Sample pooling for screening diagnostics

Abstract

The invention provides a method for sample pooling prior to diagnostic screening for non-infectious diseases. The method comprises pooling only a portion of the obtained sample and storing another portion for later use. Sample pooling represents a more time and cost efficient way of performing diagnostic screening.

Description

Technical Field

[0001] This invention relates to a method for pooling samples for screening and diagnosis of non-infectious diseases. Background Technology

[0002] Screening diagnostics are applicable for assessing disease in many asymptomatic, average-risk groups. For example, colonoscopy is a common screening diagnostic for colorectal cancer, cervical smears are used to screen for cervical cancer in average-risk groups, and mammography is used to screen for breast cancer. Generally, while screening diagnostics can and is being used to assess symptomatic individuals, the primary target group for screening diagnostics is the average-risk, asymptomatic population.

[0003] The primary goal of screening diagnosis is early detection of disease. Early detection increases the likelihood of successful treatment before significant disease progression. Screening can also inform lifestyle changes and early interventions to prevent disease and / or its progression. Screening diagnostic tests are typically not used for definitive diagnosis but rather to identify individuals for further testing to determine the presence and / or exact nature of the disease.

[0004] Cancer is an example of a disease that benefits from early detection. Many cancers can be treated and their metastasis prevented if detected early. According to Cancer Research UK, the survival rate for lung cancer when detected early is around 60%, while the survival rate for late-stage detection is less than around 10%. Similarly, the survival rate for colorectal and breast cancer when detected early is greater than 90%, while the survival rate for late-stage detection is between 10% and 30%. Therefore, it is clear that regular screening is crucial for improving survival rates for the most common cancers.

[0005] Besides cancer, screening can be used for heart disease, eye disease, liver function, kidney function, and many other conditions. However, screening is typically performed on an individual basis for each of those conditions (e.g., during an annual physical examination). Typical cancer screening is also performed on an individual basis (e.g., cervical smears, mammograms, colonoscopies).

[0006] Infectious disease screening is also conducted on an individual basis. However, in the field of infectious disease diagnosis, pooled samples are common to prevent transmission through the blood supply. For example, pooled testing for HIV-causing viruses in donated blood is common. Furthermore, over 17 million units of blood are drawn annually in the United States, and each is tested for bloodborne viruses such as Zika, West Nile, hepatitis B virus, hepatitis C virus, and HIV-1. Testing each unit individually would be extremely expensive and time-consuming, especially since only a very small percentage of units might be infected. Generally, the positivity rate in blood bank testing pools nationwide is less than 1% (although some areas may have higher rates, especially if certain viruses such as SARS-CoV-2 infect some people without causing symptoms).

[0007] However, biosample pooling for cancer detection to screen populations for early-stage cancer and / or minimal residual disease has not yet been implemented. Therefore, there is a need in the art for pooled screening diagnostics of cancer. Summary of the Invention

[0008] Generally, the present invention provides a method for screening cancer and / or minimal residual disease in cancer by combining biological samples obtained from individual donors. According to the invention, tissue or bodily fluid samples are obtained from multiple individuals and combined for the detection of biomarkers or analytes indicating cancer or cancer recurrence. In a preferred embodiment, samples are obtained from an asymptomatic, average-risk group. However, the invention is equally applicable to symptomatic groups or mixed groups. In one aspect, the invention includes combining samples from multiple individuals previously diagnosed with cancer for the detection of minimal residual disease and / or recurrence monitoring.

[0009] In a preferred method, the invention includes obtaining tissue or bodily fluid samples from multiple individuals. The samples are divided into two portions, one portion being merged with samples from other members of the multiple individuals, and the other portion being stored separately. The merged sample is then tested for one or more cancer types. If the merged sample is positive, the same one or more cancers are tested separately for each individually stored sample constituting the pool to identify individual members of the multiple individuals who tested positive for the specified screening. If the merged sample is negative for a specified screening, then all members of the multiple individuals constituting the merged sample are considered negative for the specified screening.

[0010] In another embodiment, the obtained samples are divided into three or more aliquots, and if the combined sample screening of the first aliquot from multiple patients is positive, then the second or subsequent aliquots can be further divided and screened to reduce the number of potentially positive individuals to be screened. In the case of positive combined screening, the stored individual samples from each member of multiple patients are always retained for individual testing.

[0011] Any tissue or bodily fluid sample is suitable for the practice of this invention. Exemplary samples include blood, urine, cerebrospinal fluid, lymph, and saliva. In some embodiments, the biological sample is a liquid sample that has undergone, for example, size exclusion chromatography to separate extracellular vesicles. In some other embodiments, the sample material is immobilized on a solid matrix. For example, the solid matrix may be beads, including magnetic beads, cellulose, silica, gold, or organic polymers, etc.

[0012] The method of this invention also considers the ideal size of the pool. Generally, the size of the pool will depend on the number of individuals screened, the prevalence of one or more cancer types screened in the subject population, and the sensitivity and / or specificity of the screening. While the exact size of the pool will depend on the factors mentioned above, in any case, pooling will improve efficiency in terms of the time and number of screening tests required to identify positive individuals.

[0013] Many screening assays are applicable to the practice of this invention. For example, a pooled sample is measured to detect the presence of nucleic acid or protein biomarkers indicating cancer. In one aspect, the invention contemplates the detection of cell-free nucleic acids in a sample (e.g., blood). Nucleic acid detection in a pooled sample involves DNA (including cDNA comprising RNA from the sample), hybridization-based methods, or alternatives to conventional 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 screening methods involving the detection of proteins in a pooled sample. Protein biomarkers may be present freely in the sample or on the surface of cells or extracellular vesicles. Detection of proteins, peptides, etc., is performed using methods known in the art, including, but not limited to, antibody-based detection, including labeled antibodies.

[0014] A preferred embodiment utilizes the Mercy Halo test in the pooled sample. The Mercy Halo test is a cancer detection assay that detects extracellular vesicles (EVs) shed from tumor cells and is described in commonly owned, co-pending 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, which are incorporated herein by reference. Extracellular vesicles are known to be present in high abundance, even in early-stage cancers. Extracellular vesicles carry surface proteins specific to their originating tumor cells, which are used to determine whether an individual is positive for the disease being screened. Only a small amount of blood from the individual is required for screening; therefore, after obtaining the sample, a first portion is pooled with other samples to run a pooled test, while another portion is stored for later use if a positive screening result is determined in the pooled sample. If the screening returns negative, then all individuals in the pool can be considered negative. However, if the screening returns positive, then the stored samples can be run independently to identify individuals who are positive for the disease.

[0015] Other aspects and advantages of the invention will become apparent from the following detailed description of the invention. Detailed Implementation

[0016] This invention discloses a method for merging samples from multiple individuals for diagnostic screening. Diagnostic screening is used to identify unidentified symptoms or risk markers. Diagnostic screening can be applied to individuals or to an entire population with symptoms or signs of diseases not being screened for. Methods for diagnostic screening are designed to identify conditions that may develop into diseases at some future time, enabling early intervention and management to reduce mortality and disease-related suffering. Embodiments of this invention utilize merged samples to detect disease biomarkers. The merged samples are used in a diagnostic screening method by combining samples from several individuals into a single assay. The merged samples are first measured. If negative, all members of the pool can be given a negative result, saving the cost of testing each individual one at a time. However, if the pool test is positive, each individual sample in the pool must be retested to identify which sample actually caused the positive pool test. In a preferred embodiment, a sample of tissue or bodily fluid is obtained from an individual and separated into a first portion and a second portion. The first portion is merged with samples from other individuals to produce the merged sample. The second portion is stored for later use.

[0017] Once an appropriate number of samples have been collected to generate a pooled sample, various methods can be used to determine and detect the presence of disease biomarkers in the pooled sample for positive or negative screening. Disease biomarkers include, but are not limited to, DNA, RNA, proteins, lipids, carbohydrates, single nucleotide polymorphisms (SNPs), protein-specific antigens (PSA), structural variants, specific cell types, antigens / antibodies, etc.

[0018] In a preferred embodiment, the Mercy Halo test is used to screen the pooled samples. The Mercy Halo test is designed to detect the co-occurrence of two or more disease biomarkers (e.g., cell surface proteins, nucleic acids, etc.) associated with extracellular vesicles released from tumor cells. In some embodiments, the Mercy Halo test includes isolating cancer-associated extracellular vesicles using, for example, a capture reagent (e.g., antibody-functionalized beads). In this case, the isolated extracellular vesicles are incubated with a plurality of dsDNA oligonucleotide-conjugated detection antibodies having DNA including single-stranded overhangs, such that members of the plurality of antibodies have single-stranded overhangs complementary to a second dsDNA oligonucleotide-conjugated detection antibody among the plurality of antibodies. The complementary dsDNA oligonucleotide-conjugated detection antibodies binding to the same extracellular vesicles are brought close enough to hybridize and are subsequently ligated. The ligation product is detected using qPCR, and a positive signal generated by the co-occurrence of the disease biomarkers indicates a positive screening.

[0019] Extracellular vesicles (EVs) generally consist of lipid bilayer-bound particles. Almost all cell types release these particles naturally, but unlike cells, they cannot replicate. EVs range in diameter from nearly the size of the smallest physically possible monolayer liposome (approximately 20 to 30 nanometers) to 10 micrometers or larger, but the vast majority are smaller than 200 nm. EVs can be classified into exosomes, microvesicles, and apoptotic bodies based on size and synthetic pathways. They carry proteins, nucleic acids, lipids, metabolites, and even organelles from the mother cell. Most cells studied to date are believed to release EVs, including some archaea, bacteria, fungi, and plant cells enclosed by a cell wall.

[0020] In some embodiments where the disease biomarker is or includes surface protein markers and / or vesicle intracellular protein markers, an ortho-linking assay, for example, can be used to detect the co-occurrence of two or more disease biomarkers. An ortho-linking assay may include contacting sample material comprising extracellular vesicles with an oligonucleotide-linked probe set (e.g., a detection antibody conjugated to dsDNA oligonucleotides), each probe targeting a disease biomarker. The oligonucleotide-linked probe set (e.g., a detection antibody conjugated to dsDNA oligonucleotides) comprises at least two different probes, resulting in a combination comprising extracellular vesicles and the probe set. Generally, each of the two probes comprises: (i) a target-binding portion targeting the surface protein marker and / or vesicle intracellular protein marker; and (ii) an oligonucleotide domain coupled to the target-binding portion, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. Such single-stranded overhangs of the probes are characterized in that they can hybridize with each other when the probes bind to the same extracellular vesicles. This combination, comprising extracellular vesicles and the probe set, is then maintained under conditions that allow the probe set to bind to its corresponding targets on the extracellular vesicles, such that the probes can bind to the same extracellular vesicles to form a double-stranded complex. The double-stranded complex is detected by contacting the double-stranded complex with a nuclease to produce a covalently continuous ligation product (e.g., a ligated dsDNA oligonucleotide); and by detecting the covalently continuous ligation product (e.g., the ligated dsDNA oligonucleotide). The ligation product (e.g., the ligated dsDNA oligonucleotide) is detected by amplification or sequencing. In one embodiment, the ligated dsDNA oligonucleotide serves as a template for qPCR. In one embodiment, the amplification is PCR, and can be digital PCR, qPCR, etc., in each case using a fluorescent hydrolysis probe to detect the ligation product. The presence of the covalently continuous ligation product indicates the presence of extracellular vesicles that are positive for disease markers. While the adjacent connectivity assays described above can be performed better than other adjacent connectivity assays (e.g., with higher specificity and / or sensitivity), those skilled in the art who read this disclosure will understand that other forms of adjacent connectivity assays known in the art can be used, in addition to other means for detecting extracellular vesicle-related markers.

[0021] Pooled samples that show a positive result for the presence of a cancer biomarker are identified as cancer-positive. Subsequently, stored samples for each corresponding individual can be tested to identify the specific individual who is positive for the biomarker. In another embodiment, the stored samples are further divided into equal aliquots to create smaller pools. These smaller pools are then screened for the presence of disease biomarkers to narrow down the number of potentially positive individuals to screen. In the case of positive screening, stored individual samples from each member of the multiple individuals are further tested separately.

[0022] This invention further considers detecting multiple cancer types in a single screening. For example, antibodies can be detected using multiple disease-specific biomarkers in a single screening, each antibody corresponding to a disease biomarker specific to a certain cancer type.

[0023] The number of samples in the pool can depend on the number of individuals screened, the prevalence of one or more cancer types screened in the subject population, and the sensitivity and / or specificity of the screening. Various methods can be used to determine the optimal pool size.

[0024] In some embodiments, the disease biomarker to be detected is nucleic acid. Detection is performed by any suitable method, such as PCR, qPCR, sequencing, probe hybridization, ligase chain reaction, multiplex PCR, etc. Nucleic acid can be extracted using known methods, such as suspension in extraction buffer (e.g., phosphate-buffered saline or EDTA) followed by cell lysis (e.g., using proteinase K), and extraction using commercially available kits, such as the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA).

[0025] In a preferred embodiment, the disease biomarker is a cancer biomarker, 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, sarcoma, skin cancer, and stomach cancer.

[0026] In some embodiments, the sample material is obtained from or derived from a biological source of interest (e.g., tissues, organisms, or cell cultures). In some embodiments, the source of interest may be or include cells or organisms, such as animals or humans. In some embodiments, the source of interest is or includes biological tissues or fluids. In some embodiments, biological tissues or fluids may be or include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, 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 fluid, vomitus, and / or combinations or components thereof. In some embodiments, the biofluid may be or include intracellular fluid, extracellular fluid, vesicle fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, biological tissue or sample material may be obtained, for example, by aspiration, biopsy (e.g., fine-needle or tissue biopsy), swabs (e.g., oral swabs, nasal swabs, skin swabs, or vaginal swabs), scraping, surgery, washing, or irrigation (e.g., bronchoalveolar, catheter, nose, eye, mouth, uterus, vagina, or other washing or irrigation). In some embodiments, the biological sample material is or includes body fluid sample material or body fluid-derived sample material. Examples of body fluid sample material or body fluid-derived sample material include, but are not limited to, amniotic fluid, bile, blood, breast milk, bronchoalveolar lavage fluid (BAL), cerebrospinal fluid, dialysis fluid, feces, saliva, semen, synovial fluid, tears, urine, etc. In some embodiments, body fluid sample materials or body fluid-derived sample materials applicable according to this disclosure are or include blood-derived samples, saliva-derived samples, sputum-derived samples, or pleural effusion-derived samples. In some embodiments, biological sample materials are or include liquid biopsies. In some embodiments, biological sample materials are or include cells obtained from an individual. In one example, a plasma or serum sample (4 ml) is processed to analyze extracellular vesicles and nucleic acids associated with and unrelated to extracellular vesicles.

[0027] Generally, when the sample is blood, the method disclosed herein works for approximately 4 to 10 mL of blood, as typically collected in blood collection tubes. A typical workflow involves centrifugation and capturing the supernatant plasma, which usually yields about 3 to 5 mL of plasma.

[0028] In some embodiments, extracellular vesicles in a sample may be captured or immobilized on a solid matrix prior to detection of one or more of the provided biomarkers according to this disclosure. In some embodiments, extracellular vesicles may be captured on the surface of the solid matrix via nonspecific interactions, including, for example, adsorption. In some embodiments, extracellular vesicles may be selectively captured on the surface of the solid matrix. For example, in some embodiments, the surface of the solid matrix may be coated with an agent that specifically binds to extracellular vesicles (e.g., an antibody agent that specifically targets such extracellular vesicles, for example, those associated with cancer). In some embodiments, the surface of the solid matrix may be coated with members of an affinity pair, and the entity of interest to be captured (e.g., an extracellular vesicle) may be conjugated to a complementary member of the affinity pair. In some embodiments, exemplary affinity pairs include, for example, but not limited to, biotin and avidin-like molecules, such as streptavidin. As those skilled in the art will understand, other suitable affinity pairs may also be used to facilitate the capture of the entity of interest onto the surface of the solid matrix. In some embodiments, the entity of interest can be captured on the surface of a solid matrix by applying an electric current, 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 purposes described herein, and both describe the isolation of extracellular vesicles from undiluted human blood or plasma samples using an AC-powered microarray chip device.

[0029] Solid matrices can be provided in a form suitable for capturing extracellular vesicles without interfering with downstream disposal, processing, and / or detection. For example, in some embodiments, the solid matrix may be or include beads (e.g., magnetic beads). In some embodiments, the solid matrix may be or include a surface. For example, in some embodiments, this surface may be the capture surface of a assay chamber (including, for example, tubes, pores, micropores, plates, filters, membranes, matrices, etc.). Therefore, in some embodiments, the methods described herein include capturing or immobilizing extracellular vesicles on a solid matrix prior to detecting markers provided in a sample.

[0030] In some embodiments, the sample may be processed, for example, to remove unwanted entities, such as cell debris or cells, before extracellular vesicles are captured on a solid matrix surface. For example, in some embodiments, the sample material may be centrifuged, for example, to remove cell debris, cells, and / or other particles. Alternatively or additionally, in some embodiments, the sample material may be purified or filtered based on size exclusion. Various size exclusion-based purification or filtration methods are known in the art, and those skilled in the art will understand that, in some cases, samples may be purified by column centrifugation based on specific molecular weight or particle size cutoff values. Those skilled in the art will also understand that appropriate molecular weight or particle size cutoff values ​​for purification purposes may be selected, for example, based on the size of the extracellular vesicles. For example, in some embodiments, size exclusion separation methods may be applied to sample material comprising extracellular vesicles to separate fractions of extracellular vesicles of a certain size (e.g., greater than 30 nm and not more than 1000 nm or greater than 70 nm and not more than 200 nm). Typically, the diameter of extracellular vesicles can range from 30 nm to several micrometers. See, for example, Chuo et al., “Imaging extracellular vesicles: current and emerging methods,” *Journal of Biomedical Science* 25: 91 (2018), which is incorporated herein by reference for the purposes described herein, and provides size information for different extracellular vesicle (EV) subtypes: migratory bodies (0.5 to 3 μm), microvesicles (0.1 to 1 μm), tumor bodies (1 to 10 μm), exosomes (<50 nm), small exosomes (60 to 80 nm), and large exosomes (90 to 120 nm). In some embodiments, specific EV subtypes can be isolated, for example, by one or more size exclusion separation methods for detection and assay.

[0031] This disclosure provides, in particular, insights and techniques for achieving effective cancer screening, such as for the early detection of cancer (e.g., in some embodiments, characterized by carcinoma, sarcoma, mixed type, etc.). In some embodiments, this disclosure provides techniques for early cancer detection in subjects who may be experiencing one or more cancer-related symptoms. In some embodiments, this disclosure provides techniques for early cancer detection in subjects at genetic risk for cancer. In some embodiments, this disclosure provides techniques for early cancer detection in subjects who may be at genetic risk for cancer and / or may be experiencing one or more cancer-related symptoms. In some embodiments, this disclosure provides techniques for early cancer detection in subjects who may have life history risk factors. In some embodiments, this disclosure provides techniques for screening individuals (e.g., individuals with certain risks (e.g., genetic risk, life history-related risk, or average risk)) for early cancer (e.g., in some embodiments, characterized by carcinoma, mixed type, sarcoma, etc.). In some embodiments, the provided techniques are effective for detecting early cancer (e.g., in some embodiments, characterized by carcinoma, sarcoma, melanoma, and mixed type). In some embodiments, the provided techniques are effective when applied to a group comprising or composed of individuals having one or more symptoms that may be cancer-related. In some embodiments, the provided technology is effective even when applied to populations comprising or composed of asymptomatic or symptomatic individuals (e.g., due to sufficiently high sensitivity and / or low false positive and / or false negative result rates). In some embodiments, the provided technology is effective when applied to populations comprising or composed of individuals without a genetic risk and / or life history-related risk of cancer (e.g., asymptomatic or symptomatic individuals). In some embodiments, the provided technology is effective when applied to populations comprising or composed of individuals with a genetic risk of cancer (e.g., asymptomatic or symptomatic individuals). In some embodiments, the provided technology is effective when applied to populations comprising or composed of individuals susceptible to cancer (e.g., individuals with known genetic, environmental, or empirical risks, etc.). In some embodiments, the provided technology may be or comprise one or more compositions (e.g., molecular complexes, systems, collections, combinations, kits, etc.) and / or methods (e.g., preparation, use, evaluation, etc.), as will be apparent to those skilled in the art upon reading the disclosure provided herein.

[0032] In some embodiments, the provided technology enables the detection of one or more characteristics of cancer (e.g., incidence, progression, responsiveness to therapy, recurrence, etc.) (e.g., early detection, such as in asymptomatic individuals and / or populations), and its sensitivity and / or specificity (e.g., false positive and / or false negative result rates) are suitable for making the provided technology applicable to single and / or periodic (e.g., cyclical) assessments. In some embodiments, the provided technology is suitable for use in conjunction with an individual's regular medical examinations, such as, but not limited to: physical examinations, general practitioner visits, cholesterol / lipid testing, diabetes screening (e.g., type 2 diabetes screening), colonoscopy, blood pressure screening, thyroid function testing, prostate cancer screening, mammograms, HPV / cervical smears, and / or vaccinations. In some embodiments, the provided technology is suitable for use in conjunction with treatment regimens; in some embodiments, the provided technology can improve one or more characteristics of such treatment regimens (e.g., success rate based on accepted parameters).

[0033] In some embodiments, this disclosure provides in particular the insight that screening asymptomatic individuals (e.g., regular screening before the onset of symptoms or in the absence of symptoms) can be beneficial and even crucial for the effective management (e.g., successful treatment) of cancer. In some embodiments, this disclosure provides cancer screening systems that can be implemented to detect cancer, including early-stage cancer, in asymptomatic individuals (e.g., those without a genetic and / or life history-related risk of cancer) in some embodiments. In some embodiments, the provided techniques are implemented to enable regular screening of asymptomatic individuals (e.g., those with or without a genetic risk of cancer). In some embodiments, the provided techniques are implemented to enable regular screening of symptomatic individuals (e.g., those with or without a genetic and / or life history-related risk of cancer). For example, this disclosure provides compositions (e.g., reagents, kits, components, etc.) and methods of providing and / or using them, including strategies involving the regular testing of one or more individuals (e.g., asymptomatic individuals). This disclosure defines the usefulness of such systems and provides compositions and methods for implementing them.

[0034] In some aspects, techniques are provided for classifying subjects (e.g., asymptomatic subjects) as having or being susceptible to cancer (e.g., carcinoma, sarcoma, mixed type, etc.). In some embodiments, this disclosure provides methods or assays for classifying subjects (e.g., asymptomatic subjects) as having or being susceptible to cancer (e.g., carcinoma, sarcoma, mixed type, etc.). In some embodiments, the provided methods or assays include measuring multiple different combinations of disease biomarkers from a sample material (e.g., from a blood-derived sample) of the subject to determine whether extracellular vesicles in the sample material (e.g., from a blood-derived sample) show at least one combination of biomarkers from the multiple biomarker combinations (e.g., co-occurrence of at least two biomarkers), wherein each of the multiple biomarker combinations independently comprises at least two biomarkers whose combined expression levels have been determined to be associated with at least one cancer type (including, for example, at least two cancer types).

[0035] This disclosure also provides, in particular, techniques for determining, for example, whether a subject has or is susceptible to cancer from sample material containing extracellular vesicles. For example, in some embodiments, when biological sample material from a subject in need (e.g., bodily fluid sample material, such as, but not limited to, blood-derived samples) shows that the level of extracellular vesicles expressing a combination of markers is at or above a reference threshold level (e.g., a cutoff value (e.g., determined according to this disclosure)), then the subject is classified as having or being susceptible to cancer. In some such embodiments, the reference threshold level (e.g., a cutoff value) may be determined based on a log-normal distribution around healthy subjects (e.g., those within a specified age range) and subjects optionally suffering from inflammatory conditions related to the tissue of interest but not cancerous (including, for example, atherosclerosis, heart disease, chronic kidney disease, diabetes, inflammatory bowel disease, fatty liver, chronic obstructive pulmonary disease, endometriosis, rheumatoid arthritis, obesity, pancreatitis, etc.), and the level selection required to achieve the specificity of interest, for example, based on the incidence of cancer or its subtypes (e.g., in some embodiments, cancers characterized by carcinoma, sarcoma, melanoma, and mixed types). In some embodiments, the specificity of interest may be at least 70%, including, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or higher.

[0036] In some embodiments, a reference threshold level (e.g., a cutoff value) may be determined based on the expression level (e.g., transcriptional level) of individual target biomarkers in a biomarker combination relative to their expression level in a cancer sample, thereby enabling the achievement of desired specificity and / or sensitivity (e.g., as described herein). In some embodiments, the reference threshold level (e.g., a cutoff value) may vary depending on, for example, cancer stage and / or subtype and / or patient characteristics, such as patient age, cancer risk factors (e.g., genetic risk versus average risk, life history-related risk factors), symptomatic / asymptomatic status, and combinations thereof.

[0037] In some embodiments, a subject is classified as having or being susceptible to cancer when a biosample from a subject in need shows levels of a combination of biomarkers that meet a reference threshold level. For example, in some embodiments, a subject is classified as having or being susceptible to cancer when a biosample from a subject in need (e.g., a body fluid sample, such as, but not limited to, a blood-derived sample) shows elevated levels of extracellular vesicles expressing a combination of biomarkers relative to a reference threshold level. In some embodiments, a subject in need is classified as having or being susceptible to cancer when a subject's biosample (e.g., a body fluid sample, such as, but not limited to, a blood-derived sample) shows levels of extracellular vesicles expressing a combination of biomarkers that are at least 30% or higher (including, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or higher) compared to a reference threshold level. In some embodiments, when a subject's biological sample material (e.g., bodily fluid sample material, such as but not limited to blood-derived samples) shows that the level of extracellular vesicles expressing a combination of markers is at least 2 times or higher compared to a reference threshold level, including, for example, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 250 times, at least 500 times, at least 750 times, at least 1000 times or higher, the subject in need is classified as having or being susceptible to cancer.

[0038] When biological sample material from a subject in need (e.g., bodily fluid sample material, such as, but not limited to, blood-derived samples) shows a level comparable to a reference threshold level, the subject is classified as unlikely to have or unlikely to be susceptible to cancer. In some such embodiments, the reference threshold level corresponds to the level of extracellular vesicles expressing a combination of biomarkers in comparable samples from a reference subject population (e.g., non-cancer subjects). In some embodiments, exemplary non-cancer subjects include healthy subjects (e.g., healthy subjects within a specified age range, such as under 55 years or over 55 years), subjects with non-tumor-related health conditions, illnesses, or symptoms (including, for example, subjects with symptoms of a cancerous disease or illness but not cancer), subjects with benign tumors, and combinations thereof.

[0039] In some embodiments, the oligonucleotide-linked probes provided and / or utilized herein include a target-binding portion and an oligonucleotide domain coupled to the target-binding portion. In some embodiments, the oligonucleotide domain coupled to the target-binding portion may include a double-stranded portion and a single-stranded overhang extending from at least one end of the oligonucleotide domain. In some embodiments, the oligonucleotide domain coupled to the target-binding portion may include a double-stranded portion and a single-stranded overhang extending from each end of the oligonucleotide domain.

[0040] The target-binding portion coupled to the oligonucleotide domain is an entity or agent that binds specifically to the target (e.g., a provided biomarker in a biomarker combination; those skilled in the art will understand that when the target biomarker is a specific form or part / component, the target-binding portion binds specifically to said form or part / component).

[0041] In some embodiments, the target-binding portion may be or include agents of any chemical class, such as carbohydrates, nucleic acids, lipids, metals, peptides, small molecules, and / or combinations thereof. In some embodiments, the target-binding portion may be or include an affinity agent, such as an antibody, affinity agent, aptamer, lectin, sialyl lectin, etc. In some embodiments, the target-binding portion is or includes an antibody agent, such as an antibody agent that specifically binds to a target or its epitope, such as a combination of cancer biomarkers or their epitopes. In some embodiments, an oligonucleotide-linked probe comprising an oligonucleotide domain coupled to the target-binding portion is referred to as a dsDNA oligonucleotide-conjugated detection antibody, wherein the target-binding domain comprises an antibody, and wherein the oligonucleotide domain comprises a double-stranded portion. In some embodiments, the target-binding portion is or includes a lectin or sialyl lectin that specifically binds to a carbohydrate-dependent biomarker provided herein. In some embodiments, the target-binding portion of the provided biomarker may be commercially available. In some embodiments, the target-binding portion of the provided biomarker may be designed and generated for use in the assays described herein. In some embodiments, the target-binding portion is or includes an aptamer, such as an aptamer that specifically binds to a target or its epitope, such as a provided biomarker or its epitope in a combination of cancer biomarkers. In some embodiments, the target-binding portion is or includes an affinity molecule that specifically binds to a target or its epitope, such as a provided biomarker or its epitope in a combination of cancer biomarkers. In some embodiments, such an affinity molecule may be or includes a peptide or polypeptide that binds to a target or its epitope (e.g., as described herein) and has a specificity and affinity similar to that of a corresponding antibody. In some embodiments, the target may be or includes a cancer-associated target. For example, in some such embodiments, a cancer-associated target may be or includes a target associated with more than one type of cancer (i.e., at least two or more types of cancer). In some embodiments, a cancer-associated target may be or includes a target that is generally associated with cancer. In some embodiments, a cancer-associated target may be or includes a target associated with cancer in a particular tissue, such as cancer. In some embodiments, cancer-related targets may be or include targets that are specific to a particular cancer (e.g., a specific cancer and more specifically, in some embodiments, cancers characterized by carcinoma, sarcoma, melanoma, and mixed types).

[0042] In some embodiments, the target-binding portion recognizes and specifically binds to a target present in a biological entity (including, for example, but not limited to, cells and / or extracellular vesicles). For example, in some embodiments, the target-binding portion may recognize and specifically bind to a tumor-associated antigen or its epitope. In some embodiments, the tumor-associated antigen may be or include antigens associated with cancer, such as skin cancer, brain cancer (including, for example, glioblastoma), breast cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, etc. In some embodiments, the target-binding portion may recognize tumor antigens associated with cancer (e.g., in some embodiments, characterized by carcinoma, sarcoma, melanoma, and mixed types). In some embodiments, the target-binding portion may recognize tumor antigens associated with carcinoma, sarcoma, melanoma, and mixed types in some embodiments.

[0043] In some embodiments, the target-binding portion may specifically bind to intravesical targets, such as provided intravesical proteins or RNA (e.g., mRNA). In some embodiments, the target-binding portion may specifically bind to surface targets present on / inside extracellular vesicles, such as membrane-binding peptides present on cancer-associated extracellular vesicles.

[0044] In some embodiments, the target-binding portion targets a biomarker for a specific symptom or disease (e.g., cancer), which is determined or has been determined, for example, by analyzing a population or library of patient biopsies and / or patient data (e.g., tens, hundreds, thousands, tens of thousands, hundreds of thousands or more), to identify the biomarker (e.g., predictive biomarker).

[0045] In some embodiments, the relevant biomarkers may be biomarkers identified and / or characterized, for example, through data analysis. In some embodiments, for example, a variety of data (e.g., in some embodiments, including one or more of batch RNA sequencing, single-cell RNA (scRNA) sequencing, mass spectrometry, histology, post-translational modification data, in vitro and / or in vivo experimental data) may be analyzed through machine learning and / or computational modeling to identify biomarkers (e.g., predictive biomarkers) that are highly specific to a disease or symptom (e.g., cancer).

[0046] In some embodiments, the target-binding portion targets tissue-specific targets, such as targets associated with specific tissues (e.g., 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, lungs, and skin). In some embodiments, this tissue-specific target may be associated with normal healthy tissue and / or diseased tissue (e.g., tumor). In some embodiments, the target-binding portion targets targets specifically associated with the subject's normal health condition. In some embodiments, the target-binding portion may recognize tissue-specific antigens.

[0047] In some embodiments, individual target-binding entities utilized in multiple oligonucleotide-linked probes (e.g., as described and / or utilized herein) target different targets. In some embodiments, such different targets may represent different biomarker proteins or peptides. In some embodiments, such different targets may represent different epitopes of the same biomarker protein or peptide. In some embodiments, two or more individual target-binding entities utilized in multiple oligonucleotide-linked probes (e.g., as described and / or utilized herein) may target the same target.

[0048] In some embodiments, the individual target-binding entities utilized in a probe linked by multiple oligonucleotides for detecting cancer may target different target biomarkers of a combination of cancer biomarkers.

[0049] In some embodiments, individual target-binding entities utilized in a plurality of oligonucleotide-linked probes for detecting cancer may target the same target biomarker of the same biomarker combination for cancer. In some embodiments, these target-binding entities may target the same or different epitopes of the same target biomarker of the same biomarker combination for cancer.

[0050] In some embodiments, the oligonucleotide domain used according to this disclosure (e.g., conjugable to a target binding portion) may include a double-stranded portion and single-stranded overhangs extending from one or both ends of the oligonucleotide domain. In some embodiments, where the oligonucleotide domain includes single-stranded overhangs extending from each end, the single-stranded overhangs extend from different strands of the double-stranded portion. In some embodiments, where the oligonucleotide domain includes single-stranded overhangs extending from one end of the oligonucleotide domain, the other end of the oligonucleotide domain may be a blunt end.

[0051] In some embodiments, the oligonucleotide domain may include ribonucleotides, deoxyribonucleotides, synthetic nucleotide residues capable of participating in Watson-Crick type or similar base pair interactions, and any combination thereof. In some embodiments, the oligonucleotide domain is or includes DNA. In some embodiments, the oligonucleotide domain is or includes peptide nucleic acid (PNA).

[0052] The single-stranded overhang of the oligonucleotide domain is designed to include at least a portion of a complementary nucleotide sequence to the single-stranded overhang of the probe linked to the second oligonucleotide, such that hybridization of the complementary single-stranded overhangs can form a double-stranded complex comprising the probe linked to the first oligonucleotide and the probe linked to the second oligonucleotide. In some embodiments, the nucleotide sequence of the complementary single-stranded overhang is selected to obtain optimal ligation efficiency in the presence of a suitable nucleic acid ligase. In some embodiments, the single-stranded overhang has a nucleotide sequence preferably 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, this single-stranded overhang may have a nucleotide sequence of GAGT, as described, for example, in Song et al., “Enzyme-guided DNA sewing architecture,” Scientific Reports 5: 17722 (2015), which is incorporated herein by reference for the purposes described herein. In other instances, blunt-end ligation is used.

[0053] 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 10 or more) may be conjugated or coupled to a target-binding moiety (e.g., a target-binding antibody agent).

[0054] This invention provides a method for pooling samples prior to diagnostic screening for non-infectious diseases. The method involves pooling only a portion of the obtained samples and storing the remaining portion for later use. Sample pooling represents a more time- and cost-effective way to conduct diagnostic screening. Regardless of the number of samples or assays used, sample pooling provides a more efficient and cost-effective way to screen average risk populations. To illustrate, consider the following scenario: a 0.03% prevalence of active disease means approximately 3 active cases in a population of 1,000. If 1,000 people are tested in 20 pools of 50 each, and all positive patients eventually appear individually in their respective pools, then 3 out of the 20 pools will test positive. If cases are clustered, as is typical, then the number of positive pools will be lower. In this case, 20 pools are tested, one test per pool. Subsequently, all participants in the 3 positive pools will be tested individually, for a total of 150 tests. At the end, 170 tests will have been performed to obtain results for 1,000 people. This equates to an 83% reduction in the number of tests performed compared to conventional non-pooled screening methods.

Claims

1. A method for diagnostic screening, the method comprising the following steps: The first portions of tissue or body fluid samples obtained from multiple individuals are combined to produce the combined sample; A second portion of the tissue or body fluid sample is stored separately; The merged samples were analyzed to identify the presence of disease biomarkers; If the biomarker is not detected in the merged sample, then all members of the plurality of individuals are identified as negative; as well as If the biomarker is detected in the merged samples, the determination is performed on each stored sample to identify members of the plurality of individuals who are positive for the presence of the biomarker.

2. The method according to 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 appear 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 determination is performed using size exclusion chromatography to purify extracellular vesicles extracted from the tissue or body fluid sample.

6. The method of claim 5, wherein extracellular vesicles are captured by immunoaffinity using disease-specific biomarker antibodies conjugated to magnetic beads.

7. The method of claim 6, wherein a detection antibody with complementary dsDNA oligonucleotide conjugation is used to detect up to two additional disease-specific biomarkers.

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

9. The method of claim 8, wherein the linked dsDNA oligonucleotide serves as a template for qPCR.

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