Screening samples for subclinical indicia of cancer

Abstract

Described herein is a method of cancer screening with high sensitivity and high specificity to detect indicia of subclinical cancer in potential blood and organ donors prior to donation to reduce cancer transmission between donors and recipients.

Description

[0001] SCREENING SAMPLES FOR SUBCLINICAL INDICIA OF

[0002] CANCER

[0003] Cross-Reference to Related Applications

[0004] This application claims the benefit of U.S. provisional application serial no. 63 / 610,118 filed on December 14, 2023, the entire contents of which are incorporated herein by reference.

[0005] Field of the invention

[0006] The invention relates to methods for screening blood and organ donors for subclinical indicia of cancer.

[0007] Background

[0008] There is a concern that blood transfusion might be associated with complications having delayed onset, notably transmission of chronic diseases such as cancer (Yang TO et al Annals of Oncology 28: 393-399, 2017). The increased frequency of cancer in patients treated by blood transfusion and organ transplantation has been repeatedly reported in large studies (Chapman JR et al Cold Spring Harb Perspect Med 2013;3:a015677) (Friman TK et al. Int. J. Cancer. 2022;150:1779-1791). However, the association between blood transfusion and cancer remains a debated issue and the mechanisms underlying it are unclear. There are similar concerns in organ transplantation, especially given that recipients are likely to be immunocompromised. In both cases, the donors do not present with disease, they are seemingly cancer-free. However, it is suspected that some donor samples or organs may contain subclinical indicia of cancer and therefore that cancer or cancer’s elements may be transmitted to a recipient.

[0009] Currently, blood donations are screened for various infectious diseases. These include Trypanosoma curzi, Hepatitis B, Hepatitis C, HIV, HLTV, Syphilis, Zika virus, West Nile virus, and others. In general, blood donations are screened for what are called Transfusion Transmissible Infections (often abbreviated TTI). In most countries, all donated blood is screened for TTIs at or near the time of donation using standardized laboratory procedures.

[0010] Similarly, organ donations are screened for infectious disease in addition to immune compatibility. Organ donations are typically screened for HIV, HBV, HCV, Syphilis, Cytomegalovirus, Epstein-Barr virus. In addition, certain donated organs are screened in an organ-specific manner. For example, heart transplants are typically screened for Chagas disease and organs from deceased donors are screened for toxoplasmosis.

[0011] Screening blood and organ donations for infectious disease limits the spread of known infections via the donor process. In some instances, donors are screened prior to donation. Infectious disease screens are simple laboratory tests that are highly sensitive and specific for detection of common infectious diseases, the indicia of which are present in the donor or donor sample. These tests generally use antibodies that interact with specific antigens presenting on a virus or bacterium and / or molecular tests targeting the genome of the infectious agent. While donors may present as healthy subjects, screening for infectious diseases is relatively low-cost and seeks to determine presence of analytes that are in a sample (blood or tissue typically) and are easy to detect.

[0012] Screening of blood and organ donors for subclinical signs of cancer is currently not implemented.

[0013] Summary

[0014] Described herein are methods for screening for subclinical indicia of cancer in organ and blood donors who are seemingly cancer-free. According to the described methods, a tissue or body fluid sample is obtained from a potential blood or organ donor. An assay is performed to screen for indicia of subclinical cancer in the sample. If indicia of subclinical cancer are present in the blood or organ donor, the blood or organ donation can be discarded and / or the donor can be further evaluated. Generally, subclinical disease has no recognizable clinical findings as distinguished from clinical disease, which has signs and symptoms that can be recognized. Moreover, for purposes of the invention, subclinical disease has diagnostic indicia that are present in a clinical sample at low levels, such as ctDNA, circulating tumor cells, extracellular vesicles, exosomes, and glycans and glycoproteins, blood molecular spectroscopic changes

[0015] The invention provides a significant advance in public health, as it allows for the screening of donated tissue and body fluid samples for possible presence of cancer in donors who appear healthy or who have not been diagnosed with cancer or present with no macromolecular or clinical indicia of cancer.

[0016] In one embodiment, there is provided a method of screening for blood or organ donors, the method comprising: obtaining a sample from a seemingly cancer-free blood or organ donor; assaying the sample to identify one or more indicia of subclinical cancer; and determining the risk of cancer in seemingly cancer-free blood or organ donor based on presence or absence of said one or more indicia of subclinical cancer. The sample may be blood, a tissue sample; and / or one or more of plasma, urine, saliva, sperm, feces, hair, nails, or body secretions. Various indicia of subclinical cancer include one or more of circulating tumor cells, circulating tumor microemboli, and circulating tumor DNA, circulating tumor cells DNA, extracellular vesicles, exosomes, glycans and glycoproteins.

[0017] This method can also include clinically evaluating the donor, based on presence of one or more indicia of subclinical cancer, to determine the diagnosis of cancer and, under the appropriate circumstances, excluding the donor from blood or organ donation based on presence of one or more indicia of subclinical cancer.

[0018] In one embodiment, samples from a plurality of donors or receivers are pooled and pooled samples are assayed to identify one or more indicia of subclinical cancer.

[0019] In one embodiment, the method uses a size exclusion fdtration to isolate intact circulating tumor cells and circulating tumor microemboli.

[0020] Also provided herein, in other embodiments are screening a seemingly cancer-free blood donor or organ donor, the method comprising: assaying on one or more samples obtained from one or more prospective blood donors or organ donors for one or more indicia of subclinical cancer; and excluding the one or more prospective blood donors or organ donors from blood or organ donation if the one or more indicia of subclinical cancer are identified; optionally, further clinically evaluating the risk of cancer of the one or more blood donors or organ donors if the one or more indicia of subclinical cancer are identified.

[0021] In some embodiments, the one or more subclinical indicia of cancer comprise peripheral blood assays targeting circulating tumor cells, circulating giant cells, circulating tumor clusters, circulating tumor microemboli, circulating tumor-associated cells, cancer-associated cells, subcellular elements, exosomes, circulating tumor DNA, cancer-associated proteins or cancer- associated molecules.

[0022] The assay(s) can, in certain embodiments comprise amplifying tumor-associated nucleic acid using primers specific for known or suspected tumor mutations, including single nucleotide variants, structural variants, or oncogene-related variants.

[0023] In an embodiment, the assay comprises an antibody-based assay, labeled probe detection, or next-generation sequencing of circulating tumor DNA or of circulating tumor cells DNA. In an embodiment, the assay comprises amplifying tumor-associated nucleic acid using primers specific for known or suspected tumor mutations of circulating tumor cells DNA or next-generation sequencing of circulating tumor cells DNA and of circulating tumor DNA.

[0024] Also provided herein, in other embodiments are method(s) for screening a seemingly cancer-free asymptomatic individual, the method comprising: assaying on one or more samples obtained from the asymptomatic individual for one or more indicia of subclinical cancer; and further clinically evaluating the risk of cancer of the asymptomatic individual if the one or more indicia of subclinical cancer are identified.

[0025] In an embodiment, the tissue or body fluid sample is stained for the presence of clusters of circulating tumor cells.

[0026] In an embodiment, the screening methods comprise cellular analyses, including morphological staining, immunolabeling, FISH, multiplexing, or in situ RNA or DNA detection.

[0027] In an embodiment, the screening methods comprise molecular analyses of proteins, RNAs, DNAs, peptides, amino acids, or lipids, glycans, and glycoproteins,

[0028] In an embodiment, the assay is performed on a pool of one or several samples to identify the one or more subclinical cancer markers.

[0029] In an embodiment, the samples in the pool are obtained from different individuals or from the same individual.

[0030] Description of the Drawings

[0031] FIGs. 1A and IB show examples of Circulating Tumor Cells Clusters found in the blood of blood donors using Giemsa staining. Tumor cells are isolated from blood using ISET.

[0032] Detailed Description

[0033] The disclosure herein provides one or more methods for screening blood and / or organ donors for subclinical indicia of cancer thereby to reduce the risk of cancer transmission between a donor and a recipient. Methods herein described are applicable to individual samples or pools of samples. Methods herein described are useful to identify analytes indicative that transfer of a blood sample or organ donation increases the risk of cancer in a recipient via introduction of subclinical cancer. In certain aspects, the invention comprises performing an assay for circulating tumor DNA, circulating cancer cells, or cancer-associated cells in tissue or body fluid samples obtained from a blood or tissue donor. These subclinical cancer markers are preferably identified using sequencing, probe detection, cell staining, computerized image analysis, molecular spectroscopic analyses and / or machine learning algorithms.

[0034] In another aspect, the invention provides methods for treating a patient in need of transfusion therapy with blood or blood components from a subject who has been screened for subclinical cancer in order to avoid the risk of cancer transmission from donor to recipient. In another aspect, the invention provides methods of screening potential organ donors for subclinical cancer to avoid risk of cancer transmission through organ donation and transplantation.

[0035] The invention applies equally to blood donors and organ donors. In an embodiment of the invention, blood donors are screened for subclinical indicia of cancer before being made available for transfusion. Preferred assays seek to identify circulating tumor DNA (e.g., DNA shed from, for example, apoptotic cells), circulating tumor cells (CTC), circulating tumor cells (CTC-DNA), circulating tumor microemboli, and other cancer biomarkers. Preferred assays include amplifying tumor-associated nucleic acid using primers that are specific for known or suspected tumor mutations, including single nucleotide variants, structural variants (e.g., rearrangements, deletions, breakpoint mutations and the like), and oncogene-related variants (e.g., ALU repeats, ACTB, and GAPDH). Additional assays include antibody-based assays, labeled probe detection, and next-generation sequencing of ctDNA, CTC-DNA or amplified ctDNA and CTC-DNA. Methods of the invention are useful to detect indicia of disease at levels below the limit of detection in conventional assays for macroscopic disease and clinically detectable disease.

[0036] In another embodiment, an organ is screened prior to transplantation / implantation / xenotransplantation. In one instance, a sample is obtained from donated organ tissue and screened for subclinical indicia of cancer. In another instance, a blood sample is obtained from the organ donor and the blood sample is analyzed for subclinical indicia of cancer. Whether in tissue or body fluid, the donated organ is evaluated using standard molecular biology assays and techniques. Whether tissue or body fluid, the sample is evaluated using standard molecular biology assays and techniques.

[0037] In an alternative embodiment, tissue or body fluid is stained for the presence of clusters of circulating tumor cells using, for example Giemsa stain. In one embodiment, circulating tumor cells are fluorescently labeled prior to Giemsa staining. Protocols for staining tumor cells are available and have been applied to tumor biopsy analysis. See, e.g., Arch Pathol Lab Med 2009 133(9): 1468-1471, incorporated by reference herein. Other staining techniques are useful for identification of cells in both blood and tissue samples.

[0038] In another aspect, subclinical indicia of cancer comprise peripheral blood analyses targeting circulating tumor cells, circulating giant cells, circulating tumor clusters, circulating tumor microemboli, circulating tumor-associated cells, cancer-associated cells, subcellular elements, or cancer-associated molecules. Cancer-associated cells can include endothelial cells, neutrophils, macrophages, monocytes and non-cancer giant cells. Subcellular elements can include extracellular vesicles, exosomes, platelets, platelet microparticles, and platelet extracellular vesicles. Cancer-associated molecules include DNA (including ctDNA, ctcDNA, or mitochondrial DNA), RNA (including miRNA, ncRNA, snRNA, or mRNA), lipids, glycans and glycoproteins, carbohydrates, proteins, and metabolites, including for spectroscopic analyses

[0039] Many different tissue or body fluid samples are useful in the practice of the invention. Exemplary samples include plasma, serum, whole blood, urine, saliva, feces, hair, nails, and body secretions. In certain embodiments, preferred screening methods target one or more tissue or body fluids, such as blood, urine, saliva, feces, hair, nails, and body secretions.

[0040] In some embodiments, screening methods include cellular analyses, such as morphological staining, immunolabelling, FISH, multiplexing, in situ RNA, DNA, or other known molecular detection methods. In some embodiments, screening methods include subcellular analyses, such as analyses of extracellular vesicles, exosomes, mitochondria endoplasmic reticulum or other subcellular elements. In some embodiments, screening methods comprise molecular analyses of proteins, RNAs, DNAs, peptides, amino-acids, lipids, glycans, glycoproteins, metabolites.

[0041] In one aspect, the invention points to performing a liquid biopsy assay on a pool of one or several samples to identify subclinical cancer markers. For example, different samples from the same individual may be pooled together and analyzed in the same test. Another example includes pooling samples from a plurality of individuals.

[0042] In certain other embodiments, the invention comprises screening xeno-transplants for subclinical indicia of cancer.

[0043] Analysis of subclinical indicia of cancer is performed according to the invention using a variety of techniques. The invention is the recognition that screening for subclinical indicia of cancer reduces a potential spread of cancer cells and / or cancer DNA or cancer elements from a donor to a recipient. The invention is the recognition that screening for subclinical indicia of cancer increases the probability of early cancer detection in the donor. That said, and without limiting the full scope of the invention, examples of useful methods include, glycoproteomic analyses, next-generation sequencing, fragmentomic DNA analyses probe-based detection, including labeled probes, cytopathological staining, immunolabelling, antibody staining, in situ detection of antigens and multiplexing of antigens, or in situ detection of nucleic acids, proteins, carbohydrates, metabolites, lipids, glycans and glycoproteins. In some embodiments, analysis methods include molecular isolation or extraction from cellular structures, subcellular structures, or extracellular liquids, followed by analysis methods including proteomic, next generation sequencing, transcriptomic next generation sequencing, DNA next generation sequencing, high throughput sequencing, RNA analyses (including miRNA, ncRNA, snRNA, or mRNA), or other molecular analyses known in the art. In some embodiments, analysis methods include analysis of plasma, serum or whole blood. In some embodiments, analysis methods include analysis of blood cells. In some embodiments, analysis methods include analysis of tissues.

[0044] In one aspect, the invention comprises an assay useful for target circulating tumor cells (CTCs). These are cells usually larger than about 8 pm that have been shed into the vasculature or lymphatics from a primary tumor and are present in the circulating blood. CTCs can extravasate and become seeds for the subsequent growth (metastases), a mechanism that is responsible for the vast majority of cancer-related deaths. CTCs are found in frequencies on the order of 1-10 CTC per mL of whole blood in patients with metastatic disease, making them difficult to detect and so they are not normally a target of donor screening.

[0045] Circulating tumor cells that originate from carcinomas can be classified according to the expression of epithelial markers, as well as their size and whether they are apoptotic. In general, CTCs are anoikis-resistant, which means that they can survive in the bloodstream without attaching to a substrate. Traditional CTCs are characterized by an intact nucleus; the expression of epithelial cell adhesion molecule (EpCAM) and cytokeratins, which demonstrate epithelial origin; the absence of CD45, indicating the cell is not of hematopoietic origin; and their larger size, irregular shape or subcellular morphology. Cytokeratin-negative CTCs are characterized by the lack of EpCAM or cytokeratins, which may indicate an undifferentiated phenotype (circulating cancer stem cells) or the acquisition of a mesenchymal phenotype (known as epithelial-mesenchymal transition or EMT). These populations of CTCs may be the most resistant and most prone to metastasis. They are also more difficult to isolate because they express neither cytokeratins nor CD45. Otherwise, their morphology, gene expression and genomics are similar to those of other cancer cells. Apoptotic CTCs are traditional CTCs that are undergoing apoptosis. Measuring the ratio of traditional CTCs to apoptotic CTCs — from baseline to therapy — provides clues to treatment efficacy in targeting and killing cancer cells. Small CTCs are cytokeratin-positive and CD45 -negative, but with sizes and shapes similar to white blood cells. Importantly, small CTCs have cancer-specific biomarkers that identify them as CTCs. Small CTCs have been implicated in progressive disease and differentiation into small cell carcinomas, which often require a different therapeutic course. Giant tumor cells have been associated with onset of tumors and tumor recurrence. They are also called Polyploid Giant Cancer Cells (PGCC) and can be found circulating in blood.

[0046] In another embodiment, the invention targets circulating tumor DNA (ctDNA). ctDNA originates directly from the tumor or from circulating tumor cells (CTCs) and can be found in the blood. Comparison of mutations in ctDNA and DNA extracted from primary tumors of the same patients can reveal the presence of identical cancer-relevant genetic changes. This allows for the possibility of using ctDNA as a target for blood and tissue screening.

[0047] In a preferred embodiment, the disease biomarkers useful in the invention 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.

[0048] 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, an organ, or an organism, such as an animal or human. In some embodiments, a source of interest is or comprises biological tissue or fluid. A biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cells, 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. A biological fluid may further 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 a preferred embodiment, the sample is derived from a blood draw or organ. In general, when the sample is blood, methods of the disclosure work with samples of approximately 5 to 40 mL of blood, preferably 10 or 20 mL of blood.

[0049] Isolation of cells using ISET and Cell Staining

[0050] In a preferred embodiment, cells are isolated using ISET®, a machine that uses a sizebased isolation system to extract CTCs, circulating giant cells, and circulating tumor microemboli from blood. Here, the sample can be diluted 1:10 with a fdtration buffer containing saponin, paraformaldehyde, EDTA, and bovine serum albumin. Hundred milliliters of diluted solution, corresponding to lOmL of undiluted blood, is loaded onto a polycarbonate membrane and fdtered by gentle aspiration under vacuum (created by a vacuum pump). The membrane is then washed once by aspiration with phosphate-buffered saline (PBS), disassembled from the fdtration module, and allowed to air-dry. The membrane is stained with hematoxylin and eosin (H&E) or May-Grumwald-Giemsa, washed once with PBS, and dried. For microscopic studies, the membrane is placed on a specifically designed stage without aspiration. (Vona et al. 2000 Am J Pathol). Circulating tumor cells isolated by this method are shown in FIG. 1A and IB.

[0051] In a preferred embodiment, after CTC enrichment with ISET®, downstream analyses are performed on the membrane. Analysis can include cytopathological staining, immunocytochemistry (ICC), molecular analyses, immunofluorescence (IF), or fluorescence in situ hybridization (FISH) or other cellular analyses known in the art.

[0052] Cytopathological staining can be used to identify circulating tumor cells. Because the ISET® filtration process isolates intact CTCs of all types, including Circulating Tumor Microemboli (CTM), Circulating Tumor Clusters, Circulating Giant Cells and PGCC, it is possible to validate that the classical cytopathological criteria used in exfoliative cytopathology are reliable for CTC diagnosis.

[0053] Cytopathological criteria include nuclear size equal or larger than 16 microns, irregularity of the nuclear contour, irregularity of the chromatin, presence of a visible cytoplasm, and high nuclear to cytoplasmic ratio (>0.8). (Hofman et al. 2011 Clin Cancer Res). Following CTC identification by cytopathology, further CTC characterization is possible through immunocytochemistry (ICC) to identify epithelial-mesenchymal transition (EMT) markers. (Hou et al. 2011 Am J Pathol; Lecharpentier et al. 2011 Brit J Cancer), proliferation markers (Hou et al. 2012 J Clin Oncol) and various theranostic markers. (Hofrnan et al. 2013 J Invest Dermatol; Cummings et al. 2014 BMC Cancer). ISET® enables assessment of PD-L1 expression on CTC, demonstrating concordant PD-L1 staining in tumor tissue and corresponding ISET® membranes from selected non-small cell lung cancer (NSCLC) patients. (Hie et al. 2017 Ann Oncol).

[0054] Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression.

[0055] In further embodiments, ISET® enables immunofluorescent staining of CTCs on ISET® membranes, thereby increasing efficiency and reliability of the computational analysis used in automated or semi-automated imaging methods.

[0056] Another embodiment includes using fluorescence in situ hybridization (FISH) on CTCs recovered by ISET®, enabling identification of oncogenic fusions, rearrangements, and gene amplifications with respect to therapeutic decision-making and follow-up of potential recurrence.

[0057] Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. FISH can also be used to detect and localize specific RNA targets (mRNA, IncRNA, and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

[0058] Another embodiment includes analysis of the CTC-DNA. CTC isolated on the ISET® membrane are lysed and their DNA is used for molecular analyses, including amplification, whole genome amplification and NGS analyses. CTC can be isolated in suspension using ISET® filtration. They are then centrifuged and lysed and their DNA is used for molecular analyses, including PCR amplification, whole genome amplification and NGS analyses. In a preferred embodiment, analysis can be performed on live cells. In this method, blood is fdtered and enriched using ISET®, then live CTCs are collected in suspension in 15 mL falcon tubes. Examples include in vivo CTC culture, CTC-derived xenografts, RT-PCR, RNAseq, and immunolabelling.

[0059] Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry.

[0060] There are two complex steps in the manufacture of antibody for immunolabeling. The first is producing the antibody that binds specifically to the antigen of interest and the second is fusing the tag to the antibody. Since it is impractical to fuse a tag to every conceivable antigen-specific antibody, most immunolabeling processes use an indirect method of detection. This indirect method employs a primary antibody that is antigen-specific and a secondary antibody fused to a tag that specifically binds the primary antibody. Pursuant to this indirect method, the primary antibody is added to the test system. The primary antibody seeks out and binds to the target antigen. The tagged secondary antibody, designed to attach exclusively to the primary antibody, is subsequently added.

[0061] Typical tags include: a fluorescent compound, gold beads, a particular epitope tag, or an enzyme that produces a colored compound. The association of the tags to the target via the antibodies provides for the identification and visualization of the antigen of interest in its native location in the tissue, such as the cell membrane, cytoplasm, or nuclear membrane. Under certain conditions the method can be adapted to provide quantitative information.

[0062] In certain embodiments, the invention comprises performing an assay on a pool of one or several samples to identify subclinical cancer markers. For example, different samples from the same individual may be pooled together and analyzed in the same test. Another example includes pooling samples from a plurality of individuals to be analyzed in the same test.

[0063] In some embodiments, subclinical indicia of cancer may determine clinically evaluating the donor to determine the diagnosis of cancer using clinical examination, imaging studies (MRI, ultrasound, CTscan Radiographic studies, PETscan, etc), biological analyses and / or biopsy aiming to diagnose cancer. In some embodiments, the present disclosure, among other things, provides insights that screening of asymptomatic individuals, e.g., regular screening prior to or otherwise in absence of developed symptom(s), can be beneficial, and even important for subsequent clinical investigations, cancer diagnosis and effective treatment of cancer. In some embodiments, the present disclosure provides cancer screening methods that can be implemented to detect cancer, including early-stage cancer, in asymptomatic individuals, who are seemingly cancer-free. An individual is seemingly cancer-free when that individual provides no physical and clinical manifestations of cancer as determined by a clinician during the course of medical examination and / or client testing. Provided technologies can be implemented to achieve regular screening of asymptomatic or symptomatic individuals, with or without hereditary risk(s) of developing cancer. Provided technologies can be implemented to perform regular screening tests to guide the decision to perform a biopsy or other clinical investigations (e.g.: imaging studies like MRI, ultrasound, CTscan Radiographic studies, PETscan, etc, and biological analyses) aimed to diagnose cancer.

[0064] In some embodiments, relevant subclinical indicia of cancer may be further identified and / or characterized, for example, via data analysis. In some embodiments, a diverse set of data (including but not limited to one or more of bulk DNA or RNA sequencing, single-cell DNA or RNA (scRNA) sequencing, histology, post-translational modification data, immunocytochemistry, sanger sequencing, FISH, multiplexing, immunolabeling, PCR, in vitro and / or in vivo experimental data) can be analyzed through machine learning and / or computational modeling to identify markers that are highly specific to a specific type of cancer. In some embodiments, subclinical indicia of cancer may determine further clinical investigations, such as clinical examination, imaging studies (MRI, ultrasound, CTscan Radiographic studies, PETscan, etc), biological analyses and / or biopsy aiming to diagnose cancer.

Claims

What is claimed is:

1. A method of screening for blood or organ donors, the method comprising: obtaining a sample from a seemingly cancer-free blood or organ donor; assaying the sample to identify one or more indicia of subclinical cancer; and determining the risk of cancer in seemingly cancer-free blood or organ donor based on presence or absence of said one or more indicia of subclinical cancer.

2. The method of claim 1 , wherein the sample is blood.

3. The method of claim 1, wherein the sample is a tissue sample.

4. The method of claim 1, wherein the sample is one or more of plasma, urine, saliva, sperm, feces, hair, nails, or body secretions.

5. The method of claim 1, wherein the one or more indicia of subclinical cancer is selected from the group consisting of circulating tumor cells, circulating tumor microemboli, and circulating tumor DNA, circulating tumor cells DNA, extracellular vesicles, exosomes, glycans and glycoproteins.

6. The method of claim 1, further comprising clinically evaluating the donor, based on presence of one or more indicia of subclinical cancer, to determine the diagnosis of cancer.

7. The method of claim 1, further comprising excluding the donor from blood or organ donation based on presence of one or more indicia of subclinical cancer.

8. The method of claim 1, wherein samples from a plurality of donors or receivers are pooled and pooled samples are assayed to identify one or more indicia of subclinical cancer.

9. The method of claim 1, further comprising using a size exclusion filtration to isolate intact circulating tumor cells and circulating tumor microemboli.

10. A method for screening a seemingly cancer-free blood donor or organ donor, the method comprising: assaying on one or more samples obtained from one or more prospective blood donors or organ donors for one or more indicia of subclinical cancer; and excluding the one or more prospective blood donors or organ donors from blood or organ donation if the one or more indicia of subclinical cancer are identified; optionally, further clinically evaluating the risk of cancer of the one or more blood donors or organ donors if the one or more indicia of subclinical cancer are identified.

11. The methods of claim 10, wherein the one or more subclinical indicia of cancer comprise peripheral blood assays targeting circulating tumor cells, circulating giant cells, circulating tumor clusters, circulating tumor microemboli, circulating tumor-associated cells, cancer-associated cells, subcellular elements, exosomes, circulating tumor DNA, cancer- associated proteins or cancer-associated molecules.

12. The method of claim 11, wherein the assay comprises amplifying tumor-associated nucleic acid using primers specific for known or suspected tumor mutations, including single nucleotide variants, structural variants, or oncogene-related variants.

13. The method of claim 10, wherein the assay comprises an antibody -based assay, labeled probe detection, or next-generation sequencing of circulating tumor DNA or of circulating tumor cells DNA.

14. The method of claim 10, wherein the assay comprises amplifying tumor-associated nucleic acid using primers specific for known or suspected tumor mutations of circulating tumor cells DNA or next-generation sequencing of circulating tumor cells DNA and of circulating tumor DNA.

15. A method for screening a seemingly cancer-free asymptomatic individual, the method comprising: assaying on one or more samples obtained from the asymptomatic individual for one or more indicia of subclinical cancer; andfurther clinically evaluating the risk of cancer of the asymptomatic individual if the one or more indicia of subclinical cancer are identified.

16. The method of claim 10, wherein the tissue or body fluid sample is stained for the presence of clusters of circulating tumor cells.

17. The method of claim 10, wherein the screening methods comprise cellular analyses, including morphological staining, immunolabeling, FISH, multiplexing, or in situ RNA or DNA detection.

18. The method of claim 10, wherein the screening methods comprise molecular analyses of proteins, RNAs, DNAs, peptides, amino acids, or lipids, glycans, and glycoproteins,19. The method of claim 10, wherein the assay is performed on a pool of one or several samples to identify the one or more subclinical cancer markers.

20. The method of claim 19, wherein the samples in the pool are obtained from different individuals or from the same individual.

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