seidman et al., 2003, 2004, each of which is incorporated herein by reference for the purposes described herein in its entirety and for additional information.

[0239] [Figure 7] FIG. 7 is a table showing delta Ct values ​​for certain exemplary biomarker combinations useful for distinguishing between ovarian cancer patients and control subjects (e.g., healthy female subjects and / or subjects with benign gynecological tumors and / or inflammatory conditions, including, e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.) using the exemplary assays described herein.

[0240] [Figure 8]Figure 8 shows the performance of an exemplary assay described herein, including a particular exemplary biomarker combination. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Therefore, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value.

[0241] [Figure 9] 9 is a table showing delta Ct values ​​for certain exemplary biomarker combinations useful for distinguishing between ovarian cancer patients and control subjects (e.g., healthy female subjects and / or subjects with benign gynecological tumors and / or inflammatory conditions, including, e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.) using the exemplary assays described herein. In some embodiments, certain biomarker combinations were selected and ranked by the overall mean delta Ct across three pools of subject samples from end-stage HGSOC cancer patients.

[0242] [Figure 10] 10 is a table showing delta Ct values ​​of certain exemplary biomarker combinations useful for distinguishing between ovarian cancer patients and control subjects (e.g., healthy female subjects and / or subjects with benign gynecological tumors and / or inflammatory conditions, including, e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.) using the exemplary assays described herein. In some embodiments, certain biomarker combinations were selected and ranked by the mean delta Ct of late-stage HGSOC cancer patients with low CA-125 plasma levels.

[0243] [Figure 11]Figures 11-15 show the performance of exemplary assays described herein, including certain exemplary biomarker combinations. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Thus, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value. [Figure 12] Figures 11-15 show the performance of exemplary assays described herein, including certain exemplary biomarker combinations. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Thus, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value. [Figure 13] Figures 11-15 show the performance of exemplary assays described herein, including certain exemplary biomarker combinations. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Thus, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value. [Figure 14]Figures 11-15 show the performance of exemplary assays described herein, including certain exemplary biomarker combinations. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Thus, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value. [Figure 15] Figures 11-15 show the performance of exemplary assays described herein, including certain exemplary biomarker combinations. The lower red dotted line indicates the Ct value for the healthy sample with the strongest signal, and the upper red dotted line indicates the Ct value for the 10th percentile of healthy controls. In some embodiments, benign ovarian tumor samples may be less concerned about off-target signals than healthy control subjects and / or subjects with inflammatory conditions (e.g., Crohn's disease, ulcerative colitis, endometriosis, etc.). Thus, in some such embodiments, benign ovarian tumor samples may not be included in determining the cutoff value.

[0244] [Figure 16] 16-18 show the performance of exemplary assays described herein that include certain exemplary biomarker combinations. For each plot, from left to right, the Ct values ​​for the "no EV" negative control, healthy control pool I, healthy control pool II, benign samples, early stage ovarian cancer, and late stage ovarian cancer are shown, and on the right, the Ct values ​​for the positive cell lines are shown. [Figure 17] 16-18 show the performance of exemplary assays described herein that include certain exemplary biomarker combinations. For each plot, from left to right, the Ct values ​​for the "no EV" negative control, healthy control pool I, healthy control pool II, benign samples, early stage ovarian cancer, and late stage ovarian cancer are shown, and on the right, the Ct values ​​for the positive cell lines are shown. [Figure 18]16-18 show the performance of exemplary assays described herein that include certain exemplary biomarker combinations. For each plot, from left to right, the Ct values ​​for the "no EV" negative control, healthy control pool I, healthy control pool II, benign samples, early stage ovarian cancer, and late stage ovarian cancer are shown, and on the right, the Ct values ​​for the positive cell lines are shown.

[0245] [Figure 19] Figure 19 shows exemplary receiver operating characteristic (ROC) curves for distinguishing patients with early stage I-II ovarian cancer from healthy patients / patients with benign ovarian masses. The curves were generated using Ct values ​​determined from the exemplary assay shown in Figure 1. The curves show an area under the curve (AUC) of 0.94 when utilizing the biomarker combination of sTn antigen, BST2+MUC1, and an AUC of 0.85 when utilizing plasma CA-125 levels.

[0246] [Figure 20] Figure 20 shows the performance of an exemplary assay described herein, including certain exemplary biomarker combinations. (A-D) Box plots for certain exemplary biomarker combinations. For each plot, Ct values ​​are shown, from left to right, for a "no EV" negative control, a healthy control, a benign sample, early-stage ovarian cancer, late-stage ovarian cancer, and a cell line positive control. (E-H) Corresponding receiver operating characteristic (ROC) curves for distinguishing patients with ovarian cancer (including early-stage and late-stage ovarian cancer patients) from both healthy patients and patients with benign ovarian masses.

[0247] [Figure 21] FIG. 21 shows exemplary receiver operating characteristic (ROC) curves for distinguishing patients with ovarian cancer from healthy patients (A) or from both healthy patients and patients with benign ovarian masses (B).

[0248] [Figure 22]Figure 22 shows the performance of an exemplary assay described herein that includes certain exemplary biomarker combinations. (A-G) For each plot, Ct values ​​(subtracted from 40) are shown for healthy controls, benign samples, early stage ovarian cancer, and late stage ovarian cancer.

[0249] [Figure 23] Figure 23 shows the performance of an exemplary assay described herein that includes certain exemplary biomarker combinations. (A-C) For each plot, Ct values ​​(subtracted from 40) are shown for healthy controls, benign samples, early-stage ovarian cancer, and late-stage ovarian cancer.

[0250] [Figure 24] Figure 24 shows the performance of plasma CA-125 for distinguishing early and late stage ovarian cancer from both healthy and benign samples as measured by ELISA, shown in U / mL (log2) from left to right for healthy controls, benign samples, early stage ovarian cancer, and late stage ovarian cancer.

[0251] [Figure 25-1] Figure 25 (A-D) shows the performance of an exemplary assay described herein that includes certain exemplary biomarker combinations. For each plot, from left to right, Ct values ​​are shown for healthy control, early stage ovarian cancer, late stage ovarian cancer, adenofibroma, fibroma, other, cyst, cystadenoma, no evidence of malignancy, endometriosis, leiomyoma, teratoma, and cystadenoma. [Figure 25-2] Figure 25 (A-D) shows the performance of an exemplary assay described herein that includes certain exemplary biomarker combinations. For each plot, from left to right, Ct values ​​are shown for healthy control, early stage ovarian cancer, late stage ovarian cancer, adenofibroma, fibroma, other, cyst, cystadenoma, no evidence of malignancy, endometriosis, leiomyoma, teratoma, and cystadenoma.

[0252] [Figure 26]26 shows the performance of (A) an exemplary assay described herein including the indicated biomarker combinations, and (B) CA-125 levels, for distinguishing between ovarian cancer and exemplary off-target cancers. For each plot, from left to right, Ct values ​​are shown for healthy controls, benign samples, early-stage ovarian cancer, late-stage ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, colorectal cancer (CRC), breast cancer, and bladder cancer.

[0253] [Figure 27] 27 shows the performance of (A) an exemplary assay described herein including a particular biomarker combination and (B) CA-125 levels for distinguishing between ovarian cancer and exemplary inflammatory conditions. For each plot, from left to right, Ct values ​​are shown for healthy controls, benign samples, early-stage ovarian cancer, late-stage ovarian cancer, Crohn's disease, type 2 diabetes, endometriosis, acute pancreatitis, rheumatoid arthritis, and ulcerative colitis.

[0254] [Figure 28-1] Figure 28 shows exemplary receiver operating characteristic (ROC) curves for illustrating the performance of an exemplary assay described herein including the particular set of biomarker combinations shown in Table 8 compared to a CA-125 test. (A) ROC curve for distinguishing patients with ovarian cancer (including both early-stage and late-stage patients) from both healthy patients and patients with benign ovarian masses. The McNemar p-value comparing the curve for CA-125 to the curve for the set of seven biomarker combinations at 99% specificity was <0.0001 (p-value: 4.71 x 10). (B) ROC curve for distinguishing patients with early-stage ovarian cancer (e.g., stage I and / or stage II HGSOC cases) from both healthy patients and patients with benign ovarian masses. The McNemar p-value comparing the curve for CA-125 to the curve for the set of seven biomarker combinations at 99% specificity was <0.0001 (p-value: 2.99 x 10). [Figure 28-2]Figure 28 shows exemplary receiver operating characteristic (ROC) curves for illustrating the performance of an exemplary assay described herein including the particular set of biomarker combinations shown in Table 8 compared to a CA-125 test. (A) ROC curve for distinguishing patients with ovarian cancer (including both early-stage and late-stage patients) from both healthy patients and patients with benign ovarian masses. The McNemar p-value comparing the curve for CA-125 to the curve for the set of seven biomarker combinations at 99% specificity was <0.0001 (p-value: 4.71 x 10). (B) ROC curve for distinguishing patients with early-stage ovarian cancer (e.g., stage I and / or stage II HGSOC cases) from both healthy patients and patients with benign ovarian masses. The McNemar p-value comparing the curve for CA-125 to the curve for the set of seven biomarker combinations at 99% specificity was <0.0001 (p-value: 2.99 x 10).

[0255] [Figure 29] Figure 29 shows exemplary receiver operating characteristic (ROC) curves to demonstrate the performance of exemplary assays described herein including the particular set of biomarker combinations shown in Table 8 compared to CA-125 testing. (A) ROC curve for distinguishing patients with early stage ovarian cancer (e.g., stage I and stage II HGSOC cases) from healthy patients. The McNemar p-value comparing the ROC curve for CA-125 to the ROC curve for the set of seven biomarker combinations at 99% specificity was 1. (B) ROC curve for distinguishing patients with early stage ovarian cancer (e.g., stage I and stage II HGSOC cases) from patients with benign ovarian masses. The McNemar p-value comparing the ROC curve for CA-125 to the ROC curve for the set of seven biomarker combinations at 99% specificity was <0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0256] 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.

[0257] Adnexal mass: As used herein, the term "adnexal mass" refers to a growth or lump in the tissue surrounding the uterus. In some embodiments, an adnexal mass may occur in one or more ovaries. In some embodiments, an adnexal mass may occur in one or more fallopian tubes. In some embodiments, an adnexal mass may occur in the adjacent connective tissue surrounding the uterus. In some embodiments, an adnexal mass may be a benign tumor. Examples of benign adnexal masses include, but are not limited to, adenofibromas, fibromas, ovarian cysts, cystadenomas, endometriosis, leiomyomas, tetratomies, cystadenomas, and the like. In some embodiments, an adnexal mass may be a malignant tumor.

[0258] Affinity agent: As used herein, the term "affinity agent" refers to an entity that is or includes a target-binding moiety described herein and thus binds to a target of interest (e.g., a molecular target of interest, such as a biomarker or epitope). In many embodiments, an affinity agent according to the present disclosure specifically binds to a biomarker described herein. In many embodiments, an affinity agent according to the present disclosure specifically binds to a surface biomarker described herein. In some embodiments, an affinity agent according to the present disclosure specifically binds to a carbohydrate-dependent marker described herein. In some embodiments, an affinity agent may be or include an antibody agent (e.g., an antibody or other entity that is or includes an antigen-binding portion thereof). Alternatively, or in addition, in some embodiments, an affinity agent may be selected from the group consisting of an affimer, an aptamer, a lectin, a sialic acid-binding immunoglobulin-type lectin (siglec), and combinations thereof, and / or another binding agent that may be considered a ligand. In some embodiments, the target of the affinity agent (e.g., a biomarker target) is or includes one or more polypeptide, nucleic acid, carbohydrate, and / or lipid moieties and / or entities.

[0259] 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.

[0260] 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).

[0261] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimerized, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more 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 embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementary determining regions (CDRs) or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (SMPs). ImmunoPharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®, KALBITOR®, and Affimers®, and the like. In some embodiments, the antibody may lack covalent modifications (e.g., 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.).In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs); in some embodiments, an antibody agent is or comprises a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are identical in sequence or contain one to five amino acid substitutions compared to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDR in that they exhibit at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDR in that at least one amino acid in the included CDR has been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDR in that 1 to 5 amino acids in the included CDR have been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDR in that at least one amino acid in the included CDR has been substituted compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDR, but the included CDRs have an otherwise identical amino acid sequence to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.

[0262] Antibody agents can be produced by those skilled in the art using methods known in the art and commercially available services and kits. For example, methods for preparing monoclonal antibodies are well known in the art and include hybridoma and phage display technologies. Additional antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second Edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (August 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (February 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (September 30, 2013)).

[0263] Antibodies can be generated by standard techniques, such as by immunization with an appropriate polypeptide or portion thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected host animal (e.g., mouse, rabbit, goat, horse, chicken, etc.) is immunized with an immunogenic polypeptide bearing the desired epitope, optionally haptenized to another polypeptide. Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include, but are not limited to, inorganic gels such as Freund's and aluminum hydroxide, as well as surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum is collected from the immunized animal and processed according to known procedures. If the serum containing polyclonal antibodies against the desired epitope contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are known in the art.

[0264] 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).

[0265] 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.

[0266] 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.

[0267] 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 ovarian cancer (e.g., a specific type of ovarian cancer and / or stage of ovarian cancer) if its presence correlates with the occurrence and / or susceptibility of ovarian cancer (e.g., in an entire relevant population).

[0268] Biological entity: In appropriate circumstances, as will be clear from the context to one of skill in the art, the term "biological entity" may, in some embodiments, be or include a cell or organism, e.g., an animal or human, or, in some embodiments, may be or include a biological tissue or biological fluid; for example, in some embodiments, it 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 an extracellular vesicle. In some embodiments, the biological entity is or includes 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., the protein or polypeptide is retained in its naturally occurring conformational structure). In some embodiments, the biological entity is processed, for example, by isolating it from a 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. In some embodiments, the biological entity is or includes nanoparticles having a size in the range of about 30 nm to about 1000 nm, which in some embodiments are obtained from a subject's bodily fluid sample (e.g., without limitation, a blood sample). In some embodiments, such nanoparticles may be or include protein aggregates, including, for example, 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. 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 the formation of crosslinks of proteins and / or peptides present in the cell or extracellular vesicle. In some embodiments, the biological entity is in isolated or pure form (e.g., isolated from a body fluid sample, such as a blood, serum, or plasma 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, or plasma sample).

[0269] 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 ovarian cancer. In some embodiments, biomarkers are intravesicular (e.g., protein or RNA markers present in extracellular vesicles). In some embodiments, biomarkers may be or include genetic or epigenetic signatures. In some embodiments, a biomarker may be or include a gene expression signature. In some embodiments, a "biomarker" suitable for use in accordance with the present disclosure may refer to the presence, level, and / or form of a molecular entity (e.g., an epitope) present on a target marker. For example, in some embodiments, two or more "biomarkers" (e.g., epitopes) as molecular entities may be present on the same target marker (e.g., a marker protein, e.g., a surface protein present on extracellular vesicles).

[0270] 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.

[0271] 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 ovarian cancer.

[0272] Capture assay: As used herein, the term "capture assay" refers to a process of isolating or separating a biological entity of interest from a sample (e.g., in some embodiments, a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample)). In some embodiments, the biological entity of interest is isolated or separated from a sample (e.g., in some embodiments, a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-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 assays 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, a biological entity of interest suitable for the capture assays described herein is or comprises an extracellular vesicle of interest (e.g., in some embodiments, an exosome).

[0273] 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, such as, but not limited to, a blood-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., those described herein) of a nanoparticle having a size within a range of about 30 nm to about 1000 nm, including, for example, an extracellular vesicle (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.

[0274] 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., ovarian cancer), for example, by defining one or more boundaries among two or more subsets of a population (e.g., normal healthy subjects and subjects with an inflammatory condition versus ovarian cancer 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 classification is based on two 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.

[0275] 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.

[0276] 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.

[0277] Complementary: As used herein, the term "complementary" 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. Therefore, 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.

[0278] 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 ovarian cancer 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 protein biomarker, an intravesicular protein 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 an entity of interest (e.g., a ligated template indicative of a surface protein biomarker and / or an intravesicular protein 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 an entity of interest (e.g., a surface protein biomarker, an intravesicular protein 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 protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof, can refer to absolute or relative quantification. Absolute quantification may be achieved by correlating the detected level of, or a form of measurement indicative of, an entity of interest (e.g., a surface protein biomarker, an intravesicular protein 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 protein biomarkers, intravesicular protein 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.

[0279] 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.

[0280] 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).

[0281] 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.

[0282] 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.

[0283] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding entity 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 a 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).

[0284] Extracellular vesicles: As used herein, the term "extracellular vesicles" typically refers to vesicles outside of cells, e.g., secreted by cells. Examples of secreted vesicles include, but are not limited to, exosomes, microvesicles, microparticles, ectosomes, oncosomes, and apoptotic bodies. While not 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 limiting membrane, while microvesicles typically bud from the cell surface and their size can vary from 50 nm to 1000 nm. In some embodiments, the extracellular vesicles are or include exosomes and / or microvesicles. In some embodiments, the sample containing extracellular vesicles is substantially free of apoptotic bodies. In some embodiments, the sample containing nanoparticles may include nanoparticles 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 or derived from ovarian cancer tumors, and in some embodiments, the extracellular vesicles are shed or derived from non-ovarian cancer tumors. In some embodiments, the extracellular vesicles are shed or derived from healthy tissue. In some embodiments, the extracellular vesicles are shed or derived from benign gynecological tumors. In some embodiments, the extracellular vesicles are shed or derived from tissue of a subject with symptoms associated with ovarian cancer (e.g., non-specific symptoms).

[0285] 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 polypeptide may be tumor-specific. In some embodiments, such a polypeptide may be tissue-specific (e.g., ovarian 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.

[0286] 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.

[0287] 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 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. In some embodiments, non-limiting examples of intravesicular biomarkers (e.g., intravesicular protein biomarkers) that are useful for ovarian cancer detection include CRABP2, KLK7, MIF, PRAME, S100A1, or a combination thereof.

[0288] 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 (e.g., mRNA) present within 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.

[0289] 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.

[0290] Life history-related risk factors: As used herein, the term "life history risk factors" refers to activities, experiences, medical histories, and / or exposures of an individual in their life that may directly or indirectly increase the individual's risk of a condition, such as ovarian cancer, compared to an individual who does not have such activities, experiences, medical histories, and / or exposures in their life. In some embodiments, non-limiting examples of life history-related risk factors include smoking, alcohol, drugs, carcinogens, diet, obesity, diabetes, polycystic ovary syndrome (PCOS), endometriosis, pelvic inflammatory disease (PID), nulliparity / infertility, no / poor oral contraceptive use, physical activity, sun exposure, radiation exposure, perineal talc use, hormone replacement therapy (HRT), exposure to infectious agents such as viruses, 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 lifestyle-related risk factors that contribute to cancer (eg, ovarian cancer) susceptibility is not exhaustive and is constantly evolving.

[0291] 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.

[0292] Nanoparticles: The term "nanoparticles," when used in the context of a sample for a detection assay (e.g., as described herein), refers to nanoparticles having a size range of interest, including extracellular vesicles. In some embodiments, such nanoparticles have a size range of about 30 nm to about 1000 nm. In some embodiments, nanoparticles have a size range of about 30 nm to about 750 nm. In some embodiments, nanoparticles have a size range of about 50 nm to about 750 nm. In some embodiments, nanoparticles have a size range of about 30 nm to about 500 nm. In some embodiments, nanoparticles have a size range of about 50 nm to about 500 nm. In some embodiments, nanoparticles described herein are obtained from a subject's bodily fluid sample (e.g., a blood-derived sample), for example, in some embodiments, by a size-exclusion-based method (e.g., in some embodiments, size-exclusion chromatography). In some embodiments, the nanoparticles are or comprise analyte aggregates, which in some embodiments may be or comprise protein or mucin aggregates. In some embodiments, the nanoparticles are or comprise protein multimers. In some embodiments, the nanoparticles are or comprise extracellular vesicles. In some embodiments, the nanoparticles are or comprise intact extracellular vesicles.

[0293] Non-cancer subject: As used herein, the term "non-cancer subject" generally refers to a female subject who does not have non-benign ovarian cancer. For example, in some embodiments, the non-cancer subject is a healthy female subject (e.g., a healthy female subject). In some embodiments, the non-cancer subject is a healthy female subject (e.g., a healthy female subject) under the age of 55. In some embodiments, the non-cancer subject is a healthy female subject (e.g., a healthy female subject) who is 55 years of age or older. In some embodiments, the non-cancer subject is a female subject (e.g., a female subject) who has a non-ovarian-related health disease, disorder, or condition. In some embodiments, the non-cancer subject is a female subject (e.g., a female subject) who has a benign ovarian tumor (e.g., a benign mass observed in the fallopian tubes and / or ovaries).

[0294] 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 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 2250 and 500, 2000, 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.

[0295] 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.

[0296] 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.

[0297] 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, 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.

[0298] 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.

[0299] 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.

[0300] 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 contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference or control in the context of a reference level of a target 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 reference level of a target refers to the level of the target in a subject prior to 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. One of skill in the art will recognize when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0301] 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.

[0302] 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 expected 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 nanoparticles, while in some embodiments, it may also include nucleic acids and / or proteins extracted from the sample, etc. In some embodiments, a processed sample may be obtained by subjecting a primary sample to one or more techniques, such as amplification or reverse transcription of nucleic acids, isolation and / or purification of certain components, etc.

[0303] 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.

[0304] 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. .

[0305] 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.

[0306] 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., ovarian cancer). 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).

[0307] 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 female subject, e.g., a human female subject. In some embodiments, the subject is afflicted with ovarian cancer. In some embodiments, the subject is predisposed to ovarian cancer. In some embodiments, the subject exhibits one or more symptoms or characteristics of ovarian cancer. In some embodiments, the subject exhibits one or more non-specific symptoms of ovarian cancer. In some embodiments, the subject does not exhibit any symptoms or characteristics of ovarian cancer. In some embodiments, the subject is a subject with one or more features characteristic of susceptibility to or risk of ovarian cancer. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnostic and / or therapeutic treatment will be administered. In some embodiments, the subject is a female subject (e.g., a female subject) determined to have an adnexal mass. In some embodiments, the subject is an asymptomatic subject. Such an asymptomatic subject may be a female subject (e.g., a woman) 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 previously or periodically 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 undergone cancer therapy. In some embodiments, subjects suitable for the provided technology are individuals selected based on one or more characteristics, such as age, race, geographic location, genetic history, medical history, personal history (e.g., smoking, alcohol, drugs, carcinogens, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and / or occupational hazards).

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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 binding 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 that are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.), for example, via one or more non-peptide linkages (e.g., by glycosylphosphatidylinositol (GPI) anchors or lipidation, or by non-covalent interactions). In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions that are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.) via one or more non-peptide linkages (e.g., by glycosylphosphatidylinositol (GPI) anchors or lipidation, or by non-covalent interactions). The surface protein may comprise one or more domains or regions anchored to either side of a cell membrane of a body (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 an ovarian cancer-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) of a subject suffering from or susceptible to ovarian cancer). As will be understood by one of skill in the art, detection of 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 ovarian cancer-associated extracellular vesicles from a biological sample (e.g., in some embodiments, a body fluid-derived sample such as, but not limited to, a blood-derived sample) from a subject (e.g., a blood sample or blood-derived sample). In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detection of an intravesicular portion of such a surface polypeptide or surface protein (e.g., an intravesicular epitope). 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 detection of an extravesicular portion of such a surface polypeptide or surface protein.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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 ovarian cancer, 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 ovarian cancer, or a subtype and / or disease state thereof, although one or more biomarkers in such a combination may be directed to a target that is not specific to ovarian cancer (e.g., a surface protein biomarker, an intravesicular protein biomarker, and / or an intravesicular RNA). For example, in some embodiments, a target biomarker signature may include at least one biomarker specific to ovarian cancer, or a stage and / or subtype thereof (i.e., an ovarian cancer-specific target), and may further include biomarkers that are not necessarily or completely specific to ovarian cancer (e.g., biomarkers that may also be found in some or all biological entities, such as cells, nanoparticles, 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 are together specific for the relevant target biological entity of interest (e.g., ovarian cancer cells of interest, or nanoparticles secreted by ovarian cancer cells) (i.e., sufficiently distinguish the relevant target biological entity for detection (e.g., ovarian cancer cells of interest, or nanoparticles secreted by ovarian cancer cells) from other biological entities that are not of interest for detection), such a combination of biomarkers is a useful target biomarker signature in accordance with certain embodiments of the present disclosure.

[0316] 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.

[0317] 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-ovarian cancer vs. ovarian cancer). 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.

[0318] 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.

[0319] 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.

[0320] Detailed Description of Specific Embodiments Ovarian cancer was responsible for an estimated 14,070 deaths in the United States in 2018 (Torre et al., 2018; incorporated herein by reference for purposes described herein). The majority of these deaths were due to delayed diagnosis, with the estimated 5-year survival rate for ovarian cancer being 93% for those diagnosed at their earliest stage compared with 26% for those diagnosed at their later stage (Torre et al., 2018; incorporated herein by reference for purposes described herein). High-grade serous ovarian cancer (HGSOC) accounts for 70%-80% of all ovarian cancer deaths, while other subtypes are slower-growing and prone to overdiagnosis using current technology, making detection of HGSOC particularly important (Temkin et al., 2017; incorporated herein by reference for purposes described herein). Unfortunately, despite being the fifth most lethal cancer in women of all cancers (Howlader et al., 2019; which is incorporated herein by reference for purposes described herein), there are no recommended ovarian cancer screening tools for average-risk women. Many women who are at genetic risk and / or who may be experiencing one or more symptoms of ovarian cancer (e.g., fluid in the abdominal cavity (ascites), general gastrointestinal dysfunction, constipation, bowel obstruction, nausea, vomiting, diarrhea, gastrointestinal reflux, increased abdominal size, urinary symptoms, abdominal bloating, abdominal and / or pelvic pain, fatigue, and / or shortness of breath) are currently screened with plasma CA-125 and / or transvaginal ultrasound (TVUS), which are suboptimal for screening due to their low sensitivity (approximately 20%) and insufficient specificity for stage I and II disease.For example, the Prostate, Lung, Colorectal and Ovarian Cancer Screening Randomized Trial found that plasma CA-125 and TVUS increased the number of unnecessary surgeries and provided no mortality benefit for average-risk women (Buys et al., 2011; which is incorporated herein by reference for purposes described herein). Despite this poor performance, plasma CA-125 and TVUS are now common screening tools for prioritizing postmenopausal women with nonspecific pelvic pain, which could potentially indicate ovarian cancer.

[0321] The present disclosure identifies the origin of certain prior technology challenges, including, among other things, certain conventional approaches to the detection and diagnosis of ovarian cancer. For example, the present disclosure recognizes that many conventional diagnostic assays, such as those based on bulk analysis of cell-free nucleic acids, serum proteins (e.g., CA-125), and / or 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, identifying combinations of biomarkers that are predicted to exhibit high sensitivity and specificity for ovarian cancer based on bioinformatics analysis. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that address this issue by detecting the colocalization (e.g., identified by bioinformatics analysis) of a target biomarker signature for ovarian cancer in individual nanoparticles, which includes at least one extracellular vesicle-associated surface biomarker and at least one target biomarker, including a target surface marker that may be a polypeptide or carbohydrate-dependent marker present on nanoparticles associated with ovarian cancer. In some embodiments, the target biomarker signature may further include at least one internal biomarker (e.g., an internal protein biomarker described herein and / or an RNA biomarker described herein) present on nanoparticles associated with ovarian cancer. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, among others, by detecting such ovarian cancer target biomarker signatures using a target entity detection approach developed by applicant and described in US2020 / 0299780 and WO2020 / 180741, which is based on the interaction and / or co-localization of target biomarker signatures on individual nanoparticles. The contents of each of the foregoing disclosures are incorporated herein by reference in their entirety.

[0322] In some embodiments, extracellular vesicles for detection as described herein can be isolated from a subject's bodily fluids by size-exclusion-based methods. As will be understood by those skilled in the art, in some embodiments, size-exclusion-based methods can provide samples containing nanoparticles of a desired size range, including extracellular vesicles. Thus, in some embodiments, the provided techniques of the present disclosure encompass the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for prostate cancer in individual nanoparticles of a desired size range (e.g., in some embodiments, about 30 nm to about 1000 nm), including extracellular vesicles. Those skilled in the art who read this disclosure will understand that various embodiments described herein in the context of "extracellular vesicles" (e.g., assays for detecting individual extracellular vesicles and / or the provided "extracellular vesicle-associated surface biomarkers") may also be applicable in the context of "nanoparticles" as described herein.

[0323] The present disclosure provides, among other things, insights and techniques for achieving effective ovarian cancer screening, for example, for the early detection of ovarian cancer. In some embodiments, the present disclosure provides techniques for the early detection of ovarian cancer in women who may be experiencing one or more symptoms associated with ovarian cancer. In some embodiments, the present disclosure provides techniques for the early detection of ovarian cancer in women who are at genetic risk for ovarian cancer. In some embodiments, the present disclosure provides techniques for the early detection of ovarian cancer in postmenopausal women who may be at genetic risk for ovarian cancer and / or who may be experiencing one or more symptoms associated therewith. In some embodiments, the present disclosure provides techniques for screening women at genetic or average risk for early-stage high-grade serous ovarian cancer (HGSOC). HGSOC is the most common and fatal subtype of ovarian cancer, with 84% of cases detected at an advanced stage (Torre et al., 2018, which is incorporated herein by reference for purposes described herein). In some embodiments, the provided techniques are effective in detecting early-stage ovarian cancer. In some embodiments, the provided techniques are effective even when applied to populations that include or consist of asymptomatic or symptomatic individuals (e.g., due to sufficiently high sensitivity and / or low false positive and / or false negative rates). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals without a genetic risk of developing ovarian cancer (e.g., asymptomatic or symptomatic individuals). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals with a genetic risk of developing ovarian cancer (e.g., asymptomatic or symptomatic individuals). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals who are susceptible to ovarian 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 would be apparent to one of skill in the art upon reading the disclosure provided herein.

[0324] In some embodiments, the provided technologies achieve detection (e.g., early detection, e.g., in asymptomatic individuals and / or populations) of one or more characteristics of ovarian 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 technologies to single and / or regular (e.g., periodic) assessments. In some embodiments, the provided technologies are useful in conjunction with an individual's regular screening, including, but not limited to, physical exams, general practitioner visits, cholesterol / lipid blood tests, diabetes (type 2) screening, colonoscopy, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV / Pap smears, and / or vaccinations. In some embodiments, the provided technologies are useful in conjunction with imaging tests such as abdominal / transvaginal ultrasound and / or other diagnostic assays for ovarian cancer, including serum biomarkers (e.g., CA-125).

[0325] 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 ovarian cancer. In some embodiments, the present disclosure provides ovarian cancer screening systems that can be implemented to detect early stage cancers, including early stage cancers in asymptomatic individuals (e.g., without a genetic risk for ovarian cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals (e.g., with or without a genetic risk for ovarian cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of symptomatic individuals (e.g., with or without a genetic risk for ovarian cancer). The present disclosure provides compositions (e.g., reagents, kits, components, etc.), including, for example, strategies involving periodic testing of one or more individuals (e.g., asymptomatic individuals), as well as methods of providing and / or using them. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them.

[0326] I. Ovarian cancer detection Currently, there are no FDA-approved ovarian cancer screening tests for average-risk asymptomatic women, but in the United States, the average lifetime risk of developing ovarian cancer is 1.3%, which corresponds to 1 in 78 women. The overall ovarian cancer prevalence in the United States was 5.7 per 10,000 women aged 55 to 74 (Buys et al., 2011; incorporated herein by reference for purposes described herein). In 2018, there were approximately 22,240 new cases of ovarian cancer diagnosed in the United States, and 14,070 deaths from ovarian cancer (Torre et al., 2018; incorporated herein by reference for purposes described herein). Among other factors, age and menopausal status have been identified as risk factors for ovarian cancer, with the average age of first onset being approximately 68 years.

[0327] Epithelial ovarian cancer subtypes account for 90% of all ovarian cancers. Epithelial cancers are classified as serous (52%), endometrioid (10%), mucinous (6%), or clear cell (6%) (Torre et al., 2018; incorporated herein by reference for purposes described herein). The majority of serous cancers are diagnosed at stage III (51%) or stage IV (29%), with 5-year survival rates of 42% and 26%, respectively, indicating the need for early screening. Germ cell and sex cord-stromal tumors comprise the majority of non-epithelial cancers, but account for only 3% and 2% of all ovarian cancers, respectively. Ovarian cancer affects women of all ethnicities.

[0328] The strongest risk factor for ovarian cancer is a family history of breast or ovarian cancer. The risk of developing invasive epithelial ovarian cancer is approximately 50% increased among women with a first-degree relative with a history of ovarian cancer and 10% increased among women with a first-degree relative with a history of breast cancer. It is estimated that approximately 18% of epithelial ovarian cancer cases, particularly high-grade serous carcinoma, are attributable to inherited mutations that confer increased risk. BRCA1 and BRCA2 mutations contribute to nearly 40% of ovarian cancer cases in women with a family history of the disease. Among women with BRCA1 or BRCA2 mutations, the risk of developing ovarian cancer by age 80 is 44% and 17%, respectively. Rare, moderately penetrant genetic mutations in epithelial ovarian cancer include genes involved in the Fanconi anemia / BRCA pathway, such as PALB2, BARD1, BRIP1, RAD51C, and RAD51D, as described, for example, in Matulonis et al., 2016, which is incorporated herein by reference for purposes described herein. Families with Lynch syndrome are characterized by germline mutations in DNA mismatch repair genes (e.g., MLH1, MSH2, MSH6, or PMS2). The risk of developing ovarian cancer (usually non-serous epithelial tumors) by age 70 is approximately 8% in women with Lynch syndrome, compared to 0.7% in the general population (Torre, et al., 2018; which is incorporated herein by reference for purposes described herein). Inherited mutations in other genes involved in DNA repair, such as CHEK2, MRE11A, RAD50, ATM, and TP53, also increase the risk of developing ovarian cancer. Additional common low-penetrance alleles may also be associated with epithelial ovarian cancer susceptibility, as suggested by genome-wide association studies.Such genes and loci include WNT4, RSPO1, BCL2L11, HOXD3, HAGLR, TIPARP, SYNPO2, TERT, GPX6, CHMP4C, LINC00824, COL15A1, SMC2-AS1, MLLT10, INCENP, RCCD1, ATAD5, HNF1B, PLEKHM1, SKAP1, ANKLE1, GATAD2A, Cytoband, and SNPs 2q13 rs752590, 4q32.3 rs4691139, 9p22 rs3814113, 9q34.2 rs635634, 10p11.21 rs1192691, and / or 19q13.2 rs688187 (Reid et al., 2017; which is incorporated herein by reference for the purposes described herein).

[0329] The number of young women identified as at genetic risk is expected to increase in the coming years. In December 2019, the NCCN guidelines for pancreatic cancer were updated to include a recommendation for testing all patients for germline mutations in ATM, BRCA1, BRCA2, CDKN2A, MSH2, MLH1, MSH2, EPCAM, PALB2, STK11, and TP53. Given the overlap between this list of genes and genes that confer genetic risk for ovarian cancer, it is possible that more daughters of pancreatic cancer patients will learn their genetic risk for both pancreatic and ovarian cancer and move from the general risk category to the genetic risk category. Furthermore, a recent cost-effectiveness study for breast cancer patients concluded that it would be cost-effective to screen all breast cancer patients in the United States and the United Kingdom for germline mutations in BRCA1 and / or BRCA2 and PALB2 (Sun, et al., 2019; which is incorporated herein by reference for purposes described herein). By incorporating germline genetic testing for all women with breast cancer into practice guidelines, additional risk mutation carriers whose daughters are also at genetic risk for breast and ovarian cancer will be identified. Currently, there are no recommended screening tests for ovarian cancer for women (e.g., without genetic risk). Among other things, in certain embodiments, the present disclosure provides insight into the need for the development of ovarian cancer liquid biopsy assays (e.g., as described herein) that can be utilized to provide ovarian cancer risk assessment. In certain embodiments, the assays and / or techniques described herein can provide a score compared to a reference threshold (e.g., as described herein). In certain embodiments, such a score can be or can include an ovarian cancer risk score.In some embodiments, such scores may be used in conjunction with other ovarian cancer screening assessments, such as, but not limited to, CA-125 measurements (e.g., CA-125 serum level measurements and / or TVUS) and / or ovarian cancer-related risk factors to provide an overall assessment.

[0330] The Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO), which assessed the use of transvaginal ultrasound (TVUS) and a fixed cutpoint (≥35 U / mL) of the tumor marker CA-125 for early detection, did not observe a reduction in ovarian cancer mortality after up to 19 years of follow-up. The UK Collaborative Trial of Ovarian Cancer Screening evaluated the combination of TVUS with a risk algorithm that incorporated changes in CA-125 levels and found a reduction in mortality after 15 years in average-risk women. Despite this discrepancy, the US Preventive Services Task Force (USPSTF) continues to recommend screening for ovarian cancer in the general population, concluding that there is sufficient evidence that annual screening does not reduce ovarian cancer mortality and may result in important harms, primarily surgical intervention in women without ovarian cancer.

[0331] Among other things, in certain embodiments, the present disclosure provides the insight that there is a need for the development of an ovarian cancer liquid biopsy assay for screening women with a genetic risk of ovarian cancer and / or women who may be experiencing one or more symptoms associated with ovarian cancer. In certain embodiments, the present disclosure provides the insight that there is a need for the development of an ovarian cancer liquid biopsy assay for screening symptomatic or asymptomatic women, for example, before other screening methods, such as TVUS. In certain embodiments, the present disclosure provides the insight that there is a need for the development of an ovarian cancer liquid biopsy assay for screening asymptomatic women, for example, before other screening methods, such as TVUS. In certain embodiments, the present disclosure provides the insight that there is a need for the development of an ovarian cancer liquid biopsy assay for screening women with an average risk of ovarian cancer. In certain embodiments, the present disclosure provides the insight that there is a need for the development of an ovarian cancer liquid biopsy assay for screening women with a lifestyle-related risk of ovarian cancer. In certain embodiments, the present disclosure provides insight into the need for the development of ovarian cancer liquid biopsy assays for screening postmenopausal women, for example, women who may be experiencing one or more symptoms associated with postmenopausal ovarian cancer. Despite being the fifth most lethal cancer in women (Howlader et al., 2019; incorporated herein by reference for purposes described herein), there are currently no recommended ovarian cancer screening tools for average-risk women, and current standard-of-care screening assays for stage I and II disease in women at genetic risk and / or who may be experiencing symptoms of ovarian cancer (e.g., TVUS and serum marker CA-125 levels) exhibit low sensitivity (approximately 20%) and low specificity (NCCN, 2019; Buys et al., 2011; each of which is incorporated herein by reference for purposes described herein).These low sensitivity and specificity rates pose barriers to efficient and timely diagnosis. Given the incidence of ovarian cancer in average-risk women, inadequate test specificity (e.g., <99.5%) results in false-positive results that outnumber true-positives by more than an order of magnitude. This places a significant burden on the healthcare system and on the women who are screened with false-positive results, resulting in additional testing, unnecessary surgery, and emotional / physical distress (Buys et al., 2011; which is incorporated herein by reference for purposes described herein).

[0332] In some embodiments, the present disclosure provides the insight that a particularly useful ovarian cancer screening test will be characterized by (1) ultra-high specificity (>98%), minimizing the number of false positives, and (2) high sensitivity (>40%) for stage I and II ovarian cancer (i.e., when the prognosis is most favorable). For example, in some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of >98% and a sensitivity of >50%, e.g., for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of >98% and a sensitivity of >60%, e.g., for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of >98% and a sensitivity of >70%, e.g., for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >65%, e.g., for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >60%, for example, for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test may be characterized by a specificity of at least 99% and a sensitivity of at least 70%, for example, for stage I and II ovarian cancer. In some embodiments, a particularly useful ovarian cancer screening test (e.g., comprising one or more biomarker combinations described herein, e.g., as shown in Table 8) may be characterized by a specificity of at least 90% (including, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) and a sensitivity of at least 80% (including, e.g., at least 85%, at least 90%, at least 95%, or higher), for example, for stage I and II ovarian cancer.In some embodiments, particularly useful ovarian cancer screening tests may be characterized by a specificity of about 90% to about 100% and a sensitivity of about 80% to about 100%, or about 80% to about 95%.

[0333] In some embodiments, the present disclosure provides insight that ovarian cancer screening tests (e.g., comprising, in some embodiments, a combination of one or more biomarkers described herein, e.g., as shown in Table 8) that are particularly useful for distinguishing between benign adnexal masses and ovarian cancer may be characterized by (1) high specificity (>90%), which minimizes the number of false positives, and (2) high sensitivity (>65%), which minimizes the number of false negatives. For example, in some embodiments, an ovarian cancer screening test (e.g., comprising, in some embodiments, a combination of one or more biomarkers described herein, e.g., as shown in Table 8) that is particularly useful for distinguishing between benign adnexal masses and ovarian cancer may be characterized by a specificity of at least 90% (including, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) and a sensitivity of at least 65% (including, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher). In some embodiments, ovarian cancer screening tests that are particularly useful for distinguishing between benign adnexal masses and ovarian cancer (e.g., in some embodiments, comprising one or more biomarker combinations described herein, e.g., as shown in Table 8) may be characterized by a specificity of about 90% to about 100% and a sensitivity of about 60% to about 100%, or about 65% to about 95%, or about 70% to about 95%.

[0334] In some embodiments, the present disclosure provides insight that an ovarian 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 sensitivity of such an assay compared to that achieved by a single set of biomarker combinations. For example, in some embodiments, an ovarian cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve at least 98% specificity and at least 50% sensitivity. In some embodiments, an ovarian cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve at least 98% specificity and at least 60% sensitivity. In some embodiments, an ovarian cancer screening test comprising a combination of at least two orthogonal biomarkers can achieve at least 99% specificity and at least 70% sensitivity.

[0335] In some embodiments, the present disclosure provides insight that a particularly useful ovarian cancer screening test can be characterized by an acceptable positive predictive value (PPV) at an economically justifiable cost. PPV is the probability that a patient has the disease after a positive test and is affected by sensitivity, specificity, and / or disease prevalence. One clinician consensus regarding the minimum PPV required for ovarian cancer screening is 10% (Nossov et al., 2008; incorporated herein by reference for purposes described herein). A PPV of 10% results in nine false positives for every true positive. These false positives place a significant burden on the healthcare system and on both the false positives and the women screened, resulting in additional testing, unnecessary surgery, and emotional and physical distress (Buys et al., 2011; incorporated herein by reference for purposes described herein). In some embodiments, the assays described herein are particularly useful for early ovarian 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 98% for women at genetic risk for ovarian cancer, or at a specificity cutoff of at least 99.5% for women experiencing one or more symptoms associated with ovarian cancer.

[0336] In some embodiments, the assays described herein may be useful for early ovarian cancer detection achieving a PPV of greater than 2% or higher, including, for example, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 15%, greater than 20%, or greater than 25% or higher. In some such embodiments, the assays described herein may achieve a specificity cutoff of at least 95% or higher (e.g., a specificity cutoff of at least 98% for women at genetic risk for ovarian cancer, or a specificity cutoff of at least 99.5% for women experiencing one or more symptoms associated with ovarian cancer).

[0337] In some embodiments, the assays described herein (e.g., in some embodiments, comprising a combination of one or more biomarkers described herein, e.g., as shown in Table 8) may be useful for distinguishing between benign adnexal masses and ovarian cancer, achieving a positive predictive value (PPV) of greater than 65% or higher (including, e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or higher), and / or a negative predictive value (NPV) of greater than 90% (including, e.g., greater than 95%, greater than 96%, greater than 97%, greater than 98%, or higher).

[0338] Several different biomarker classes, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), bulk proteins, and extracellular vesicles (EVs), have been investigated for ovarian cancer liquid biopsy assays. EVs are particularly promising due to their abundance and stability in the bloodstream compared to ctDNA and CTCs, suggesting improved sensitivity for early-stage cancer. EVs also contain cargo (i.e., proteins, RNA, and metabolites) of the same cellular origin, providing superior specificity over bulk protein measurements. While the diagnostic utility of EVs has been investigated, much of this work has focused on bulk EV measurements or low-throughput single-EV analysis.

[0339] II. PROVIDED BIOMARKERS AND / OR TARGET BIOMARKER SIGNATURES FOR THE DETECTION OF OVARIAN CANCER The present disclosure provides, among other things, various target biomarkers or combinations thereof (e.g., target biomarker signatures) for ovarian cancer. Such target biomarker signatures that are predicted to exhibit high sensitivity and specificity for ovarian cancer are discovered through a wide range of bioinformatics analysis and biological approaches, including, for example, in some embodiments, computer analysis of diverse sets of data, including, for example, in some embodiments, 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, by machine learning and / or computational modeling. In some embodiments, the biomarker combinations described herein have been demonstrated to achieve at least 99% specificity with a certain sensitivity (e.g., in some embodiments, at least 70% sensitivity) when used to distinguish ovarian cancer samples from reference samples (e.g., normal healthy samples and / or benign tumor samples).

[0340] In some embodiments, a target biomarker signature for ovarian cancer includes at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) target biomarkers, including at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., surface polypeptides present on extracellular vesicles associated with ovarian cancer; "extracellular vesicle-associated surface biomarkers"), as well as one or more surface protein biomarkers, such that such surface The combination of biomarkers and such target biomarkers provides a target biomarker signature for ovarian cancer that provides (a) high specificity (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or higher, e.g., at least 99%, or at least 99.5%) that minimizes the number of false positives, and (b) high sensitivity (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, such target biomarker signatures described herein provide a specificity in the range of 90%-100% and a sensitivity in the range of 65%-100%. In some embodiments, such target biomarker signatures described herein provide a specificity in the range of 90%-100% and a sensitivity in the range of 70%-95%. In some embodiments, such target biomarker signatures described herein are particularly useful for detecting stage I and II ovarian cancer, which have the most favorable prognosis. In some embodiments, such target biomarker signatures described herein are particularly useful for distinguishing between benign adnexal masses and ovarian cancer.

[0341] In some embodiments, a target biomarker signature for ovarian cancer comprises at least one surface biomarker (e.g., a surface polypeptide and / or carbohydrate-dependent marker present on the surface of extracellular vesicles associated with ovarian cancer) and at least one target biomarker comprising one or more surface protein biomarkers, such that the combination of such surface biomarkers and such target biomarkers presents a target biomarker signature for ovarian cancer that provides a positive predictive value (PPV) of at least 15% or higher, at least 20% or higher, at least 25% or higher, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or higher. In some embodiments, a target biomarker signature for ovarian cancer includes at least one surface biomarker (e.g., a surface polypeptide and / or carbohydrate-dependent marker present on the surface of extracellular vesicles associated with ovarian cancer) and at least one target biomarker including one or more surface protein biomarkers, such that the combination of such surface biomarkers and such target biomarkers represents a target biomarker signature for ovarian cancer that provides a positive predictive value (PPV) of greater than 2% or higher, including, for example, greater than 3%, greater than 5%, greater than 7%, greater than 10%, greater than 15% or higher, greater than 20% or higher, greater than 25% or higher, and / or greater than 30% or higher. In some embodiments, such target biomarker signatures described herein provide a PPV within the range of 70%-100% or within the range of 70%-90%.

[0342] In some embodiments, the present disclosure recognizes that, in certain embodiments, sensitivity and specificity rates for women with different ovarian cancer risk levels may vary depending on the risk tolerance guidelines set forth by their physicians and / or interested medical associations. In some embodiments, lower specificity and / or sensitivity may be used to screen patients at higher risk for ovarian cancer (e.g., patients with lifestyle-related risk factors, symptomatic patients, or patients with a family history of ovarian cancer, etc.) compared to those for patients at lower risk for ovarian cancer. For example, in some embodiments, a biomarker combination described herein useful for detecting ovarian 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 ovarian 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.

[0343] In certain embodiments, women with a genetic risk of ovarian cancer may best be served with a 99.5% specificity rate and 70% sensitivity, or a 98% specificity rate and 80% sensitivity, or a 99% specificity rate and 70% sensitivity. In certain embodiments, postmenopausal asymptomatic women may best be served with a 99.5% specificity rate and 70% sensitivity, or a 98% specificity rate and 80% sensitivity, or a 99% specificity rate and 70% sensitivity. In certain embodiments, postmenopausal symptomatic women may best be served with a 99.5% specificity rate and 70% sensitivity, or a 98% specificity rate and 80% sensitivity, or a 99% specificity rate and 70% sensitivity. In certain embodiments, women with a life history risk may best perform at a specificity rate of 99.5% and a sensitivity of 70%, or a specificity rate of 98% and a sensitivity of 80%, or a specificity rate of 99% and a sensitivity of 70%. In some embodiments, the techniques and / or assays described herein for detecting ovarian cancer in symptomatic women may have lower sensitivity and / or specificity requirements than those for detecting ovarian cancer in asymptomatic women. In some embodiments, the assays described herein for detecting ovarian cancer in symptomatic women may have a set specificity rate that is less than 99.5%, including, for example, a specificity rate of less than 99%, less than 95%, less than 90%, or less than 85%. In some embodiments, the assays described herein for detecting ovarian cancer in symptomatic women may have a set sensitivity rate that is less than 80%, including, for example, a sensitivity rate of less than 70%, or less than 60%.

[0344] 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.

[0345] 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.

[0346] In certain embodiments, a target biomarker signature for ovarian cancer includes at least one extracellular vesicle-associated surface biomarker (e.g., a surface polypeptide and / or carbohydrate-dependent marker present on nanoparticles associated with ovarian cancer) and at least one target biomarker including one or more surface protein biomarkers, such that the combination of such extracellular vesicle-associated surface biomarkers and such target biomarkers is specific for ovarian cancer. In some embodiments, the extracellular vesicle-associated surface biomarkers and / or target surface biomarkers include a basal cell adhesion molecule polypeptide encoded by the basal cell adhesion molecule (BCAM) gene, a bone marrow stromal cell antigen 2 polypeptide encoded by the bone marrow stromal cell antigen 2 (BST2) gene, a claudin 3 polypeptide encoded by the claudin 3 (CLDN3) gene, mucin 16, a truncated mucin 16 polypeptide partially encoded by the cell surface-associated (MUC16) gene, and a folate receptor alpha (FOLR1) gene. The antigen may be selected from alpha polypeptides, mesothelin polypeptides encoded by the mesothelin (MSLN) gene, mucin 1, mucin 1 polypeptides encoded by the cell surface-associated (MUC1) gene, mucin 16, mucin 16 polypeptides encoded by the cell surface-associated (MUC16) gene, sodium-dependent phosphate transport protein 2B polypeptides encoded by the solute carrier family 34 (sodium phosphate) member 2 (SLC34A2) gene, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0347] In some embodiments, the extracellular vesicle-associated surface biomarker and / or target surface biomarker can be selected from bone marrow stromal cell antigen 2 polypeptide encoded by the bone marrow stromal cell antigen 2 (BST2) gene, folate receptor alpha polypeptide encoded by the folate receptor alpha (FOLR1) gene, mesothelin polypeptide encoded by the mesothelin (MSLN) gene, mucin 1, mucin 1 polypeptide encoded by the cell surface-associated (MUC1) gene, mucin 16, mucin 16 polypeptide encoded by the cell surface-associated (MUC16) gene, sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof. In some embodiments, the mucin 16 polypeptide is an intact polypeptide. In some embodiments, the mucin 16 polypeptide is a truncated polypeptide.

[0348] In certain embodiments, the target biomarker signature for ovarian cancer is or includes one or more (e.g., at least one, at least two, at least three, or more) of the following surface biomarkers: basal cell adhesion molecule (BCAM) polypeptide, bone marrow stromal cell antigen 2 (BST2) polypeptide, claudin-3 (CLDN3) polypeptide, truncated mucin-16 (truncated MUC16) polypeptide, folate receptor alpha (FOLR1) polypeptide, mesothelin (MSLN) polypeptide, mucin-1 (MUC1) polypeptide, mucin-16 (MUC16) polypeptide, sodium-dependent phosphate transport protein 2B (SLC34A2) polypeptide, sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof.

[0349] In certain embodiments, the target biomarker signature for ovarian cancer is or includes one or more (e.g., at least one, at least two, at least three, or more) of the following surface biomarkers: bone marrow stromal cell antigen 2 (BST2) polypeptide, folate receptor alpha (FOLR1) polypeptide, mesothelin (MSLN) polypeptide, mucin-1 (MUC1) polypeptide, mucin-16 (MUC16) polypeptide, sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and combinations thereof. In some embodiments, the mucin 16 polypeptide is a truncated polypeptide.

[0350] In certain embodiments, a target biomarker signature for detecting ovarian cancer (e.g., HGSOC) comprises a combination of at least two target surface biomarkers, which combination can be selected from the following: a MUC16 polypeptide and a FOLR1 polypeptide; or an SLC34A2 polypeptide and a FOLR1 polypeptide; or an SLC34A2 polypeptide and a MUC16 polypeptide; or a BST2 polypeptide and a FOLR1 polypeptide; or a CA19-9 antigen and a BST2 polypeptide; or a CA19-9 antigen and a CLDN3 polypeptide; or a CA19-9 antigen and a SLC34A2 polypeptide; or a MUC1 polypeptide and a BCAM polypeptide; or a MUC1 polypeptide and a BST2 polypeptide; or a MUC1 polypeptide and an MSLN polypeptide; or a MUC1 polypeptide and an sTn antigen; or a MUC16 polypeptide and a BCAM polypeptide; or a MUC16 polypeptide and a MUC1 polypeptide; or a MUC16 polypeptide and a sTn antigen; or a sTn antigen and a FOLR1 polypeptide; or a T antigen and a BST2 polypeptide; or a combination thereof. In certain embodiments, the target biomarkers described in the foregoing combinations may be used as targets for capture probes and / or targets for detection probes in the assays described herein.

[0351] In certain embodiments, a target biomarker signature for ovarian cancer detection (e.g., HGSOC) comprises a combination of at least three target surface biomarkers, the combination being one of: CA19-9 antigen and BST2 polypeptide and MUC16 polypeptide; MUC1 polypeptide and BCAM polypeptide and BST2 polypeptide; or MUC1 polypeptide and BST2 polypeptide and FOLR1 polypeptide; or MUC1 polypeptide and BST2 polypeptide and sTn antigen; or MUC1 polypeptide and MSLN polypeptide and sTn antigen; or MUC16 polypeptide and FOLR1 polypeptide and SLC34A2 polypeptide; or MUC16 polypeptide and MUC1 polypeptide and sTn antigen; or a MUC16 polypeptide and an MSLN polypeptide and an sTn antigen; or a MUC16 polypeptide and an SLC34A2 polypeptide and an sTn antigen; or an sTn antigen and a FOLR1 polypeptide and a MUC16 polypeptide; or an sTn antigen and a FOLR1 polypeptide and an MUC16 polypeptide; or an sTn antigen and a FOLR1 polypeptide and an MSLN polypeptide; or an sTn antigen and a FOLR1 polypeptide and a MUC1 polypeptide; or an sTn antigen and a MUC1 antigen and an SLC34A2 antigen; or an sTn antigen and a MUC16 polypeptide and a truncated MUC16 polypeptide; or an sTn antigen and a truncated MUC16 polypeptide and an MSLN polypeptide; or an sTn antigen and a FOLR1 polypeptide and an SLC34A2 polypeptide; or a combination thereof. In certain embodiments, the target biomarkers described in the foregoing combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0352] In some embodiments, a target biomarker signature may include a combination of targets described in Table 1, where the targets may be used in a capture probe and / or a detection probe. In some embodiments, a target biomarker signature may include a target for a capture probe and at least one or more (e.g., including at least two or more) targets for a detection probe (e.g., Detection Probe 1 and / or Detection Probe 2) described in Table 1. By way of example only, in some embodiments, a target biomarker signature may include MUC16 (a target for a capture probe described in Table 1), sTn antigen (a target for Detection Probe 1 or 2 described in Table 1), and FOLR1 (a target for Detection Probe 1 or 2 described in Table 1). In some embodiments, a target biomarker signature may include targets for a combination of capture probes and detection probes described in Table 1. Those of skill in the art reading this disclosure will recognize that the targets for "Detection Probe 1" and "Detection Probe 2" of a given combination may be used interchangeably. [Table 1-1] [Table 1-2]

[0353] In some embodiments, certain biomarker combinations described in Table 1 that may be particularly useful for ovarian cancer detection (e.g., have higher sensitivity, specificity, and / or PPV) may be subjected to one or more rounds of screening using advanced-stage (e.g., late-stage, e.g., stage III and / or IV) ovarian cancer samples (e.g., pooled or individual samples) and healthy control samples (e.g., pooled or individual samples) as references. In some embodiments, selected combinations can be further tested using early-stage ovarian cancer samples (e.g., stage I and / or II, optionally distinguished by low or high CA-125 content), benign gynecological tumor plasma samples (e.g., as described herein), non-ovarian cancer samples (e.g., as described herein), and / or any combination thereof. In some embodiments, the performance of a biomarker combination can be determined by calculating the difference (e.g., based on Ct) between the assay signals of healthy samples (e.g., pooled and / or individual samples) and ovarian cancer samples (e.g., pooled and / or individual samples).

[0354] In some embodiments, certain biomarker combinations for ovarian cancer detection can be selected with a delta Ct greater than the inter-assay variability. For example, in some embodiments, biomarker combinations with a delta Ct greater than 2.0 (corresponding to a 4-fold difference) or 1.0 (corresponding to a 2-fold difference) are considered to provide particularly effective diagnostic utility (e.g., provide a signal greater than the inter-assay variability). For example, see Examples 2-3, which provide exemplary analyses of certain combinations described herein.

[0355] In certain embodiments, the target biomarker signature for ovarian cancer distinguishes between late-stage ovarian cancer samples and control samples (e.g., compared to healthy samples, compared to benign gynecological tumor samples, and / or compared to other cancer samples). In certain embodiments, the target biomarker signature for ovarian cancer distinguishes between early-stage ovarian cancer samples (e.g., having low plasma CA-125 and / or high plasma CA-125) and control samples (e.g., compared to healthy samples, compared to benign gynecological tumor samples, and / or compared to other cancer samples). In some embodiments, assays directed to detecting the target biomarker signature for ovarian cancer may comprise a combination of capture probes and detection probes as described in Table 1.

[0356] In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to FOLR1 and MUC16, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to FOLR1 and FOLR1, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC16 and MUC16, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to BST2 and BST2, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to BST2 and MUC16, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to CLDN3 and CLDN3, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to SLC34A2 and SLC34A2, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to BCAM and BCAM, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to BCAM and BST2, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to BST2 and FOLR1, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to BST2 and MUC1, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to BST2 and sTn antigen, respectively. In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to MSLN and MUC1, respectively.In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MSLN and sTn antigens, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to sTn antigens and sTn antigens, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to FOLR1 and SLC34A2, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC1 and MUC1, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC1 and MUC16, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC1 and sTn antigens, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC16 and sTn antigens, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to SLC34A2 and sTn antigen, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to FOLR1 and MSLN, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC16 and MSLN, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to FOLR1 and MUC1, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC1 and SLC34A2, respectively. In certain embodiments, at least two of the plurality of detection probes may have their target binding entities directed to MUC16 and truncated MUC16, respectively.In certain embodiments, at least two detection probes of the plurality may have their target binding entities directed to truncated MUC16 and MSLN, respectively.

[0357] In certain embodiments where the target biomarker signature comprises a combination of SLC34A2 and FOLR1, the capture probes have their target binding entities directed to SLC34A2, and at least two detection probes have their target binding entities directed to FOLR1 and FOLR1, respectively. In certain embodiments where the target biomarker signature comprises a combination of SLC34A2 and MUC16, the capture probes have their target binding entities directed to SLC34A2, and at least two detection probes have their target binding entities directed to MUC16 and MUC16, respectively.

[0358] In certain embodiments in which the target biomarker signature comprises a combination of BST2 and FOLR1, the capture probes have their target binding entities directed to BST2 and at least two detection probes have their target binding entities directed to FOLR1 and FOLR1, respectively.

[0359] In certain embodiments where the target biomarker signature comprises a combination of CA19-9 antigen and BST2, the capture probes have their target binding entities directed to the CA19-9 antigen, and at least two detection probes have their target binding entities directed to BST2 and BST2, respectively. In certain embodiments where the target biomarker signature comprises a combination of CA19-9 antigen, BST2, and MUC16, the capture probes have their target binding entities directed to the CA19-9 antigen, and at least two detection probes have their target binding entities directed to BST2 and MUC16, respectively. In certain embodiments where the target biomarker signature comprises a combination of CA19-9 antigen and CLDN3, the capture probes have their target binding entities directed to the CA19-9 antigen, and at least two detection probes have their target binding entities directed to CLDN3 and CLDN3, respectively. In certain embodiments in which the target biomarker signature comprises a combination of CA19-9 antigen and SLC34A2, the capture probe has its target binding entity directed to the CA19-9 antigen, and at least two detection probes have their target binding entities directed to SLC34A2 and SLC34A2, respectively.

[0360] In certain embodiments where the target biomarker signature comprises a combination of MUC1 and BCAM, the capture probes have their target binding entities directed to MUC1, and at least two detection probes have their target binding entities directed to BCAM and BCAM, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1, BCAM, and BST2, the capture probes have their target binding entities directed to MUC1, and at least two detection probes have their target binding entities directed to BCAM and BST2, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1 and BST2, the capture probes have their target binding entities directed to MUC1, and at least two detection probes have their target binding entities directed to BST2 and BST2, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1, BST2, and FOLR1, the capture probes have their target binding entity directed to MUC1, and at least two detection probes have their target binding entities directed to BST2 and FOLR1, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1 and BST2, the capture probes have their target binding entity directed to MUC1, and at least two detection probes have their target binding entities directed to BST2 and MUC1, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1, BST2, and sTn antigen, the capture probes have their target binding entity directed to MUC1, and at least two detection probes have their target binding entities directed to BST2 and sTn antigen, respectively. In certain embodiments in which the target biomarker signature comprises a combination of MUC1 and MSLN, the capture probes have their target binding entities directed to MUC1, and at least two detection probes have their target binding entities directed to MSLN and MUC1, respectively.In certain embodiments where the target biomarker signature comprises a combination of MUC1 and MSLN and sTn antigens, the capture probes have their target binding entities directed to MUC1 and at least two detection probes have their target binding entities directed to MSLN and sTn antigens, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC1 and sTn antigens, the capture probes have their target binding entities directed to MUC1 and at least two detection probes have their target binding entities directed to sTn antigen and sTn antigen, respectively.

[0361] In certain embodiments where the target biomarker signature comprises a combination of MUC16 and FOLR1, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to FOLR1 and MUC16, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and BCAM, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to BCAM and BCAM, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and FOLR1, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to FOLR1 and FOLR1, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16, FOLR1, and SLC34A2, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to FOLR1 and SLC34A2, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and MUC1, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to MUC1 and MUC1, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and MUC1, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to MUC1 and MUC1, respectively. In certain embodiments in which the target biomarker signature comprises a combination of MUC16 and MUC1 and sTn antigens, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to MUC1 and sTn antigens, respectively.In certain embodiments where the target biomarker signature comprises a combination of MUC16 and sTn antigens, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to MUC16 and sTn antigens, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16, MSLN, and sTn antigens, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to MSLN and sTn antigens, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and SLC34A2, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to SLC34A2 and SLC34A2, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and SLC34A2 and sTn antigens, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to SLC34A2 and sTn antigens, respectively. In certain embodiments where the target biomarker signature comprises a combination of MUC16 and sTn antigens, the capture probes have their target binding entities directed to MUC16, and at least two detection probes have their target binding entities directed to sTn antigen and sTn antigen, respectively.

[0362] In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, BST2, and MUC1, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to BST2 and MUC1, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, FOLR1, and MUC16, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to FOLR1 and MUC16, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, FOLR1, and MSLN, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to FOLR1 and MSLN, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen and MUC1 and MSLN, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to MUC1 and MSLN, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen and MUC16 and MSLN, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to MUC16 and MSLN, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen and FOLR1 and MUC1, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to FOLR1 and MUC1, respectively. In certain embodiments in which the target biomarker signature comprises a combination of sTn antigen and FOLR1, the capture probes have their target binding entities directed to sTn antigen, and at least two detection probes have their target binding entities directed to FOLR1 and FOLR1, respectively.In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, MUC1, and SLC34A2, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to MUC1 and SLC34A2, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, MUC16, and truncated MUC16, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed to MUC16 and truncated MUC16, respectively. In certain embodiments where the target biomarker signature comprises a combination of sTn antigen, truncated MUC16, and MSLN, the capture probes have their target binding entities directed to the sTn antigen, and at least two detection probes have their target binding entities directed ...

Claims

1. (a) providing a biological sample comprising nanoparticles isolated from a body fluid-derived sample (e.g., a blood-derived sample) of a subject, the nanoparticles having a size in the range of about 30 nm to about 1000 nm; (b) detecting co-localization of at least one set of surface biomarkers on the surface of the nanoparticles, the at least one set comprising at least two surface biomarkers whose combined expression levels have been determined to be associated with ovarian cancer, the surface biomarkers being selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof; and (c) comparing the detected colocalization level with said determined level. wherein the detected colocalization level at or above the determined level indicates that the subject has or is susceptible to ovarian cancer.

2. 10. The method of claim 1, wherein the detecting on the surface comprises analyzing nanoparticles separated from other components of the sample by affinity capture that targets at least one of the surface biomarkers on their surface.

3. 3. The method of claim 1, wherein the step of detecting on the surface comprises contacting the nanoparticles with at least one set of detection probes each directed to at least one of the surface biomarkers, the set comprising at least a first detection probe for a first surface biomarker and a second detection probe for a second surface biomarker, the first surface biomarker and the second surface biomarker being the same or different.

4. the first detection probe comprises a first target-binding moiety for the first surface biomarker and a first oligonucleotide domain coupled to the first target-binding moiety, the first oligonucleotide domain comprising a first double-stranded portion and a first single-stranded overhang extending from one end of the first oligonucleotide domain; 4. The method of claim 3, wherein the second detection probe comprises a second target-binding moiety for the second surface biomarker and a second oligonucleotide domain coupled to the second target-binding moiety, the second oligonucleotide domain comprising a second double-stranded portion and a second single-stranded overhang extending from one end of the second oligonucleotide domain, the second single-stranded overhang comprising a nucleotide sequence complementary to at least a portion of the first single-stranded overhang, thereby being capable of hybridizing to the first single-stranded overhang.

5. 5. The method of claim 4, wherein the first single-stranded overhang and / or the second single-stranded overhang is 4 nucleotides in length.

6. 6. The method of claim 5, wherein the first single-stranded overhang or the second single-stranded overhang has a nucleotide sequence of GAGT.

7. 5. The method of claim 4, wherein the first oligonucleotide domain and the second oligonucleotide domain have a combined length such that when the first and second surface biomarkers are simultaneously present on the nanoparticle and the probes of the set of detection probes bind to their respective surface biomarkers on the nanoparticle, the first single-stranded overhang and the second single-stranded overhang can hybridize together and form a double-stranded complex.

8. 8. The method of claim 7, further comprising contacting the double-stranded complex with a nucleic acid ligase to create a ligated template comprising a strand of the first double-stranded portion and a strand of the second double-stranded portion.

9. 9. The method of claim 8, wherein the nucleic acid ligase is or comprises a DNA ligase (e.g., T4 or T7 DNA ligase).

10. 4. The method of claim 3, wherein the first surface biomarker and the second surface biomarker are the same target biomarker.

11. 10. The method of claim 1, wherein the step of detecting on the surface further comprises the steps of amplifying a product associated with the co-localization and detecting the presence of the amplified product.

12. 12. The method of claim 11, wherein the amplifying step is or comprises a quantitative polymerase chain reaction.

13. The method of claim 1 , wherein the step of detecting on a surface comprises immobilizing nanoparticles on a solid substrate.

14. 14. The method of claim 13, wherein the solid substrate is or comprises a bead.

15. The method of claim 14, wherein the beads are magnetic beads.

16. 14. The method of claim 13, wherein the solid substrate is or comprises a surface.

17. 17. The method of claim 16, wherein the surface is a capture surface of a filter, matrix, membrane, plate, tube, and / or well.

18. 2. The method of claim 1, wherein steps (b) and (c) are repeated for multiple (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more) sets of surface biomarkers, each set comprising at least two surface biomarkers whose combined expression levels have been determined to be associated with ovarian cancer, and wherein the surface biomarkers are selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof.

19. 20. The method of claim 18, further comprising combining the results of the comparing steps to determine a score for each set of surface biomarkers.

20. The method described in claim 19, wherein the score determined to be associated with ovarian cancer indicates that the subject has or is susceptible to ovarian cancer.

21. 2. The method of claim 1, wherein the surface biomarkers are selected from (i) polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof.

22. The at least one set or at least one of the multiple sets of surface biomarkers comprises a combination of: (i) the sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; (ii) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; and (iii) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; (iv) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; (v) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; (vi) a sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and (vii) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN The method of claim 1 , wherein the compound is selected from the group consisting of:

23. The at least one set or at least one of the multiple sets of surface biomarkers comprises a combination of: (i) the sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; (ii) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; (iii) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; (iv) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; (v) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; (vi) a sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and (vii) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN The method of claim 1 , comprising:

24. 2. The method of claim 1, wherein the determined level is determined by comparing the combined expression levels in ovarian cancer-associated extracellular vesicles to extracellular vesicles in a comparable sample from a population of non-cancer subjects.

25. 25. The method of claim 24, wherein the population of non-cancer subjects comprises one or more of the following subject populations: healthy subjects, subjects diagnosed with benign tumors, and subjects with a non-ovarian-related disease, disorder, and / or condition.

26. The method of claim 1 , wherein the nanoparticles have a size in the range of about 50 nm to about 500 nm.

27. The method of claim 1 , wherein the nanoparticle is or comprises an extracellular vesicle.

28. The method of claim 1 , wherein the nanoparticles are isolated from a body fluid-derived sample (e.g., a blood-derived sample) by size exclusion.

29. A kit for detecting ovarian 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 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; an 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 within the range of about 30 nm to about 1000 nm. Each of these includes At least the first surface biomarker and the second surface biomarker form a target biomarker signature determined to be associated with ovarian cancer, wherein the first and second surface biomarkers are each independently selected from: (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof. At least one set of detection probes Kit including:

30. 30. The kit of claim 29, wherein the first and / or second surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is an intact MUC16 polypeptide.

31. 30. The kit of claim 29, wherein the first and / or second surface biomarker is a polypeptide encoded by the human gene MUC16, and the polypeptide is a truncated MUC16 polypeptide.

32. 32. The kit of any one of claims 29 to 31, wherein the target binding moieties of at least two detection probes each target the same target surface biomarker of the target biomarker signature.

33. 30. The kit of claim 29, wherein the oligonucleotide domains of the at least two detection probes are different.

34. 32. The kit of any one of claims 29 to 31, wherein the target binding moieties of at least two detection probes are each directed to a distinct target surface biomarker of the target biomarker signature.

35. 30. The kit of claim 29, further comprising at least one additional reagent (e.g., a ligase, a fixative, and / or a permeabilization agent).

36. 30. The kit of claim 29, comprising at least two sets (e.g., including at least three sets) of detection probes, each set comprising at least two detection probes each directed to a target surface biomarker of a distinct target biomarker signature for ovarian cancer.

37. (a) a first capture agent comprising a target capture moiety; (b) a second capture agent comprising a target capture moiety; (c) at least two sets of detection probes, the detection probes comprising: (i) a target binding moiety for a target 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; an 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; at least two sets of detection probes, each comprising 30. The kit of claim 29, comprising:

38. (a) a first capture agent comprising a target capture moiety; (b) a second capture agent comprising a target capture moiety; (c) a third capture agent comprising a target capture moiety; (d) at least three sets of detection probes, the detection probes comprising: (i) a target binding moiety for a target 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; an 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; At least three sets of detection probes, each comprising 30. The kit of claim 29, comprising:

39. 30. The kit of claim 29, wherein the nanoparticles have a size in the range of about 50 nm to about 500 nm.

40. 30. The kit of claim 29, wherein the nanoparticle is or comprises an extracellular vesicle (e.g., an exosome).

41. 30. The kit of claim 29, wherein the nanoparticles are isolated from a body fluid-derived sample (e.g., a blood-derived sample) by size exclusion.

42. (a) nanoparticles having a size within the range of about 30 nm to about 1000 nm and comprising on their surface at least a first surface biomarker and a second surface biomarker, the combination of which has been determined to be a target biomarker signature for ovarian cancer, wherein the first surface biomarker and the second surface biomarker are each independently selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof; (b) a solid substrate comprising a target capture moiety directed to the first surface biomarker, wherein the target capture moiety binds to the first surface biomarker of the nanoparticle, thereby immobilizing the nanoparticle on the solid substrate; and (c) at least a first detector probe and a second detector probe, each of which is bound to said nanoparticle, each detector probe comprising: (i) a target binding moiety directed to 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; the single-stranded overhang portions of the first and second detection probes hybridize to each other; a first detection probe and a second detection probe comprising: A complex containing

43. 43. The conjugate of claim 42, wherein the first surface biomarker and the second surface biomarker are different.

44. 44. The conjugate of claim 42 or 43, wherein the first surface biomarker and the second surface biomarker are each independently selected from: (i) polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and / or combinations thereof.

45. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers including: (i) a polypeptide encoded by the human gene SLC34A2; and (ii) a polypeptide encoded by the following human genes: FOLR1, MUC16, and combinations thereof.

46. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers comprising: (i) a polypeptide encoded by the human gene MUC16; and (ii) at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MUC1, MUC16, MSLN, SLC34A2, or a combination thereof; and / or (ii) a carbohydrate-dependent marker comprising a sialyl-Tn (sTn) antigen.

47. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers comprising: (i) a polypeptide encoded by the human gene BST2; and (ii) a polypeptide encoded by the human gene FOLR1.

48. 43. The complex of claim 42, wherein the target biomarker signature comprises: (i) a carbohydrate-dependent marker comprising sialyl Lewis A antigen (also known as CA19-9); and (ii) one or more surface biomarkers comprising at least one polypeptide encoded by the following human genes: BST2, CLDN3, SLC34A2, or a combination thereof.

49. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers including (i) a polypeptide encoded by the human gene MUC1; and (ii) at least one polypeptide encoded by the following human genes: BCAM, BST2, FOLR1, MSLN, MUC1, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

50. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers including (i) a polypeptide encoded by the human gene MUC16; and (ii) at least one polypeptide encoded by the following human genes: BCAM, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof; and / or (ii) at least one of the following carbohydrate-dependent markers: sialyl-Tn (sTn) antigen.

51. 43. The complex of claim 42, wherein the target biomarker signature comprises one or more surface biomarkers comprising: (i) a carbohydrate-dependent marker comprising a sialyl-Tn (sTn) antigen; and (ii) at least one polypeptide encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, MUC16, SLC34A2, or a combination thereof.

52. 43. The complex of claim 42, wherein the target binding moieties of at least two detection probes each target the same target surface biomarker of the target biomarker signature.

53. 53. The complex of claim 52, wherein the oligonucleotide domains of the at least two detection probes are different.

54. 43. The complex of claim 42, wherein the target binding moieties of the at least two detection probes are each directed to a distinct target biomarker in the target biomarker signature.

55. 43. The complex of claim 42, wherein the solid substrate comprises a magnetic bead.

56. 43. The conjugate of claim 42, wherein the target capture moiety is or comprises an antibody agent.

57. 43. The conjugate of claim 42, wherein the nanoparticle is or comprises an extracellular vesicle (e.g., an exosome).

58. 43. The conjugate of claim 42, wherein the nanoparticles are isolated from a bodily fluid sample (e.g., a blood sample) obtained from a subject.

59. 43. The conjugate of claim 42, wherein the nanoparticles are isolated from a subject's bodily fluid sample (e.g., a blood sample) by size exclusion.

60. 60. The conjugate of claim 58 or 59, wherein the subject is a human subject.

61. 43. The complex of claim 42, wherein the formation of the complex is indicative of an ovarian cancer-associated nanoparticle.

62. 43. The complex of claim 42, wherein the single-stranded overhang portions of the first and second detection probes are at least partially complementary.

63. 43. The complex of claim 42, wherein the nanoparticles have a size in the range of about 50 nm to about 500 nm.

64. Each set of probes comprises (a) a biomarker binding moiety having a size within the range of about 300 nm to about 1000 nm, which specifically binds to a surface biomarker on a nanoparticle found in a sample from a cancer subject; and (b) an oligonucleotide domain, wherein the oligonucleotide domains of the probes in the set are arranged and constructed such that when the probes bind to their target biomarkers, the oligonucleotide domains hybridize to each other to form ligatable hybrids only when the target biomarkers are in close proximity to each other, and the target biomarkers are selected from (i) polypeptides encoded by the following human genes: BCAM, BST2, CLDN3, FOLR1, MSLN, MUC1, MUC16, SLC34A2; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, Thomsen-Friedenreich (T,TF) antigen, Tn antigen, sialyl Lewis 43. A set of probes for use in the method of claim 1, the kit of claim 29, or the complex of claim 42, comprising oligonucleotide domains each independently selected from: A antigen (also known as CA19-9), and / or combinations thereof.

65. A method for analyzing colocalization levels as an indicator for distinguishing between benign adnexal masses and ovarian cancer, comprising: (a) detecting, in a blood-derived sample from a female subject determined to have an adnexal mass, the co-localization of at least one combination of biomarkers on the surface of nanoparticles having a size in the range of about 300 nm to about 1000 nm, the combination of biomarkers comprising at least one capture biomarker and at least one detection biomarker, wherein the at least one capture biomarker and the at least one detection biomarker are: (i) Polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; and (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and (iii) combinations thereof each independently selected from: (b) comparing the detected colocalization level with a reference level; wherein the detected colocalization level that is equal to or comparable to the reference level indicates that the adnexal mass in the female subject is likely benign; and the detected colocalization level that is greater than the reference level indicates that the adnexal mass is cancerous.

66. 66. The method of claim 65, wherein the nanoparticle is or comprises an extracellular vesicle.

67. A method described in claim 65 or 66, wherein the specificity of the indicator for distinguishing between benign adnexal masses and ovarian cancer is within the range of 90% to 100% and the sensitivity is within the range of 65% to 95%.

68. 66. The method of claim 65, wherein the female subject has been determined to have an elevated serum CA-125 level (e.g., greater than 25 U / mL).

69. A method for analyzing colocalization levels as an indicator for detecting early stage ovarian cancer, comprising: (a) detecting, in a blood-derived sample from a female subject, co-localization of at least one combination of biomarkers on the surface of nanoparticles having a size in the range of about 300 nm to about 1000 nm, wherein the at least one combination of biomarkers comprises at least one capture biomarker and at least one detection biomarker, wherein the at least one capture biomarker and the at least one detection biomarker are: (i) Polypeptides encoded by the following human genes: BST2, FOLR1, MSLN, MUC1, and MUC16; (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl Lewis A antigen (also known as CA19-9), and (iii) combinations thereof each independently selected from: (b) comparing the detected colocalization level to a reference level. wherein a detected co-localization level that is equal to or comparable to the reference level indicates that the female subject is negative for ovarian cancer; and wherein the detected co-localization level that is greater than the reference level indicates that the female subject is likely to have or be susceptible to ovarian cancer.

70. 70. The method of claim 69, wherein the nanoparticle is or comprises an extracellular vesicle.

71. A method described in claim 69 or 70, wherein the specificity of the indicator for detecting early ovarian cancer is within the range of 90% to 100% and the sensitivity is within the range of 80% to 95%.

72. 70. The method of claim 69, wherein the female subject has been determined to have normal plasma or serum CA-125 levels (e.g., less than or equal to 25 U / mL).

73. The detecting step comprises detecting co-localization of the at least one combination of biomarkers on the surface of the nanoparticle, wherein the at least one combination of biomarkers is selected from the following: (i) the sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; (ii) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; (iii) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; (iv) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; (v) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; (vi) a sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and (vii) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN 70. The method of claim 65 or 69, wherein the method is selected from one of the following:

74. The detecting step comprises detecting, on the surface of the nanoparticles, a combination of the following biomarkers: (i) the sialyl Lewis A antigen (also known as CA19-9) and the polypeptide encoded by the human gene BST2; (ii) a polypeptide encoded by the human gene MUC1 and a polypeptide encoded by the human gene BST2; (iii) the polypeptide encoded by the human gene MUC16 and the sialyl-Tn (sTn) antigen; (iv) a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene FOLR1; (v) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene FOLR1, and a polypeptide encoded by the human gene MUC1; (vi) a sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene BST2, and a polypeptide encoded by the human gene MUC1; and (vii) sialyl-Tn (sTn) antigen, a polypeptide encoded by the human gene MUC16, and a polypeptide encoded by the human gene MSLN 70. The method of claim 65 or 69, comprising detecting co-localization of each of:

75. the detecting step (a) capturing said nanoparticles from said blood-derived sample using a capture probe that selectively interacts with said at least one capture biomarker on said nanoparticles; (b) contacting the captured nanoparticles with at least one set of at least two detection probes, each of which selectively interacts with the at least one detection biomarker on the nanoparticles; and (c) detecting a product formed when said at least two detection probes of said set are in sufficient proximity on said individual nanoparticles.

70. The method of claim 65 or 69, comprising:

76. 76. The method of claim 75, wherein the capture probe comprises a target capture moiety that binds to the capture biomarker.

77. 77. The method of claim 76, wherein the target capture moiety is or comprises an antibody agent directed against the capture biomarker.

78. 76. The method of claim 75, wherein the capture biomarker is or comprises sialyl Lewis A antigen (also known as CA19-9), a polypeptide encoded by the human gene MUC1, a polypeptide encoded by the human gene MUC16, or a sialyl Tn (sTn) antigen.

79. 76. The method of claim 75, wherein the capture probe is or comprises a solid substrate comprising the target capture moiety conjugated to a solid substrate.

80. 80. The method of claim 79, wherein the solid substrate comprises magnetic beads.

81. The at least two detection probes are (i) a target binding moiety directed to one of the at least two detectable biomarkers; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain; The single-stranded overhang portions of the detection probes are characterized in that they can hybridize to each other when the detection probes are bound to the same nanoparticle.

76. The method of claim 75, each comprising:

82. 76. The method of claim 75, wherein the product is formed when the at least two detection probes of the set are in sufficient proximity on the individual nanoparticles such that the single-stranded overhang portions of the at least two detection probes of the set hybridize to one another to form a double-stranded complex.

83. 83. The method of Claim 82, wherein the formed product comprises a template that is ligated upon contacting the double-stranded complex with a nucleic acid ligase.

84. 76. The method of claim 75, wherein the target binding moieties of the at least two detection probes are each directed to the same detection biomarker.

85. 85. The method of claim 84, wherein the oligonucleotide domains of the at least two detection probes are different.

86. 85. The method of claim 84, wherein the same detectable biomarker is or comprises a polypeptide encoded by the human gene BST2.

87. 87. The method of claim 86, wherein the target capture portion of the capture agent is or comprises at least one antibody agent directed against the sialyl Lewis A antigen (also known as CA19-9) or against a polypeptide encoded by the human gene MUC1.

88. 85. The method of claim 84, wherein the same detectable biomarker is or comprises a sialyl-Tn (sTn) antigen.

89. 89. The method of claim 88, wherein the target capture portion of the capture agent is or comprises at least one antibody agent directed against a polypeptide encoded by the human gene MUC16.

90. 76. The method of claim 75, wherein the target binding moieties of the at least two detection probes are each directed to a distinct detection biomarker.

91. 91. The method of claim 90, wherein the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene BST2 and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene FOLR1.

92. 92. The method of claim 91, wherein the target capture portion of the capture agent is or comprises at least one antibody agent directed against a polypeptide encoded by the human gene MUC1.

93. 91. The method of claim 90, wherein the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene BST2 and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene MUC1.

94. 91. The method of claim 90, wherein the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene FOLR1, and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene MUC1.

95. 91. The method of claim 90, wherein the target binding portion of the first detection probe is directed to a polypeptide encoded by the human gene MUC16 and the target binding portion of the second detection probe is directed to a polypeptide encoded by the human gene MSLN.

96. 94. The method of claim 93, wherein the target capture portion of the capture agent is or comprises at least one antibody agent directed against a sialyl-Tn (sTn) antigen.

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