Pooling samples for screening diagnostics

Abstract

The invention provides a method for pooling samples prior to a diagnostic screen for a non-infectious disease. The method includes pooling only a portion of the obtained samples and storing the other portion for later use. Pooling samples represents a more time and cost-effective way to perform diagnostic screening.

Description

Field of the invention The invention relates to methods for pooling samples for screening diagnostics for non-infectious diseases. Background Screening diagnostics are useful to assess disease in many asymptomatic, average-risk populations. For example, colonoscopy is a common screening diagnostic for colorectal cancer, the Pap smear is used to screen average-risk populations for cervical cancer, and mammography is used to screen for breast cancer. In general, while screening diagnostics can be, and are, used to assess symptomatic individuals, the primary target population for screening diagnostics is of average risk and asymptomatic. A primary goal of screening diagnostics is the early detection of disease. Early detection increases the likelihood of successful treatment before a disease has advanced significantly. Screening can also inform lifestyle changes and early intervention to prevent disease and / or its advancement. A screening diagnostic test is generally not used for definitive diagnosis, but rather to identify individuals for further testing to determine the presence and / or exact nature of disease. Cancer is an example of a disease that benefits from early detection. If detected early, many cancers can be treated and prevented from metastasizing. According to Cancer Research UK, early detection of lung cancer results in about 60% survival; whereas late-stage detection is less than about 10% survival. Similarly, early-stage detection of colorectal cancer and breast cancer results in greater than 90% survival versus between 10 and 30% survival for late-state detection. Thus, it is clear that regular screening is critical to improve survivability of the most common cancers. In addition to cancer, screening diagnostics are available for heart disease, eye diseases, liver function, kidney function and many others. Typically, however, screening is done on an individual-by-individual basis for each of those conditions (e g., during an annual physical). Typical cancer screening is also done on an individual basis (e.g., Pap smear, mammography, colonoscopy). Infectious disease screening is also performed on an individual basis. However, in the field of infectious disease diagnostics, pooled samples are common to prevent transmission through the blood supply. For example, pooled testing for the virus that causes HIV in donated blood is common. Moreover, each year, more than 17 million units of blood are drawn in the US, and each one is examined for the blood-borne viruses Zika, West Nile, hepatitis B, hepatitis C, and HIV-1. It would be extremely costly and time-consuming to test every unit individually, especially since only a very small proportion of units are likely to be affected. Generally speaking, the positive rate for blood banking testing pools is less than 1% nationwide (though certain regions may have higher rates, particularly if a specific virus, like SARS-CoV-2, infects some people without symptoms). However, pooling of biological samples for cancer detection has not been done to screen populations for early cancer and / or minimal residual disease. There is, therefore, a need in the art for pooled screening diagnostics for cancer. Summary In general, the invention provides methods for screening for cancer and / or minimal residual disease in cancer by pooling biological samples obtained from individual donors. According to the invention, a tissue or body fluid sample is obtained from multiple individuals and combined for detection of biomarkers or analytes indicative of cancer or cancer recurrence. In a preferred embodiment, samples are obtained from an asymptomatic, average risk population. However, the invention is equally applicable to symptomatic populations or mixed populations. In one aspect, the invention comprises pooling samples from a plurality of individuals who have previously been diagnosed with cancer for the purpose of detecting minimal residual disease and / or recurrence monitoring. In preferred methods, the invention comprises obtaining tissue or body fluid samples from a plurality of individuals. The samples are divided into two portions, one of which is pooled with samples from other members of the plurality and one of which is stored individually. Then the pooled sample is tested for one or more types of cancer. If the pooled sample is positive, then each individual stored sample that comprised the pool is tested separately for the same cancer or cancers to identify individual members of the plurality who screen positive. If the pooled sample is negative for the indicated screen, then all members of the plurality who make up that pooled sample are considered negative for the indicated screen. In another embodiment, obtained samples are divided into three or more aliquots and if a pooled sample of first aliquots from the plurality of patients is screened positive, second or subsequent aliquots can be further divided and screened to narrow the number of potentially positive individual screens, always keeping a stored individual sample from each member of the plurality for individual testing in the event of a positive pooled screen. Any tissue or body fluid sample is useful in practice of the invention. Exemplary samples include blood, urine, cerebrospinal fluid, lymph, and saliva. In certain embodiments, the biological sample is a liquid sample that has been subjected to, for example, size exclusion chromatography to isolate extracellular vesicles. In certain other embodiments, the sample material is immobilized on a solid substrate. For example, the solid substrate can be a bead, including magnetic beads, cellulose, silica, gold, or an organic polymer, among others. Methods of the invention also contemplate an ideal size of the pool. In general, the size of the pool will depend on the number of individuals being screened, the prevalence of the type or types of cancer being screened in a subject population, and the sensitivity and / or specificity of the screen. While the exact size of the pool will depend on the factors mentioned in the previous sentence, in any case pooling will result in efficiencies in terms of the timing and number of screening tests that need to be run to identify positive individuals. Numerous screening assays are useful in practice of the invention. For example, pooled sample are assayed to detect the presence of nucleic acid or protein biomarkers indicative of cancer. In one aspect, the invention contemplates the detection of cell-free nucleic acids in the sample, such as blood. Nucleic acid detection in pooled samples involves amplification of DNA (including cDNA from RNA in the sample), hybridization-based approaches or alternatives to traditional PCR, including but not limited to rolling circle amplification, qPCR, loop-mediated isothermal amplification, ligase chain reaction, and CRISPR-based methods. In other aspects, the invention provides methods for screening that involve the detection of proteins in the pooled sample. Protein biomarkers can exist free in the sample or on the surface of cells or extracellular vesicles. The detection of proteins, peptides and the like is accomplished using methods known in the art, including but not limited to, antibody- based detection, including labeled antibodies. A preferred embodiment utilizes the Mercy Halo test in pooled samples. The Mercy Halo test is a cancer-detection assay that detects extracellular vesicles (EVs) that have been shed from tumor cells and is described in co-pending, co-owned patent applications, including serial numbers 18 / 465,361, 18 / 015,051, 17 / 793,382, 17 / 493,259, 17 / 435,697, 17 / 204,773, and 16 / 805,637, incorporated by reference herein. Extracellular vesicles are known to exist in high-abundance, even in early-stage cancer. They carry surface proteins unique to their tumor cell of origin which are used to determine if an individual is positive for the screened disease. Only a small amount of blood is required from the individual to perform the screen, so upon obtaining a sample, a first portion is pooled with other samples to run the pooled test while another portion is stored for later use in the event a positive screen is determined on the pooled sample. If the screen comes back negative, then all individuals in the pool can be considered negative. However, if the screen comes back positive, then the stored samples may be run independently to determine the individual(s) who are positive for the disease. Other aspects and advantages of the present invention are apparent upon consideration of the following detailed description thereof. Detailed Description The present invention discloses methods for pooling samples from a plurality of individuals for diagnostic screening. Diagnostic screening is used to look for as-yet-unrecognized conditions or risk markers. Diagnostic screening can be applied to individuals or to a whole population without symptoms or signs of the disease being screened. Methods for diagnostic screening are designed to identify conditions which could at some future point turn into disease, thus enabling earlier intervention and management in the hope to reduce mortality and suffering from a disease. Embodiments of the present invention utilize pooled samples to detect disease biomarkers. Pooled samples are used for diagnostic screening approaches by combining samples from some number of individuals into one assay. The pooled sample is assayed first. If negative, all members of the pool can be given a negative result, saving the cost of testing each individual one at a time. If the pool tests positive, however, then each individual sample within the pool must be re-tested to identify who within the pool is actually causing the pool test to be positive. In a preferred embodiment, samples of a tissue or body fluid are obtained from an individual and separated into a first portion and a second portion. The first portion is pooled with samples from other individuals to create the pooled sample. The second portion is stored for later use. Once the appropriate number of samples have been gathered to create the pooled sample, various methods may be used for performing an assay and detecting a positive or negative screen for the presence of a disease biomarker in the pooled sample. Disease biomarkers include, but are not limited to, DNA, RNA, protein, lipids, carbohydrates, single-nucleotide polymorphisms (SNPs), protein specific antigens (PSA), structural variants, specific cell types, antigens / antibodies, and the like. In a preferred embodiment, the pooled sample is screened using the Mercy Halo test. The Mercy Halo test is designed to detect the co-occurrence of two or more disease biomarkers (e.g., cell-surface proteins, nucleic acids, and the like) that are associated with extracellular vesicles released from tumor cells. In certain embodiments, the Mercy Halo test comprises isolation of extracellular vesicles associated with cancer using, for example, a capture reagent, such as antibody-functionalized beads. In that case, the isolated extracellular vesicles are incubated with a plurality of dsDNA oligo-conjugated detection antibodies having DNA comprising a singlestrand overhang, such that members of the plurality have a single-stranded overhang that is complementary to that of a second dsDNA oligo-conjugated detection antibodies in the plurality. Complementary dsDNA oligo-conjugated detection antibodies bound to the same extracellular vesicle are sufficiently close to hybridize and then be ligated. The ligation products are detected using qPCR and a positive signal resulting from the co-occurrence of disease markers is indicative of a positive screen. Extracellular vesicles (EVs) generally include lipid bilayer-bound particles that are naturally released from almost all types of cells but, unlike a cell, cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 2030 nanometers) to as large as 10 microns or more, although the vast majority of EVs are smaller than 200 nm. EVs can be divided according to size and synthesis route into exosomes, microvesicles and apoptotic bodies. They carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. Most cells that have been studied to date are thought to release EVs, including some archaeal, bacterial, fungal, and plant cells that are surrounded by cell walls. In some embodiments in which a disease biomarker is or comprises a surface protein marker and / or an intravesicular protein marker, the co-occurrence of two or more disease biomarkers may be detected using, for example, a proximity ligation assay. The proximity ligation assay may comprise contacting sample material comprising extracellular vesicles with a set of oligo-linked probes (e.g., dsDNA oligo-conjugated detection antibodies), each directed to a disease marker. The set of oligo-linked probes (e.g., dsDNA oligo-conjugated detection antibodies) comprises at least two distinct probes so that a combination comprising the extracellular vesicles and the set of probes is generated. Generally, the two probes each comprise: (i) a target binding moiety directed to a surface protein marker and / or an intravesicular protein marker; 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 extended from one end of the oligonucleotide domain. Such single-stranded overhang portions of the probes are characterized in that they can hybridize to each other when the probes are bound to the same extracellular vesicle. Such a combination comprising the extracellular vesicles and the set of probes is then maintained under conditions that permit binding of the set of probes to their respective targets on the extracellular vesicles such that the probes can bind to the same extracellular vesicle to form a double-stranded complex. The double-stranded complex is detected by contacting it with a nucleic acid ligase to generate a covalently contiguous ligation product (e.g., ligated dsDNA oligos); and detecting the covalently contiguous ligation product (e.g., ligated dsDNA oligos). The ligation product (e.g., ligated dsDNA oligos) is detected by amplification or sequencing. In a certain embodiment, the ligated dsDNA oligos serve as templates for qPCR. In a certain embodiment, the amplification is PCR and may be digital PCR, qPCR and the like, in each case that detects the ligation product using fluorescent hydrolysis probes. The presence of a covalently contiguous ligation product is indicative of the presence of extracellular vesicles that are positive for a marker of a disease. While a proximity ligation assay described above may perform better, e.g., with higher specificity and / or sensitivity than other proximity ligation assays, a person skilled in the art reading the present disclosure will appreciate that other forms of proximity ligation assays that are known in the art may be used in addition to other means for detecting extracellular vesicle-related markers. A pooled sample determined to be positive for the presence of cancer biomarkers is determined to be positive for cancer. Subsequently, the stored samples of each of the corresponding individuals may be tested to determine the specific individual(s) positive for the biomarker. In another embodiment, the stored samples are further divided into aliquots to create smaller pools. These smaller pools are then screened for the presence of the disease biomarker to narrow the number of potentially positive individual screens. In case of a positive screen, a stored individual sample from each member of the plurality is further individually tested. The present invention further contemplates detecting multiple cancer types in a single screen. For example, multiple disease-specific biomarker detection antibodies may be used in a single screen, each corresponding to a disease biomarker that is specific to a certain cancer type. The number of samples in a pool may depend on the number of individuals being screened, the prevalence of the type or types of cancer being screened in a subject population, and the sensitivity and / or specificity of the screen. Various methods may be employed to determine an optimal pool size. In certain embodiments, the disease biomarker to be detected is a nucleic acid. Detection is accomplished by any suitable method, for example PCR, qPCR, sequencing, probe hybridization, ligase chain reaction, multiplex PCR, and others. Nucleic acid can be extracted using known methods, such as suspension in an extraction buffer (e g., phosphate-buffered saline or EDTA) followed by cell lysis (e.g., with proteinase K) and nucleic acid is extracted using commercially-available kits, such as the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia CA). In a preferred embodiment, the disease biomarkers are markers for cancer, such as bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, Hodgkin lymphoma, kidney cancer, liver cancer, lung cancer, multiple myeloma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcomas, skin cancer, and stomach cancer. In some embodiments, sample material is obtained or derived from a biological source (e g., a tissue or organism or cell culture) of interest. In some embodiments, a source of interest may be or comprise a cell or an organism, such as an animal or human. In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravesicular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological tissue or sample material may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample material is or comprises a bodily fluid sample material or a bodily fluid-derived sample material. Examples of a bodily fluid sample material or a bodily fluid-derived sample material include, but are not limited to an amniotic fluid, bile, blood, breast milk, bronchoalveolar lavage fluid (BAL), cerebrospinal fluid, dialysate, feces, saliva, semen, synovial fluid, tears, urine, etc. In some embodiments, a bodily fluid sample material or a bodily fluid-derived sample material that may be useful in accordance with the present disclosure is or comprises a blood-derived sample, a saliva-derived sample, a sputum-derived sample, or a pleural effusion-derived sample. In some embodiments, a biological sample material is or comprises a liquid biopsy. In some embodiments, a biological sample material is or comprises cells obtained from an individual. In one example, a blood plasma or serum sample (4ml) is processed for analysis of extracellular vesicles and nucleic acids, both those associated with an extracellular vesicle and those not associated with the extracellular vesicle. In general, when the sample is blood, methods of the disclosure work with samples of approximately 4 to 10 mL of blood, as is commonly collected in blood collection tubes. Typical workflows involve centrifugation and capture of the supernatant plasma, which typically yields about 3 to 5 mL of plasma. In some embodiments, extracellular vesicles in a sample may be captured or immobilized on a solid substrate prior to detecting one or more provided markers in accordance with the present disclosure. In some embodiments, extracellular vesicles may be captured on a solid substrate surface by non-specific interaction, including, e.g., adsorption. In some embodiments, extracellular vesicles may be selectively captured on a solid substrate surface. For example, in some embodiments, a solid substrate surface may be coated with an agent that specifically binds to extracellular vesicles (e.g., an antibody agent specifically targeting such extracellular vesicles, e.g., associated with cancer). In some embodiments, a solid substrate surface may be coated with a member of an affinity binding pair and an entity of interest (e.g., extracellular vesicles) to be captured may be conjugated to a complementary member of the affinity binding pair. In some embodiments, an exemplary affinity binding pair includes, e.g., but is not limited to biotin and avidin-like molecules such as streptavidin. As will be understood by those of skilled in the art, other appropriate affinity binding pairs can also be used to facilitate capture of an entity of interest to a solid substrate surface. In some embodiments, an entity of interest may be captured on a solid substrate surface by application of a current, e.g., as described in Ibsen et al. ACS Nano., 11: 6641-6651 (2017) and Lewis et al. ACS Nano., 12: 3311-3320 (2018), both of which are incorporated herein by reference for the purpose described herein, and both of which describe use of an alternating current electrokinetic microarray chip device to isolate extracellular vesicles from an undiluted human blood or plasma sample. A solid substrate may be provided in a form that is suitable for capturing extracellular vesicles and does not interfere with downstream handling, processing, and / or detection. For example, in some embodiments, a solid substrate may be or comprise a bead (e.g., a magnetic bead). In some embodiments, a solid substrate may be or comprise a surface. For example, in some embodiments, such a surface may be a capture surface of an assay chamber (including, e.g., a tube, a well, a microwell, a plate, a filter, a membrane, a matrix, etc.). Accordingly, in some embodiments, a method described herein comprises, prior to detecting provided markers in a sample, capturing or immobilizing extracellular vesicles on a solid substrate. In some embodiments, a sample may be processed, e.g., to remove undesirable entities such as cell debris or cells, prior to capturing extracellular vesicles on a solid substrate surface. For example, in some embodiments, such a sample material may be subjected to centrifugation, e.g., to remove cell debris, cells, and / or other particulates. Additionally, or alternatively, in some embodiments, such a sample material may be subjected to size-exclusion-based purification or filtration. Various size-exclusion-based purification or filtration are known in the art and those skilled in the art will appreciate that in some cases, a sample may be subjected to a spin column purification based on specific molecular weight or particle size cutoff. Those skilled in the art will also appreciate that appropriate molecular weight or particle size cutoff for purification purposes can be selected, e.g., based on the size of extracellular vesicles. For example, in some embodiments, size-exclusion separation methods may be applied to sample materials comprising extracellular vesicles to isolate a fraction of extracellular vesicles of a certain size (e.g., greater than 30 nm and no more than 1000 nm, or greater than 70 nm and no more than 200 nm). Typically, extracellular vesicles may range from 30 nm to several micrometers in diameter. See, e.g., Chuo et al., “Imaging extracellular vesicles: current and emerging methods” Journal of Biomedical Sciences 25: 91 (2018) which is incorporated herein by reference for the purpose described herein, which provides information of sizes for different extracellular vesicle (EV) subtypes: migrasomes (0.5-3 pm), microvesicles (0.1-1 pm), oncosomes (1-10 pm), exomeres (<50 nm), small exosomes (60-80 nm), and large exosomes (90-120 nm). In some embodiments, specific EV subtype(s) may be isolated, for example, in some embodiments by one or more sizeexclusion separation methods, for detection assay. The present disclosure, among other things, provides insights and technologies for achieving effective cancer screening, e.g., for early detection of cancer (e.g., in some embodiments characterized by carcinoma, sarcoma, mixed types, etc.). In some embodiments, the present disclosure provides technologies for early detection of cancer in subjects who may be experiencing one more symptoms associated with cancer. In some embodiments, the present disclosure provides technologies for early detection of cancer in subjects who are at hereditary risks for cancer. In some embodiments, the present disclosure provides technologies for early detection of cancer in subjects who may be at hereditary risk and / or experiencing one or more symptoms associated with cancer. In some embodiments, the present disclosure provides technologies for early detection of cancer in subjects who may have life-history risk factors. In some embodiments, the present disclosure provides technologies for screening individuals, e.g., individuals with certain risks (e.g., hereditary risk, life history associated risk, or average risk) for early stage cancer (e.g., in some embodiments characterized by carcinoma, sarcoma, mixed types, etc.)). In some embodiments, provided technologies are effective for detection of early stage cancer (e.g., in some embodiments characterized by carcinoma, sarcoma, melanoma, and mixed types). In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals having one or more symptoms that may be associated with cancer. In some embodiments, provided technologies are effective even when applied to populations comprising or consisting of asymptomatic or symptomatic individuals (e.g., due to sufficiently high sensitivity and / or low rates of false positive and / or false negative results). In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals (e.g., asymptomatic or symptomatic individuals) without hereditary risk, and / or life-history related risk of developing cancer. In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals (e.g., asymptomatic or symptomatic individuals) with hereditary risk for developing cancer. In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals susceptible to cancer (e.g., individuals with a known genetic, environmental, or experiential risk, etc.). In some embodiments, provided technologies may be or include one or more compositions (e.g., molecular complexes, systems, collections, combinations, kits, etc.) and / or methods (e.g., of making, using, assessing, etc.), as will be clear to one skilled in the art reading the disclosure provided herein. In some embodiments, provided technologies achieve detection (e.g., early detection, e.g., in asymptomatic individual(s) and / or population(s)) of one or more features (e.g., incidence, progression, responsiveness to therapy, recurrence, etc.) of cancer, with sensitivity and / or specificity (e.g., rate of false positive and / or false negative results) appropriate to permit useful application of provided technologies to single-time and / or regular (e.g., periodic) assessment. In some embodiments, provided technologies are useful in conjunction with an individual’s regular medical examinations, such as but not limited to: physicals, general practitioner visits, cholesterol / lipid blood tests, diabetes screening (e.g., diabetes (type 2) screening), colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV / Pap smears, and / or vaccinations. In some embodiments, provided technologies are useful in conjunction with treatment regimen(s); in some embodiments, provided technologies may improve one or more characteristics (e.g., rate of success according to an accepted parameter) of such treatment regimen(s). In some embodiments, the present disclosure, among other things, provides insights that screening of asymptotic individuals, e.g., regular screening prior to or otherwise in absence of developed symptom(s), can be beneficial, and even important for effective management (e.g., successful treatment) of cancer. In some embodiments, the present disclosure provides cancer screening systems that can be implemented to detect cancer, including early-stage cancer, in some embodiments in asymptomatic individuals (e.g., without hereditary, and / or life-history associated risks in cancer). In some embodiments, provided technologies are implemented to achieve regular screening of asymptomatic individuals (e.g., with or without hereditary risk(s) in cancer). In some embodiments, provided technologies are implemented to achieve regular screening of symptomatic individuals (e.g., with or without hereditary and / or life-history associated risk(s) in cancer). The present disclosure provides, for example, compositions (e.g., reagents, kits, components, etc.), and methods of providing and / or using them, including strategies that involve regular testing of one or more individuals (e.g., asymptomatic individuals). The present disclosure defines usefulness of such systems and provides compositions and methods for implementing them. In some aspects, provided are technologies for use in classifying a subject (e.g., an asymptomatic subject) as having or being susceptible to cancer (e.g., carcinoma, sarcoma, mixed types, etc.). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or being susceptible to cancer (e.g., carcinoma, sarcoma, mixed types, etc.). In some embodiments, a provided method or assay comprises assaying sample material (e.g., from a blood-derived sample) from a subject for a plurality of distinct disease marker combinations to determine in the sample material (e.g., from a blood-derived sample) whether extracellular vesicles display at least a marker combination from the plurality (e.g., co-occurrence of at least two markers), wherein the plurality of marker combinations each independently comprises at least two markers, whose combined expression level has been determined to be associated with at least one type of cancer (including, e.g., at least two types of cancer). The present disclosure, among other things, also provides technologies for determining whether a subject as having or being susceptible to cancer, for example, from a sample material that includes extracellular vesicles. For example, in some embodiments, when a biological sample material (e.g., a bodily fluid sample material from, e.g., but not limited to, a blood-derived sample) from a subject in need thereof shows a level of marker combination-expressing extracellular vesicles that is at or above a reference threshold level, e.g., cutoff value (e.g., as determined in accordance with the present disclosure), then the subject is classified as having or being susceptible to cancer. In some such embodiments, a reference threshold level (e.g., cutoff value) may be determined based on a log-normal distribution around healthy subjects (e.g., of specified age ranges), and optionally subjects with inflammatory conditions that are associated with tissues of interest but that are not cancerous (including, e.g., atherosclerosis, heart disease, chronic kidney disease, diabetes, inflammatory bowel disease, fatty liver disease, chronic obstructive pulmonary disease, endometriosis, rheumatoid arthritis, obesity, pancreatitis etc.) and selection of a level that is necessary to achieve the specificity of interest, e.g., based on prevalence of cancer or a subtype thereof (e.g., in some embodiments characterized by carcinoma, sarcoma, melanoma, and mixed types). In some embodiments, specificity of interest may be at least 70%, including, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or higher. In some embodiments, a reference threshold level (e.g., a cutoff value) may be determined based on expression level (e.g., transcript level) of individual target marker(s) of a marker combination in normal healthy tissues vs. in cancer samples such that the specificity and / or sensitivity of interest (e.g., as described herein) can be achieved. In some embodiments, a reference threshold level (e.g., a cutoff value) may vary dependent on, for example, cancer stages and / or subtypes and / or patient characteristics, for example, patient age, risks factors for cancer (e.g., hereditary risk vs. average risk, life-history-associated risk factors), symptomatic / asymptomatic status, and combinations thereof. In some embodiments, when a biological sample material from a subject in need thereof shows a level of marker combination that satisfies a reference threshold level, then the subject is classified as having or being susceptible to cancer. For example, in some embodiments, when a biological sample material (e.g., a bodily fluid sample material from, e.g., but not limited to, a blood-derived sample) from a subject in need thereof shows an elevated level of marker combination-expressing extracellular vesicles relative to a reference threshold level, then the subject is classified as having or being susceptible to cancer. In some embodiments, a subject in need thereof is classified as having or being susceptible to cancer when the subject’s biological sample (e.g., a bodily fluid sample such as, e.g., but not limited to a blood-derived sample) shows a level of marker combination-expressing extracellular vesicles that is at least 30% or higher, including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher, as compared to a reference threshold level. In some embodiments, a subject in need thereof is classified as having or being susceptible to cancer when the subject’s biological sample material (e.g., a bodily fluid sample material, e.g., but not limited to, from a blood-derived sample) shows a level of marker combination-expressing extracellular vesicles that is at least 2-fold or higher, including, e g., at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750fold, at least 1000-fold, or higher, as compared to a reference threshold level. When a biological sample material (e.g., a bodily fluid sample material from, e.g., but not limited to, a blood-derived sample) from a subject in need thereof shows a comparable level to a reference threshold level, then the subject is classified as not likely to have or as not likely to be susceptible to cancer. In some such embodiments, a reference threshold level corresponds to a level of extracellular vesicles that express a marker combination in comparable samples from a population of reference subjects, e.g., non-cancer subjects. In some embodiments, exemplary non-cancer subjects include healthy subjects (e.g., healthy subjects of specified age ranges, such as e.g., below age 55 or above age 55), subjects with non-tumor related health diseases, disorders, or conditions (including, e.g., subjects having symptoms of cancerous diseases or disorders but not cancer), subjects having benign tumors, and combinations thereof. In some embodiments, an oligo-linked probe as provided and / or utilized herein comprises a target-binding moiety and an oligonucleotide domain coupled to the target-binding moiety. In some embodiments, an oligonucleotide domain coupled to a target-binding moiety may comprise a double-stranded portion and a single-stranded overhang extended from at least one end of the oligonucleotide domain. In some embodiments, an oligonucleotide domain coupled to a targetbinding moiety may comprise a double-stranded portion and a single-stranded overhang extended from each end of the oligonucleotide domain. A target-binding moiety that is coupled to an oligonucleotide domain is an entity or an agent that specifically binds to a target (e.g., a provided marker of a marker combination; those skilled in the art will appreciate that, where the target marker is a particular form or moiety / component, the target-binding moiety specifically binds to that form or moi ety / component). In some embodiments, a target-binding moiety may be or comprise an agent of any chemical class such as, for example, a carbohydrate, a nucleic acid, a lipid, a metal, a polypeptide, a small molecule, etc., and / or a combination thereof. In some embodiments, a target-binding moiety may be or comprise an affinity agent such as an antibody, affimer, aptamer, lectin, siglec, etc. In some embodiments, a target-binding moiety is or comprises an antibody agent, e.g., an antibody agent that specifically binds to a target or an epitope thereof, e.g., a provided marker of a marker combination for cancer or an epitope thereof. In some embodiments, an oligo-linked probe comprising an oligonucleotide domain coupled to a targetbinding moiety wherein the target-binding domain comprises an antibody and wherein the oligonucleotide domain comprises a double-stranded portion is referred to as a dsDNA oligoconjugated detection antibody. In some embodiments, a target-binding moiety is or comprises a lectin or siglec that specifically binds to a carbohydrate-dependent marker as provided herein. In some embodiments, a target-binding moiety for a provided marker may be a commercially available. In some embodiments, a target-binding moiety for a provided marker may be designed and created for the purpose of use in assays as described herein. In some embodiments, a targetbinding moiety is or comprises an aptamer, e.g., an aptamer that specifically binds to a target or an epitope thereof, e.g., a provided marker of a marker combination for cancer or an epitope thereof. In some embodiments, a target-binding moiety is or comprises an affimer molecule that specifically binds to a target or an epitope thereof, e.g., a provided marker of a marker combination for cancer or an epitope thereof. In some embodiments, such an affimer molecule can be or comprise a peptide or polypeptide that binds to a target or an epitope thereof (e.g., as described herein) with similar specificity and affinity to that of a corresponding antibody. In some embodiments, a target may be or comprise a target that is associated with cancer. For example, in some such embodiments, a cancer-associated target can be or comprise a target that is associated with more than one cancer (i.e., at least two or more cancers). In some embodiments, a cancer-associated target can be or comprise a target that is typically associated with cancers. In some embodiments, a cancer-associated target can be or comprise a target that is associated with cancers of a specific tissue, e.g., cancer. In some embodiments, a cancer-associated target can be or comprise a target that is specific to a particular cancer, e.g., a particular cancer and more specifically in some embodiments characterized by carcinoma, sarcoma, melanoma, and mixed types. In some embodiments, a target-binding moiety recognizes and specifically binds to a target present in a biological entity (including, e.g., but not limited to cells and / or extracellular vesicles). For example, in some embodiments, a target-binding moiety may recognize and specifically bind to a tumor-associated antigen or epitope thereof. In some embodiments, a tumor-associated antigen may be or comprise an antigen that is associated with a cancer such as, for example, skin cancer, brain cancer (including, e.g., glioblastoma), breast cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, etc. In some embodiments, a target-binding moiety may recognize a tumor antigen associated with cancer (e.g., in some embodiments characterized by carcinoma, sarcoma, melanoma, and mixed types). In some embodiments, a target-binding moiety may recognize a tumor antigen associated with in some embodiments characterized by carcinoma, sarcoma, melanoma, and mixed types. In some embodiments, a target-binding moiety may specifically bind to an intravesicular target, e.g., a provided intravesicular protein or RNA (e.g., mRNA). In some embodiments, a target-binding moiety may specifically bind to a surface target that is present on / within extracellular vesicles, e.g., a membrane-bound polypeptide present on cancer-associated extracellular vesicles. In some embodiments, a target-binding moiety is directed to a marker for a specific condition or disease (e.g., cancer), which marker is or has been determined, for example, by analyzing a population or library (e.g., tens, hundreds, thousands, tens of thousands, hundreds of thousands, or more) of patient biopsies and / or patient data to identify such a marker (e.g., a predictive marker). In some embodiments, a relevant marker may be one identified and / or characterized, for example, via data analysis. In some embodiments, for example, a diverse set of data (e.g., in some embodiments comprising one or more of bulk RNA sequencing, single-cell RNA (scRNA) sequencing, mass spectrometry, histology, post-translational modification data, in vitro and / or in vivo experimental data) can be analyzed through machine learning and / or computational modeling to identify markers (e.g., predictive markers) that are highly specific to a disease or condition (e.g., cancer). In some embodiments, a target-binding moiety is directed to a tissue-specific target, for example, a target that is associated with a specific tissue such as, for example, brain, breast, colon, ovary and / or other tissues associated with a female reproductive system, pancreas, prostate and / or other tissues associated with a male reproductive system, liver, lung, and skin. In some embodiments, such a tissue-specific target may be associated with a normal healthy tissue and / or a diseased tissue, such as a tumor. In some embodiments, a target-binding moiety is directed to a target that is specifically associated with a normal healthy condition of a subject. In some embodiments, a target-binding moiety may recognize a tissue specific antigen. In some embodiments, individual target binding entities utilized in a plurality of oligolinked probes (e.g., as described and / or utilized herein) are directed to different targets. In some embodiments, such different targets may represent different marker proteins or polypeptides. In some embodiments, such different targets may represent different epitopes of the same marker proteins or polypeptides. In some embodiments, two or more individual target binding entities utilized in a plurality of oligo-linked probes (e.g., as described and / or utilized herein) may be directed to the same target. In some embodiments, individual target binding entities utilized in a plurality of oligolinked probes for detection of cancer may be directed to different target markers of a marker combination for cancer. In some embodiments, individual target binding entities utilized in a plurality of oligolinked probes for detection of cancer may be directed to the same target marker of a marker combination for cancer. In some embodiments, such target binding entities may be directed to the same or different epitopes of the same target marker of such a marker combination for cancer. In some embodiments, an oligonucleotide domain for use in accordance with the present disclosure (e.g., that may be coupled to a target-binding moiety) may comprise a double-stranded portion and a single-stranded overhang extended from one or both ends of the oligonucleotide domain. In some embodiments, where an oligonucleotide domain comprises a single-stranded overhang extended from each end a single-stranded overhang is extended from a different strand of a double-stranded portion. In some embodiments, where an oligonucleotide domain comprises a single-stranded overhang extended from one end of the oligonucleotide domain the other end of the oligonucleotide domain may be a blunt end. In some embodiments, an oligonucleotide domain may comprise ribonucleotides, deoxyribonucleotides, synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions, and any combinations thereof. In some embodiments, an oligonucleotide domain is or comprises DNA. In some embodiments, an oligonucleotide domain is or comprises peptide nucleic acid (PNA). A single-stranded overhang of an oligonucleotide domain is designed to comprise a nucleotide sequence that is complementary to at least a portion of a single-stranded overhang of a second oligo-linked probe such that a double-stranded complex comprising a first oligo-linked probe and a second oligo-linked probe can be formed through hybridization of the complementary single-stranded overhangs. In some embodiments, nucleotide sequences of complementary single-stranded overhangs are selected for optimal ligation efficiency in the presence of an appropriate nucleic acid ligase. In some embodiments, a single-stranded overhang has a nucleotide sequence preferentially selected for efficient ligation by a specific nucleic acid ligase of interest (e.g., a DNA ligase such as a T4 or T7 ligase). For example, such a singlestranded overhang may have a nucleotide sequence of GAGT, e.g., as described in Song et al., “Enzyme-guided DNA sewing architecture” Scientific Reports 5: 17722 (2015), which is incorporated herein by reference for the purpose described herein. In other examples, blunt-end ligation is used. In some embodiments, a plurality of oligonucleotides (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least ten, or more) can be coupled or conjugated to a target-binding moiety (e.g., a target binding antibody agent). The present invention provides a method for pooling samples prior to a diagnostic screen for a non-infectious disease. The method includes pooling only a portion of the obtained samples and storing the other portion for later use. Pooling samples represents a more time and costeffective way to perform diagnostic screening. Regardless of the number of samples or assay used, pooling samples provides a more efficient and cost-effective way to screen average risk populations. To illustrate, consider the following: A 0.03% active disease prevalence implies approximately 3 active cases in a 1,000 person population. If 1,000 people are tested in 20 pools of 50 each, and all the positive patients end up alone in their pools, then 3 of the 20 pools will test positive. If the cases are clustered, as is typical, the number of positive pools would be lower. In this case, 20 pools are tested with one test each. Then, all of the participants in the 3 positive pools would be tested individually, totaling 150 tests. In the end, 170 tests would have been performed to get results for 1,000 people. This amounts to an 83 percent reduction in the number of tests performed when compared to traditional, non-pooling screening methods.

Claims

1. A method for diagnostic screening, the method comprising the steps of:pooling a first portion of a tissue or body fluid sample obtained from a plurality of individuals to create a pooled sample;separately storing a second portion of the tissue or body fluid sample;performing an assay on the pooled sample to identify presence of a disease biomarker;identifying all members of the plurality as negative if the biomarker is not detected in the pooled sample; andperforming the assay on each stored sample if the biomarker is detected in the pooled sample, thereby to identify members of the plurality who are positive for presence of the biomarker.

2. The method of claim 1, wherein the tissue or body fluid sample is blood, serum, or plasma.

3. The method of claim 1, wherein the biomarker is a cell-surface protein that can occur on extracellular vesicles released from tumor cells.

4. The method of claim 1, wherein the disease is cancer.

5. The method of claim 1, wherein the assay performed uses size exclusion chromatographyto purify extracellular vesicles extracted from the tissue or body fluid samples.

6. The method of claim 5, wherein extracellular vesicles are subjected to immunoaffinity capture with disease-specific biomarker antibodies conjugated to magnetic beads.

7. The method of claim 6, wherein up to two additional disease-specific biomarkers are detected using complementary dsDNA oligo-conjugated detection antibodies.

8. The method of claim 7, wherein DNA ligase ligates the complementary dsDNA oligos between biomarkers in proximity on the same extracellular vesicle.

9. The method of claim 8, wherein the ligated dsDNA oligos serve as templates for qPCR.

10. The method of claim 1, wherein the size of the pool is determined based on the diseasebeing screened for.