Methods for analyzing pluripotent stem cell biomarkers and embodiments thereof

Abstract

Disclosed herein are in vitro methods for detecting the presence of metabolically altered cells. Also disclosed herein are in vitro methods for detecting the presence of stationary cells. Disclosed herein are in vitro methods for detecting and predicting the presence of cancer. Disclosed herein are in vitro methods for monitoring response to anti-cancer therapy. The present disclosure analyzes the expression of at least one biomarker of pluripotent stem cells for detecting or predicting or monitoring cancer. Disclosed herein are related uses of at least one biomarker of pluripotent stem cell markers and methods of treatment including in vitro detection or prediction methods.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980038677.5, entitled "Method for Analyzing Biomarkers of Pluripotent Stem Cells and its Embodiments" (the corresponding PCT application was filed on June 13, 2019, with application number 201980038677.5 and invention title 'Method for Analyzing Biomarkers of Pluripotent Stem Cells and its Embodiments'). Invention Field

[0002] This disclosure broadly relates to the field of pluripotent stem cell biomarkers and specifically provides an in vitro method for detecting and predicting cancer by using pluripotent stem cell biomarkers in blood samples. Background of the Invention

[0003] Cancer is a major health problem worldwide, causing millions of deaths. It is estimated that approximately 11 million people worldwide are diagnosed with cancer each year, and this number is projected to rise to over 16 million by 2020 (Ferlay et al., International journal of cancer 136.5 E359-E386. 2015). The bioheterogeneity of this disease and the vast population it affects raise critical questions about when to treat it, who will be treated, and which therapies will be used. These important questions can only be addressed by developing more precise and informative biomarkers.

[0004] Cancer treatment largely depends on the stage of diagnosis. With advancements in technology globally, the chances of treating cancer have increased in recent decades. The checkpoint is the stage at which the disease is detected. If cancer is detected at a late stage, there are certain situations where it becomes untreatable. If cancer is diagnosed at a very early stage, the chances of successful treatment increase. In this regard, detection methods play a crucial role, and the use of specific biomarkers is an active area of ​​research.

[0005] The nonspecific nature of cancer symptoms makes diagnosis difficult. In some cases, the disease progresses gradually. Therefore, early signs and symptoms of cancer are often overlooked by patients, providing an opportunity for the cancer to spread without any medical intervention. By the time patients seek medical help, the cancer may be beyond the reach of available clinical treatments. Furthermore, the lack of readily available biomarkers is another obstacle to cancer treatment.

[0006] Cancer can be detected in several ways, including medical imaging, tissue biopsy, and liquid biopsy. Once a possible cancer is detected, diagnosis is usually made by microscopic examination of a tissue sample from a tissue biopsy. Early detection and diagnosis of cancer are essential when it comes to treatment outcomes and survival, especially when it comes to highly malignant tumors. For decades, the only method used to detect cancer in humans was to surgically remove a small portion of the tumor and examine it under a microscope to look for cancer cells in the tissue. Unfortunately, this surgical intervention could only be performed when the tumor had reached a certain size or begun to cause functional disorders. Because this method can only be used when clinical symptoms are present, it cannot be considered the best method for early cancer diagnosis, or in other words, it cannot be used as a routine screening test before any clinical signs of cancer appear. Therefore, there is a need to develop a simple yet highly sensitive and specific cancer detection system and method to overcome the above and other problems.

[0007] Biomarkers are important not only for diagnostic purposes but also for prognostic purposes. Identifying the correct biomarkers allows for a very detailed evaluation of cancer progression and the effectiveness and success of chemotherapy drugs. There is an urgent need to identify reliable biomarkers for different cancers and to develop effective methods for detecting and predicting cancer and / or metabolic alterations and / or quiescent cells. Invention Overview

[0008] In one aspect of this disclosure, an in vitro method for detecting the presence of metabolically altered cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of metabolically altered cells.

[0009] In a second aspect of this disclosure, an in vitro method for detecting the presence of quiescent cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to analyze the determination of the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample detects the presence of quiescent cells.

[0010] In a third aspect of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample detects cancer.

[0011] In a fourth aspect of this disclosure, an in vitro method for predicting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, predicts cancer.

[0012] In a fifth aspect of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point during anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in a reference sample to monitor the response to cancer treatment.

[0013] In a sixth aspect of this disclosure, an in vitro method for detecting a positive response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample-I prior to administration of anticancer therapy; (b) obtaining a blood sample-II after administration of anticancer therapy; (c) enriching pluripotent stem cells from blood sample-I to obtain a mixture-I containing said pluripotent stem cells; (d) enriching pluripotent stem cells from blood sample-II to obtain a mixture-II containing said pluripotent stem cells; (e) obtaining nucleic acid-I from mixture-I; (f) obtaining nucleic acid-II from mixture-II; (g) independently determining the expression level of at least one biomarker of pluripotent stem cells using nucleic acid-I and nucleic acid-II; and (h) comparing the expression level of at least one biomarker from pluripotent stem cells of nucleic acid-II with the expression level of at least one biomarker from pluripotent stem cells of nucleic acid-I, wherein a reduced expression level of at least one biomarker from pluripotent stem cells of nucleic acid-II compared to the expression level of at least one biomarker from pluripotent stem cells of nucleic acid-I indicates a positive response to cancer treatment.

[0014] In a seventh aspect of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker.

[0015] In an eighth aspect of this disclosure, the use of pluripotent stem cell biomarkers for the detection of cancer from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0016] In a ninth aspect of this disclosure, the use of pluripotent stem cell biomarkers for predicting cancer from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0017] In a tenth aspect of this disclosure, the use of pluripotent stem cell biomarkers for grading cancer stages from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0018] In the eleventh aspect of this disclosure, the use of pluripotent stem cell biomarkers for monitoring the progress of anticancer therapy from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0019] In a twelfth aspect of this disclosure, a method for treating cancer is provided, the method comprising: (a) obtaining a blood sample from a subject; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample detects cancer; and (f) administering an anticancer therapy to the subject for treating cancer.

[0020] These and other features, aspects, and advantages of this subject matter will be better understood by referring to the following description and the appended claims. This overview is provided to present the selection of concepts in a simplified form. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter.

[0021] Brief description of the attached figures

[0022] The following figures form part of and are included in this specification to further illustrate aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to the accompanying figures in conjunction with the detailed description of the specific embodiments presented herein.

[0023] Figure 1 The embodiments described herein illustrate the fold change values ​​of Sox2 obtained by analyzing 20 different samples.

[0024] Figure 2 The invention describes an implementation of the present disclosure, which involves analyzing fold changes in Nanog values ​​obtained from 20 different samples.

[0025] Figure 3 The embodiments described herein illustrate the fold change values ​​of Oct-4a obtained by analyzing 20 different samples.

[0026] Figure 4 The embodiments described herein illustrate the fold change values ​​of Sirt 1 obtained by analyzing 20 different samples.

[0027] Figure 5 The embodiments described herein illustrate the fold change values ​​of Sirt 6 obtained by analyzing 20 different samples.

[0028] Figure 6 The embodiments described herein illustrate the fold change values ​​of NFκB obtained by analyzing 20 different samples.

[0029] Figure 7 The embodiments described herein illustrate the fold change values ​​of Oct-4 obtained by analyzing 20 different samples.

[0030] Figure 8 The invention describes an implementation of the present disclosure, which involves analyzing the fold change values ​​of p53 obtained from 20 different samples.

[0031] Figure 9A representative first figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0032] Figure 10 A representative second figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0033] Figure 11 A representative third figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0034] Figure 12 A representative fourth figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0035] Figure 13 A representative fifth figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0036] Figure 14 A representative sixth figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0037] Figure 15 A representative seventh figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0038] Figure 16 Figure 8, a representative embodiment of the present disclosure, illustrates the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0039] Figure 17 A representative ninth figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0040] Figure 18 A representative tenth figure depicts an embodiment according to this disclosure, showing the fold change in at least one biomarker of pluripotent stem cells disclosed in this study using a cohort of 1000 samples. The green scale below indicates consistency with the actual clinical state obtained by independent reviewers in a blinded study.

[0041] Detailed description of the invention

[0042] Those skilled in the art will understand that the present invention is affected by variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all such steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any or more such steps or features.

[0043] definition

[0044] For convenience, certain terms used in this specification and embodiments are described herein before further describing this disclosure. These definitions should be interpreted in light of the remainder of this disclosure and as understood by those skilled in the art. The terms used herein have meanings recognized and known to those skilled in the art; however, for convenience and completeness, specific terms and their meanings are set forth below.

[0045] The articles “a”, “an”, and “the” are used to refer to one or more (i.e., at least one) of the grammatical objects of the article.

[0046] The terms “comprise” and “comprising” are used in a sense of inclusion or openness, meaning that additional elements may be included. They are not intended to be interpreted as “consisting only of…”.

[0047] In this specification, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply inclusion of the stated element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps.

[0048] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0049] Ratios, concentrations, quantities, and other numerical data may be presented in range form herein. It should be understood that such range form is used solely for convenience and brevity and should be flexibly interpreted to include not only the explicitly stated limits of the range, but also all individual values ​​or subranges contained within that range, as if each value and subrange were explicitly stated. For example, a ratio of approximately 1:1 to 1:20 should be interpreted to include not only the explicitly stated limits of approximately 1:1 to approximately 1:20, but also subranges such as 1:2 to 1:10, 1:2 to 1:15, etc., and individual quantities, including fractions, within specified ranges such as 1:2.5 and 1:16.3.

[0050] The term "cancer" refers to a physiological condition in animals characterized by unregulated cell growth. As used in this disclosure, the term "cancer" is intended to include benign and malignant cancers, dormant tumors, or micrometastases. Types of cancer include, but are not limited to, carcinomas, lymphomas, germ cell tumors (including medulloblastomas and retinoblastomas), sarcomas (including liposarcomas and synovial cell sarcomas), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemias or malignant lymphomas. More specific examples of cancer include breast cancer, liver cancer, ovarian cancer, lung cancer, leukemia, prostate cancer, lymphoma, pancreatic cancer, cervical cancer, colon cancer, osteosarcoma, testicular cancer, thyroid cancer, stomach cancer, Ewing sarcoma, bladder cancer, gastrointestinal stromal tumor (GIST), kidney cancer (e.g., renal cell carcinoma), squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, breast cancer (including metastatic breast cancer), bladder cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, prostate cancer, vulvar cancer, thyroid cancer, and liver cancer. Carcinoma, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, biliary tract tumors, head and neck cancer, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; large-volume disease NHL, mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with phakomatoses, edema (e.g., edema associated with brain tumors), and McGill syndrome.

[0051] The term "advanced cancer" or "late-stage cancer" refers to cancer that originates locally or has spread beyond the site of origin through metastasis. The terms "Stage I," "Stage II," "Stage III," and "Stage IV" are well-known terms used to refer to the grade of cancer that has affected a patient. The term "precancerous" is used to refer to a stage of cancer where symptoms are not yet visible. The precancerous stage cannot be detected by PET scans. The term "early detection" has been used, when associated with cancer, to describe the detection of stage I or II cancer. The term "early onset" refers to a stage of cancer that has not yet been detected by any of the conventional methods known in the art. The terms "detections" or "detection" refer to tests performed in vitro on a patient / subject using a sample from the patient / subject.

[0052] The term "predicts" or "prediction" refers to the probability that something will happen in the future or in the appropriate course of time.

[0053] The term "blood sample" refers to a whole blood sample obtained from a subject. The scope of the methods disclosed herein begins at the stage where a blood sample has been obtained, regardless of the source of the sample, and the methods do not involve any invasive techniques or manipulation of the subject. The term "blood sample" also includes any form of processed blood sample. By "processing," this disclosure is intended to cover any method used for enriching a specific cell population or simple treatment to enable the blood sample to be used for testing by "in vitro" methods.

[0054] The term "in vitro" refers to a task, method, or experiment performed or occurring elsewhere outside of a test tube, petri dish, or living organism.

[0055] The term "reference" refers to at least one selected from: (a) a blood sample obtained before administration of anticancer therapy; (b) a blood sample obtained before the time point at which the blood sample under study was obtained; (c) a blood sample obtained after the time point at which the blood sample under study was obtained; and (d) a blood sample obtained from a control. The term "reference level" refers to the expression of at least one biomarker of pluripotent stem cells obtained from a reference. The term "control sample" refers to a blood sample obtained from a non-cancer subject, and the term "expression of at least one biomarker of pluripotent stem cells in a control sample" refers to the expression of at least one biomarker of pluripotent stem cells as disclosed herein, and the expression is studied using similar steps as disclosed herein for studying the expression of biomarkers in blood samples. It should be understood that the steps for processing and analyzing the expression of biomarkers in control samples are similar to the steps for performing and analyzing the expression of biomarkers in blood samples, and the expression level of the biomarker obtained from the blood sample is compared to the expression level obtained from the control sample.

[0056] The term "expression level" refers to a specific level of expression of a nucleic acid. Nucleic acid can be DNA or RNA. DNA is intended to include cDNA, and RNA is intended to include all types of RNA, including mRNA. The term "increased expression level" refers to an increase in the expression or level of a biomarker in an individual relative to a control, such as one or more individuals without a disease or condition (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or the level of a biomarker in a sample obtained prior to administration of therapy.

[0057] The term "metabolic altered cell" refers to any cell that has undergone metabolic changes to a form that would not normally be considered to exist in the environment. This change can manifest as increased proliferation.

[0058] The term "quiescent cell" refers to a quiescent cell that does not proliferate according to a regulated cell division cycle.

[0059] The term "pluripotent stem cell" refers to cells that have the ability to self-replicate and generate all types of cells in a subject.

[0060] As used herein, the term "biomarker" refers to a biomolecule that is a nucleic acid and is used to characterize a particular cell population. This term is intended to encompass nucleic acids in both DNA and RNA forms. The term "biomarker of pluripotent stem cells" refers to any biomarker that can be used to characterize a pluripotent stem cell population.

[0061] The term "subject" refers to any mammal whose blood sample has been collected for analysis using the in vitro methods described in this disclosure. Examples are based on humans used as subjects.

[0062] The term "cancer-free" refers to a subject who has not been diagnosed with cancer. The term "positive response" as used in this article refers to a subject's positive response to anti-cancer therapy, meaning that the therapy is effective in reducing the cancer cell population. The term "negative response" as used in this article refers to a subject whose anti-cancer therapy does not reduce the cancer cell population or cure cancer.

[0063] The term "invasive" refers to any technique that involves accessing a living body through an incision or by inserting instruments.

[0064] The term "at least one biomarker of pluripotent stem cells" refers to genes selected from the following: OCT-4 (octamer-binding transcription factor 4), Sox-2 (sex-determining region Y-box 2), Nanog, p53, NFκB, Sirt-1 (Sirtuin 1), Sir-6 (Sirtuin 6), NAD (nicotinamide adenine dinucleotide), RAS, ERC, erbB-2 (Erb-B2 receptor tyrosine kinase 2), ABL (Abelson's leukemia), subsets of the above, and combinations thereof. Subsets of Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, NAD, RAS, ERC, erbB-2, ABL, and combinations thereof are also part of this disclosure.

[0065] The term "cancer-associated biomarkers" encompasses all known cancer-associated biomarkers in the field of cancer research. This article provides a non-limiting list of cancer-associated biomarkers, which includes ABL1, EVI1, MYC, APC, IL2, TNFAIP3, ABL2, EWSR1, MYCL1, ARHGEF12, JAK2, TP53, AKT1, FEV, MYCN, ATM, MAP2K4, TSC1, AKT2, FGFR1, NCOA4, BCL11B, MDM4, TSC2, ATF1, FGFR1OP, NFKB2, BLM, MEN1, VHL, BCL11A, and FGFR2. , NRAS, BMPR1A, MLH1, WRN, BCL2, FUS, NTRK1, BRCA1, MSH2, WT1, BCL3, GOLGA5, NUP214, BRCA2, NF1, BCL6, GOPC, PAX8, CARS , NF2, BCR, HMGA1, PDGFB, CBFA2T3, NOTCH1, BRAF, HMGA2, PIK3CA, CDH1, NPM1, CARD11, HRAS, PIM1, CDH11, NR4A3, CBLB, IRF 4. PLAG1, CDK6, NUP98, CBLC, JUN, PPARG, CDKN2C, PALB2, CCND1, KIT, PTPN11, CEBPA, PML, CCND2, KRAS, RAF1, CHEK2, PTEN , CCND3, LCK, REL, CREB1, RB1, CDX2, LMO2, RET, CREBBP, RUNX1, CTNNB1, MAF, ROS1, CYLD, SDHB, DDB2, MAFB, SMO, DDX5, SDH D, DDIT3, MAML2, SS18, EXT1, SMARCA4, DDX6, MDM2, TCL1A, EXT2, SMARCB1, DEK, MET, TET2, FBXW7, SOCS1, EGFR, MITF, TFG, FH, STK11, ELK4, MLL, TLX1, FLT3, SUFU, ERBB2, MPL, TPR, FOXP1, SUZ12, ETV4, MYB, USP6, GPC3, SYK, ETV6, IDH1, TCF3, and combinations thereof. The list provided herein refers to cancer-related biomarkers that are well-known and commonly used by those skilled in the art. Abbreviations are interpreted as those known to those skilled in the art.

[0066] The term "administration" refers to the method of giving a patient a dose of a compound (e.g., a VEGF antagonist and / or a PD-L1 axis binding antagonist) or a composition (a pharmaceutical composition comprising a VEGF antagonist and / or a PD-L1 axis binding antagonist). Administration can be intramuscular, intravenous, intradermal, percutaneous, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, local, intratumoral, intraperitoneal, subcutaneous, subconjunctival, intracystic, mucosal, intraperitoneal, intraumbilical, intraocular, intraoral, intravaginal, intravitreal, or orally.

[0067] The term "cancer therapy" refers to any therapy known in the art for curing / treating cancer.

[0068] The term "chemotherapeutic agent" refers to compounds that can be used to treat cancer.

[0069] According to this disclosure, the term "enrichment" refers to a method for isolating a desired cell population in such a way that the desired cell population exists in a higher population concentration in the isolated mixture, which would facilitate the analysis of biomarkers in such population. In this context, "enrichment" means referring to the process of increasing the concentration of a specific type of pluripotent stem cells to enable studies to analyze the expression levels of at least one biomarker of pluripotent stem cells and / or to analyze mutations in nucleic acids obtained from such pluripotent stem cell populations.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the contents of this disclosure, preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference.

[0071] Current methods for detecting cancer have several drawbacks associated with them. The limitations related to positron emission tomography (PET) and circulating tumor cells (CTC) are described below.

[0072] Limitations of PET scans

[0073] Positron emission tomography (PET), nearly 45 years since its initial development, has become an established nuclear imaging modality, proving particularly useful in cancer diagnosis. PET utilizes a tracer molecule called 18F-2-fluoro-2-deoxy-D-glucose (FDG) (an analogue of glucose). Imaging glucose metabolism rates with FDG leverages the observation that malignant cells exhibit a higher rate of aerobic glycolysis than normal tissue (Griffeth 2005, BUMC Proceedings, 18:321-330). Therefore, malignant cells utilize more glucose to meet their energy needs. FDG is currently the only agent approved by the Food and Drug Administration (FDA) for use in oncology research. Fortunately, although FDG is not a perfect imaging agent (some tumors show poor FDG affinity, and some benign processes show high FDG affinity), it does perform well in most clinically significant malignant tumors, with the major exception of prostate cancer.

[0074] Several factors can make the interpretation of PET studies challenging. Among these, key factors in routine practice are the variable physiological uptake of FDG in normal tissues, FDG uptake associated with inflammation or infection, the occasional presence of malignant lesions with low affinity for FDG, unusual tumor sites, limited resolution of small lesions, altered biodistribution of FDG associated with hyperglycemia or hyperinsulinemia, and bone marrow activation and motion artifacts commonly encountered in cancer patients. Unfortunately, glucose uptake is ubiquitous in all body cells except malignant cells. Physiological uptake in some normal tissues can be highly variable. While FDG accumulates to predictable levels in many cases, there are others where its uptake is unpredictable. For example, the brain typically shows strong FDG uptake because it is the sole metabolic organ for glucose, while myocardial uptake is strong in patients who are not fasting but highly variable in those who are. Adipose tissue typically shows minimal FDG uptake, but certain adipose deposits that play a role in thermogenesis (so-called "brown fat") can be significantly activated in patients with colds or stress. Sometimes, even relatively predictable activity can be confounding. For example, unlike glucose, FDG is not well reabsorbed by the proximal tubules of the kidney. Therefore, strong activity can be predicted in the kidneys and bladder. However, focal accumulations of FDG with high excretory activity in the ureters can be confused with hypermetabolic iliac lymph node metastases. Inflammatory cells, particularly macrophages, can sometimes accumulate FDG to considerable extent, thus sites of inflammation or infection are sometimes visible on PET. Granulomatous conditions, such as sarcoidosis, fungal infections, tuberculosis, and Mycobacterium avium ( ), are also associated with FDG accumulation. Mycobacterium aviumIntracellular infections can cause particular problems in PET evaluation of lung lesions or lymph nodes. Even inflammation associated with treatment procedures (such as surgery or radiation therapy) can cause significant uptake. When clinically possible, it is generally wise to wait at least 3 months after completion of radiation therapy before performing a PET study to avoid confounding effects due to inflammatory uptake of FDG. Therefore, it is not feasible to perform PET studies within short intervals to examine the response of patients undergoing cancer treatment; a minimum timeframe of 3 months must be waited to check whether the patient has responded to the treatment. If the patient does not respond, the loss of valuable time can be fatal for the patient.

[0075] Some malignant lesions have low affinity for FDG, such as the prostate, bronchoalveolar carcinoma, low-grade sarcoma, certain low-grade non-Hodgkin lymphomas, and even some well-differentiated lung adenocarcinomas, which can show poor FDG concentrations. Most neuroendocrine tumors are rarely seen on FDG-PET. Small lesions or unusual presentations / locations always make tumor detection and staging more difficult. PET alone sometimes cannot localize small tumors or confirm whether FDG uptake at unusual sites reflects tumor or non-tumor activity.

[0076] Limitations of circulating tumor cells

[0077] Despite a large body of scientific publications on CTC detection in cancer patients, this biomarker is not used by physicians in routine clinical practice. This can be explained by the vast number of methods available for CTC detection and the difficulty physicians and biologists face in choosing the optimal method (Huang T et al., Biosens Bioelectron 2014; 51:213-8). In this context, it is noteworthy that the only FDA-approved CTC detection method, the CellSearch method (Janssen Diagnostics Company, USA), has been approved for CTC detection in patients with metastatic breast cancer, prostate cancer, and colon cancer. Conversely, CTCs have been reported to cause false positives and false negatives in the detection of breast, prostate, and colon cancer (Lori M. Millner et al., Ann Clin Lab Sci. 2013 43(3)). Because CTCs undergoing epithelial-mesenchymal transition do not express epithelial biomarkers, CellSearch systems will certainly miss detecting CTC subsets of interest in several cancer patients (Hofman VJ et al., Am J Clin Pathol 2011; 135). Direct techniques for CTC detection in cancer are certainly highly attractive, but results obtained by different teams may require validation in independent and large multicenter studies. Many other methods for CTC characterization are currently being developed, such as those that allow for the functional evaluation of CTCs and characterization of malignant cell subsets (Yao X et al., Integr Biol (Camb) 2014; 6: 388-98). At present, these new methods seem difficult to translate into routine clinical practice. These methods lack multicenter evaluation procedures, making it difficult to evaluate their reproducibility, sensitivity, and specificity.

[0078] Existing technologies for cancer detection are based on the isolation of specific cancer biomarkers from affected tissue, thus involving invasive methods of isolating affected tissue to detect the presence of cancer. Currently available technologies offer the detection of cancers involving invasive techniques, and for obvious reasons, these techniques cannot be performed frequently on patients to check for the presence of cancer. Therefore, existing technologies are disadvantageous in terms of practical difficulty, cannot accurately predict the likelihood of cancer with a single treatment of a patient, and are unsuitable for subsequent treatment. Existing technologies can only be used to detect certain types of cancer, and once they have reached an advanced stage, patients with advanced disease have a very low chance of survival with treatment, which is one of the biggest drawbacks of existing technologies.

[0079] There is a deficiency in the availability of simple tests that can detect all types of cancer at an early stage and can be performed frequently to monitor the stages of cancer. This paper discloses a method for detecting and predicting cancer by analyzing blood samples from subjects of interest. This paper discloses a method for detecting the cancer grade of a subject using a simple blood sample. This disclosure accurately detects the type of cancer using a simple blood sample without using any invasive techniques such as biopsies. The method of this disclosure predicts the likelihood of a subject having cancer using only a blood sample. This disclosure even detects precancerous stages before any symptoms begin to appear, thus providing a significant advantage over conventional detection methods that only detect cancer when it has reached a certain level in the body. This paper also discloses a method for enriching pluripotent stem cells (PSCs), which can be further used to evaluate stem cell biomarkers on the PSCs. Therefore, this paper discloses a simple, effective, and highly accurate method for detecting and predicting cancer by analyzing simple blood samples from subjects.

[0080] This document discloses an in vitro method for detecting the presence of cancer, predicting the chance of developing cancer, grading cancer stages, monitoring cancer progression, monitoring response to anti-cancer treatment, and conducting follow-up examinations to confirm whether the cancer has been eradicated in the subject of interest. The in vitro method disclosed herein also provides precise information about specific types of cancer using only blood samples without involving any invasive techniques. The method disclosed herein provides information about the type of cancer well before the manifestations of the condition can be detected by known techniques such as PET scans, thus detecting the presence of specific types of cancer without the need for a biopsy. Furthermore, in order for physicians to target specific tissues of an organ for biopsy, patients need to exhibit specific symptoms; however, in the case of cancer, symptoms may appear at an advanced stage, reducing the patient's chances of survival. The in vitro method of this disclosure, using only blood samples, not only detects the presence of cancer but also detects the primary site and type of cancer well before symptoms appear. The method disclosed herein provides all the aforementioned uses implemented using only blood samples. The simplicity of this method results in the benefit of being able to use it frequently while treating cancer patients, unlike the use of PET scans which can be performed at minimum intervals of 6 months. The method disclosed herein is an in vitro method and does not involve any invasive techniques. The method disclosed in this disclosure can detect / predict any cancer in a subject by analyzing a blood sample, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control detects / predicts cancer. The method disclosed herein enables the detection of the presence of metabolically altered cells / quiescent cells by analyzing blood samples, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control detects the presence of metabolically altered cells.The method disclosed herein enables monitoring of response to cancer treatment by analyzing blood samples, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in a reference for monitoring the response to cancer treatment.

[0081] This document discloses a method for detecting the presence of cancer from blood samples and for detecting specific types of cancer, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control indicates the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker.

[0082] The methods disclosed herein are used to detect and predict precancerous stages, stage I, stage II, stage III, and stage IV cancers, wherein the cancers are selected from the non-restrictive group consisting of: ovarian cancer, breast cancer, prostate cancer, lung cancer, liver cancer, colon cancer, leukemia, lymphoma, bladder cancer, kidney cancer, thyroid cancer, and pancreatic cancer. Other types of cancer may also be included in this disclosure.

[0083] This disclosure is not limited in scope to the specific embodiments described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0084] Although the subject matter has been described with reference to specific embodiments, this description is not intended to be limiting. Various modifications to the disclosed embodiments, as well as alternative embodiments of the subject matter, will become apparent to those skilled in the art upon reference to the description of the subject matter. Therefore, it is conceivable that such modifications may be made without departing from the spirit or scope of the inventive subject matter as defined.

[0085] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker in a control sample, wherein an increase in the expression level of at least one biomarker in the sample compared to the expression level of at least one biomarker in the control sample detects the presence of metabolically altered cells.

[0086] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of metabolically altered cells, and wherein the method further comprises analyzing the nucleic acids by performing sequence-based assays.

[0087] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased at least 2-fold compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 3-fold compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 5-fold compared to a control.

[0088] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells, as described herein, is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased by 10-20 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 20-30 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 30-40 times compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 40-50 times compared to a control.

[0089] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein at least one biomarker for pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4a. In yet another embodiment, at least one biomarker for pluripotent stem cells is Oct-4b.

[0090] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein at least one biomarker for pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Sox-2. In another embodiment, at least one biomarker for pluripotent stem cells is Nanog. In another embodiment, at least one biomarker for pluripotent stem cells is p53. In another embodiment, at least one biomarker for pluripotent stem cells is Sirt-1. In another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-6. In another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-3.

[0091] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein nucleic acids are obtained from the mixture by any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0092] In one embodiment of this disclosure, an in vitro method for detecting the presence of cells with metabolic alterations as described herein is provided, wherein the determination of the expression of at least one biomarker by analyzing nucleic acids is performed using a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0093] In one embodiment of this disclosure, an in vitro method for detecting the presence of cells with metabolic alterations, as described herein, is provided, wherein a control is the expression level of at least one biomarker from pluripotent stem cells obtained from cancer-free subjects.

[0094] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells.

[0095] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells, wherein the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and combinations thereof.

[0096] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells as described herein is provided, wherein the method is independent of invasive techniques.

[0097] In one embodiment of this disclosure, an in vitro method for detecting the presence of metabolically altered cells, as described herein, is provided, wherein the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA.

[0098] In one embodiment of this disclosure, an in vitro method for detecting the presence of quiescent cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample detects the presence of quiescent cells.

[0099] In one embodiment of this disclosure, an in vitro method for detecting the presence of quiescent cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to analyze the determination of the expression level of at least one biomarker of pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of pluripotent stem cells in the sample with the expression level of at least one biomarker of pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of quiescent cells in the sample compared with the expression level of at least one biomarker of quiescent cells in the control sample detects the presence of quiescent cells, and wherein the method further comprises analyzing the nucleic acids by performing sequence-based assays.

[0100] In one embodiment of this disclosure, an in vitro method for detecting the presence of quiescent cells is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker in a control sample, wherein an increase in the expression level of at least one biomarker in the sample compared to the expression level of at least one biomarker in the control sample detects the presence of quiescent cells, and wherein the increase is at most 1.9-fold. In another embodiment, the increase is in the range of 0.1-1.9-fold. In another embodiment, the increase is in the range of 0.2-1.8-fold.

[0101] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample detects cancer.

[0102] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the control sample detects cancer, and wherein an increase of 10-20 times in the expression level compared with the control detects stage I cancer.

[0103] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the control sample detects cancer, and wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the range of 20-30 times compared with the control detects stage II cancer.

[0104] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the control sample detects cancer, and wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the range of 30-40 times compared with the control detects stage III cancer.

[0105] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the control sample detects cancer, and wherein an increase of 40-fold or more in the expression level compared with the control detects stage IV cancer.

[0106] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample compared with the expression level of at least one biomarker of the control sample detects cancer, and wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the range of 6-10 times compared with the control detects a precancerous stage.

[0107] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) analyzing the expression level of at least one biomarker of the pluripotent stem cells using the nucleic acids; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects cancer, and wherein the method further comprises analyzing the nucleic acids by performing sequence-based assays. In another embodiment of this disclosure, the type of cancer is detected by analyzing nucleic acids using sequence-based assays.

[0108] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample detects cancer compared to the expression level of at least one biomarker of the pluripotent stem cells in the control sample is at least 2-fold increased compared to the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is at least 3-fold increased compared to the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is at least 5-fold increased compared to the control.

[0109] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects cancer, and wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by at least 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold compared with the control.

[0110] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) analyzing the expression level of at least one biomarker of the pluripotent stem cells using the nucleic acids; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared to the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects cancer, and wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by 10-20 times compared to the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is increased by 20-30 times compared to the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is increased by 30-40 times compared to the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is increased by 40 times or more compared to the control.

[0111] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects cancer, and wherein the at least one biomarker of the pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof. In another embodiment, the at least one biomarker of the pluripotent stem cells is Oct-4 and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4a. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4b.

[0112] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) analyzing the expression level of at least one biomarker of pluripotent stem cells using the nucleic acids; and (e) comparing the expression level of at least one biomarker of pluripotent stem cells in the sample with the expression level of at least one biomarker of pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of pluripotent stem cells in the control sample, detects cancer, and wherein the at least one biomarker of pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, a subset thereof, and a combination thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Sox-2 and a subset thereof. In yet another embodiment, at least one biomarker of pluripotent stem cells is Nanog and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is p53 and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-1 and a subset thereof. In another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-6 and a subset thereof. In yet another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-3 and a subset thereof.

[0113] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects cancer, and wherein the at least one biomarker of the pluripotent stem cells is selected from NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof. In another embodiment, the at least one biomarker of the pluripotent stem cells is NAD and a subset thereof. In another embodiment, the at least one biomarker of the pluripotent stem cells is RAS and a subset thereof. In another embodiment, the at least one biomarker of the pluripotent stem cells is ERC and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is erbB-2 and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is ABL and a subset thereof.

[0114] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein nucleic acids are obtained from a mixture by means of any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0115] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA.

[0116] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker by analyzing nucleic acids is performed using a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0117] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein a control is the expression level of at least one biomarker from pluripotent stem cells obtained from cancer-free subjects.

[0118] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein the expression level of the at least one biomarker in the control sample is compared with the expression level of the at least one biomarker in the control sample. In a specific embodiment, an increase in the expression level of at least one biomarker in the sample detects cancer, and wherein enriching the pluripotent stem cells from the blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells, wherein the treatment of the second mixture comprises at least one method selected from: (1) extraction; (2) washing; (3) centrifugation and combinations thereof. In another embodiment, centrifugation may comprise sequential centrifugation to obtain pluripotent stem cells.

[0119] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker.

[0120] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein the expression level of the at least one biomarker in the control sample is compared with the expression level of the at least one biomarker in the control sample. (f) The increased expression level of the at least one biomarker in the sample detects the presence of cancer; (c) Sequence-based determination of the nucleic acid and analysis of mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, wherein the at least one biomarker of pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, the at least one biomarker of pluripotent stem cells is Oct-4 and a subset thereof. In another embodiment, the at least one biomarker of pluripotent stem cells is Oct-4a. In another embodiment, the at least one biomarker of pluripotent stem cells is Oct-4b.

[0121] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to analyze the determination of the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, wherein the at least one biomarker of the pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, a subset thereof, or a combination thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Sox-2 and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Nanog and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is p53 and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-1 and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Sirt-6 and a subset thereof. In yet another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-3 and a subset thereof.

[0122] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to analyze the determination of the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, wherein the at least one biomarker of the pluripotent stem cells is selected from NAD, RAS, ERC, erbB-2, ABL, a subset thereof, or a combination thereof. In another embodiment, the at least one biomarker of the pluripotent stem cells is NAD and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is RAS and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is ERC and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is erbB-2 and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is ABL and a subset thereof.

[0123] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based sequencing on the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, wherein the sequence-based sequencing is whole-genome sequencing or transcriptome sequencing of the nucleic acids. Any sequencing technology known in the art is contemplated to be used for sequencing. In one embodiment, next-generation sequencing (NGS) is used for sequence-based sequencing.

[0124] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the nucleic acids are obtained from the mixture by a method selected from any of the following: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0125] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to analyze the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the determination of the expression of the at least one biomarker using the nucleic acids is performed by a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0126] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to analyze the determination of the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the control is the expression level of at least one biomarker of pluripotent stem cells obtained from a cancer-free subject.

[0127] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker of the pluripotent stem cells in a control sample, wherein the expression level of the at least one biomarker of the pluripotent stem cells in the sample is significantly higher than the expression level of the at least one biomarker of the pluripotent stem cells in the control sample. The increased expression level of the nucleic acid detects the presence of cancer; (f) the nucleic acid is sequence-based and mutations in at least one cancer-related biomarker are analyzed, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, and wherein enriching the pluripotent stem cells from the blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells. In another embodiment, the treatment of the second mixture comprises at least one method selected from: (1) extraction; (2) washing; (3) centrifugation and combinations thereof. In another embodiment, the at least one salt solution is sodium chloride, and the at least one neutral buffer is Ficollhypaque solution.

[0128] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the at least one cancer-related biomarker... The presence of mutations in the sample indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarkers, and the enrichment of the pluripotent stem cells from the blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; (iii) centrifuging the second mixture at a speed ranging from 1000 to 6000 rpm for a period of time from 5 to 20 minutes to obtain a supernatant and a precipitate; (iv) washing and extracting the precipitate or supernatant to obtain a third mixture; and (v) centrifuging the third mixture sequentially at varying speeds from 1000 to 10,000 rpm for 2 to 8 rounds to obtain enriched pluripotent stem cells, wherein at least one salt solution is sodium chloride, and at least one neutral buffer is a Ficoll hypaque solution.

[0129] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; (iii) centrifuging the second mixture at a speed ranging from 1000 to 6000 rpm for a period of 5 to 20 minutes to obtain a supernatant and a precipitate; (iv) washing and extracting the precipitate or supernatant to obtain a third mixture; and (v) centrifuging the third mixture sequentially at varying speeds of 1000 to 10,000 rpm for 2 to 8 cycles to obtain enriched pluripotent stem cells, wherein at least one salt solution is sodium chloride and at least one neutral buffer is a Ficoll hypaque solution.

[0130] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of the at least one biomarker in a control sample, wherein an increase in the expression level of the at least one biomarker in the sample compared to the expression level of the at least one biomarker in the control sample detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, and wherein the cancer-related biomarker is selected from cancer-related biomarkers known in the art.In another embodiment, the cancer-related biomarkers are selected from ABL1, EVI1, MYC, APC, IL2, TNFAIP3, ABL2, EWSR1, MYCL1, ARHGEF12, JAK2, TP53, AKT1, FEV, MYCN, ATM, MAP2K4, TSC1, AKT2, FGFR1, NCOA4, BCL11B, MDM4, TSC2, ATF1, FGFR1OP, NFKB2, BLM, MEN1, VHL, BCL11A, FGFR2, NRAS, BMPR1A, MLH1, WRN, BCL2, FUS, NTRK1, BRCA1, MSH2, WT1, BCL3, GOLGA5, NUP214, BRCA2, NF1, BCL6, GOPC, PAX8, CARS, NF2, BCR, HMGA1, PDGFB, CBFA2T3, NOTCH1, BRAF, HMGA2, PIK3CA, CDH1, NPM1, CARD11, HRAS, PIM1, CDH11, NR4A3, CBLB, IRF4, PL AG1, CDK6, NUP98, CBLC, JUN, PPARG, CDKN2C, PALB2, CCND1, KIT, PTPN11, CEBPA, PML, CCND2, KRAS, RAF1, CHEK2, PTEN, CC ND3, LCK, REL, CREB1, RB1, CDX2, LMO2, RET, CREBBP, RUNX1, CTNNB1, MAF, ROS1, CYLD, SDHB, DDB2, MAFB, SMO, DDX5, SDHD, D Combinations of DIT3, MAML2, SS18, EXT1, SMARCA4, DDX6, MDM2, TCL1A, EXT2, SMARCB1, DEK, MET, TET2, FBXW7, SOCS1, EGFR, MITF, TFG, FH, STK11, ELK4, MLL, TLX1, FLT3, SUFU, ERBB2, MPL, TPR, FOXP1, SUZ12, ETV4, MYB, USP6, GPC3, SYK, ETV6, IDH1, TCF3, and above are expected. It is anticipated that the detected cancer type should be based on the cancer-related biomarkers of the analyzed mutations and when the desired mutation has been identified. It should also be understood that, according to this embodiment, information about the cancer type and stage can be obtained using only blood samples without the need for biopsy or other invasive procedures.

[0131] In one embodiment of this disclosure, an in vitro method for detecting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, detects the presence of cancer; and (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker, and wherein the expression level of the at least one biomarker of the pluripotent stem cells is increased at least 2-fold compared with the control. In another embodiment, the expression level of at least one biomarker of the pluripotent stem cells is increased at least 3-fold compared with the control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 5-fold compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased in the range of 10-20-fold compared to a control. In an alternative embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased in the range of 20-30-fold compared to a control. In another alternative embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased in the range of 30-40-fold compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased in the range of 40-fold or higher compared to a control.

[0132] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the method is independent of invasive techniques.

[0133] In one embodiment of this disclosure, an in vitro method for predicting cancer is provided, the method comprising: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker of the pluripotent stem cells in a control sample, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells in the sample, compared with the expression level of at least one biomarker of the pluripotent stem cells in the control sample, predicts cancer.

[0134] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased at least 2-fold compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 3-fold compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 5-fold compared to a control. In alternative embodiments, the expression level of at least one biomarker of pluripotent stem cells is increased by 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold compared to a control.

[0135] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased by 10-20 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 20-30 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 30-40 times compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 40-50 times compared to a control.

[0136] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein at least one biomarker for pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4a. In yet another embodiment, at least one biomarker for pluripotent stem cells is Oct-4b.

[0137] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein at least one biomarker for pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Sox-2 and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Nanog and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is p53 and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-1 and a subset thereof. In another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-6 and a subset thereof. In another alternative embodiment, at least one biomarker for pluripotent stem cells is Sirt-3 and a subset thereof.

[0138] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein at least one biomarker for pluripotent stem cells is selected from NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is NAD and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is RAS and a subset thereof. In another embodiment, at least one biomarker for pluripotent stem cells is ERC and a subset thereof. In an alternative embodiment, at least one biomarker for pluripotent stem cells is erbB-2 and a subset thereof. In yet another alternative embodiment, at least one biomarker for pluripotent stem cells is ABL and a subset thereof.

[0139] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein nucleic acids are obtained from a mixture by means of any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkali extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0140] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker is performed using a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0141] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein a control is the expression level of at least one biomarker from pluripotent stem cells obtained from cancer-free subjects.

[0142] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells.

[0143] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells, wherein the at least one salt solution is sodium chloride and the neutral buffer is a Ficoll hypaque solution. In another embodiment, the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and combinations thereof.

[0144] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the method predicts all types of cancer known in the art.

[0145] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the method is independent of invasive techniques.

[0146] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the method further includes analyzing nucleic acids by performing sequence-based assays.

[0147] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the method further includes analyzing the nucleic acid by performing sequence-based assays, and wherein the type of cancer is detected by analyzing the nucleic acid by sequence-based assays.

[0148] In one embodiment of this disclosure, an in vitro method for evaluating the efficacy of a chemotherapeutic agent is provided, the method comprising: (a) obtaining a blood sample at a time point following administration of the chemotherapeutic agent; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in a reference for evaluating the efficacy of the chemotherapeutic agent.

[0149] In one embodiment of this disclosure, an in vitro method for evaluating the efficacy of a chemotherapeutic agent as described herein is provided, wherein the reference is selected from at least one of: (i) a blood sample obtained prior to administration of the chemotherapeutic agent; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); (iii) a blood sample obtained at a subsequent time point compared to the time point mentioned in step (a); and (iv) a blood sample obtained from a cancer-free subject. In another embodiment of this disclosure, the reference is a blood sample obtained prior to administration of the chemotherapeutic agent. In yet another embodiment, the reference is a blood sample obtained at a previous time point compared to the time point mentioned in step (a). In an alternative embodiment, the reference is a blood sample obtained from a cancer-free subject. In yet another alternative embodiment, the reference is a blood sample obtained at a subsequent time point compared to the time point mentioned in step (a).

[0150] In one embodiment of this disclosure, an in vitro method for evaluating the efficacy of a chemotherapeutic agent as described herein is provided, wherein a decrease in the expression level of at least one biomarker of pluripotent stem cells, compared to a reference level, indicates a positive response to cancer treatment, and wherein the reference is selected from (i) a blood sample obtained prior to administration of the chemotherapeutic agent; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); and (iii) a blood sample obtained from a cancer-free subject. In another embodiment of this disclosure, the reference is a blood sample obtained prior to administration of the chemotherapeutic agent. In yet another embodiment, the reference is a blood sample obtained at a previous time point compared to the time point mentioned in step (a). In yet another embodiment, the reference is a blood sample obtained from a cancer-free subject.

[0151] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to cancer treatment.

[0152] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to cancer treatment, wherein the reference is selected from at least one of: (i) a blood sample obtained before administration of anticancer therapy; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); (iii) a blood sample obtained at a subsequent time point compared to the time point mentioned in step (a); and (iv) a blood sample obtained from a cancer-free subject. In another embodiment of this disclosure, the reference is a blood sample obtained before administration of anticancer therapy. In another embodiment, the reference is a blood sample obtained at a previous time point compared to the time point mentioned in step (a). In an alternative embodiment, the reference is a blood sample obtained from a cancer-free subject. In another alternative embodiment, the reference is a blood sample obtained at a subsequent time point compared to the time point mentioned in step (a).

[0153] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) analyzing the expression level of at least one biomarker of the pluripotent stem cells using the nucleic acids; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to cancer treatment, wherein a decrease in the expression level of at least one biomarker of the pluripotent stem cells compared to the reference level indicates a positive response to the cancer treatment, and wherein the reference is selected from at least one of: (i) a blood sample obtained before administration of anticancer therapy; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); and (iii) a blood sample obtained from a cancer-free subject. In another embodiment of this disclosure, the reference is a blood sample obtained before administration of anticancer therapy. In another implementation, the reference is a blood sample obtained at a previous time point compared to the time point mentioned in step (a). In an alternative implementation, the reference is a blood sample obtained from a cancer-free subject.

[0154] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to cancer treatment, wherein a decrease in the expression level of at least one biomarker of the pluripotent stem cells compared to a reference level indicates a positive response to the cancer treatment, and wherein the reference is selected from at least one of: (i) a blood sample obtained prior to administration of anticancer therapy; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); and (iii) a blood sample obtained from a cancer-free subject, wherein the expression level is reduced by at least two-fold compared to the reference level. In another embodiment, the expression level is reduced by at least three-fold compared to the reference level. In another embodiment, the expression level is reduced by at least four times compared to the reference level. In an alternative embodiment, the expression level is reduced by at least five times compared to the reference level.

[0155] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; and (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to cancer treatment, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells compared to the reference level indicates a negative response to the cancer treatment, and wherein the reference is selected from at least one of: (i) a blood sample obtained prior to administration of anticancer therapy; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); and (iii) a blood sample obtained from a cancer-free subject.

[0156] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment as described herein is provided, wherein at least one biomarker of the pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, or a combination thereof. In another embodiment, at least one biomarker of the pluripotent stem cells is Oct-4. In another embodiment, at least one biomarker of the pluripotent stem cells is Oct-4a. In yet another embodiment, at least one biomarker of the pluripotent stem cells is Oct-4b.

[0157] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment, as described herein, is provided, wherein at least one biomarker of pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Sox-2 and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Nanog and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is p53 and a subset thereof. In an alternative embodiment, at least one biomarker of pluripotent stem cells is Sirt-1 and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Sirt-6 and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Sirt-3 and a subset thereof.

[0158] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment, as described herein, is provided, wherein at least one biomarker of the pluripotent stem cells is selected from NAD, RAS, ERC, erbB-2, ABL, a subset thereof, or a combination thereof. In another embodiment, at least one biomarker of the pluripotent stem cells is NAD and a subset thereof. In another embodiment, at least one biomarker of the pluripotent stem cells is RAS and a subset thereof. In another embodiment, at least one biomarker of the pluripotent stem cells is ERC and a subset thereof. In an alternative embodiment, at least one biomarker of the pluripotent stem cells is erbB-2 and a subset thereof. In a second alternative embodiment, at least one biomarker of the pluripotent stem cells is ABL and a subset thereof.

[0159] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment as described herein is provided, wherein nucleic acids are obtained from the mixture by means of any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkali extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0160] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment as described herein is provided, wherein the determination of the expression of at least one biomarker by analyzing nucleic acids is performed using a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0161] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment, as described herein, is provided, wherein a control is the expression level of at least one biomarker from pluripotent stem cells obtained from cancer-free subjects.

[0162] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells. In another embodiment, the at least one salt solution is sodium chloride, and the neutral buffer is a Ficoll-Hyaque solution.

[0163] In one embodiment of this disclosure, an in vitro method for monitoring response to cancer treatment as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells, wherein the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and combinations thereof.

[0164] In one embodiment of this disclosure, an in vitro method for monitoring the response to cancer treatment, as described herein, is provided, wherein the method does not rely on invasive techniques.

[0165] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment is provided, the method comprising: (a) obtaining a blood sample-I prior to administration of anticancer therapy; (b) obtaining a blood sample-II after administration of anticancer therapy; (c) enriching pluripotent stem cells from blood sample-I to obtain a mixture-I containing the pluripotent stem cells; (d) enriching pluripotent stem cells from blood sample-II to obtain a mixture-II containing the pluripotent stem cells; (e) obtaining nucleic acid-I from mixture-I; (f) obtaining nucleic acid-II from mixture-II; (g) independently determining the expression level of at least one biomarker of pluripotent stem cells using nucleic acid-I and nucleic acid-II; and (h) comparing the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-II with the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-I, wherein a decrease in the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-II compared to the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-I indicates a positive response to cancer treatment.

[0166] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-II is reduced by at least 2-fold compared to the expression level of at least one biomarker of pluripotent stem cells in nucleic acid-I. In another embodiment, the reduction is at least 3-fold. In another embodiment, the reduction is at least 4-fold. In yet another embodiment, the reduction is at least 5-fold.

[0167] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein at least one biomarker for pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4. In another embodiment, at least one biomarker for pluripotent stem cells is Oct-4a. In yet another embodiment, at least one biomarker for pluripotent stem cells is Oct-4b.

[0168] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein at least one biomarker of pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Sox-2 and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is Nanog and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is p53 and a subset thereof. In an alternative embodiment, at least one biomarker of pluripotent stem cells is Sirt-1 and a subset thereof. In another alternative embodiment, at least one biomarker of pluripotent stem cells is Sirt-6 and a subset thereof. In another alternative embodiment, at least one biomarker of pluripotent stem cells is Sirt-3 and a subset thereof.

[0169] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein at least one biomarker of pluripotent stem cells is selected from NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. In another embodiment, at least one biomarker of pluripotent stem cells is NAD and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is RAS and a subset thereof. In another embodiment, at least one biomarker of pluripotent stem cells is ERC and a subset thereof. In an alternative embodiment, at least one biomarker of pluripotent stem cells is erbB-2 and a subset thereof. In yet another alternative embodiment, at least one biomarker of pluripotent stem cells is ABL and a subset thereof.

[0170] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment as described herein is provided, wherein nucleic acids are obtained from the mixture by means of any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkali extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0171] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein the determination of the expression of at least one biomarker by analyzing nucleic acids is performed using a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0172] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells. In another embodiment, the at least one salt solution is sodium chloride, and the neutral buffer is a Ficoll-hyaque solution.

[0173] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (i) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (ii) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (iii) treating the second mixture to obtain enriched pluripotent stem cells, wherein the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and combinations thereof.

[0174] In one embodiment of this disclosure, an in vitro method for detecting a positive response to cancer treatment, as described herein, is provided, wherein the method does not rely on invasive techniques.

[0175] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for the detection of cancer from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0176] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for the detection of cancer from blood samples is provided. The pluripotent stem cell biomarkers are selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. The blood sample is processed using a method comprising the steps of: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) processing the second mixture to obtain a treated second mixture containing pluripotent stem cells, wherein the pluripotent stem cell biomarkers are analyzed from the treated second mixture obtained from the blood sample.

[0177] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for predicting cancer from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0178] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for predicting cancer from a blood sample is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets of the above, and combinations thereof, wherein the blood sample is processed using a method comprising the steps of: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) processing the second mixture to obtain a treated second mixture containing pluripotent stem cells, and wherein the pluripotent stem cell biomarkers are analyzed from the treated second mixture obtained from the blood sample.

[0179] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for grading cancer stages from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof.

[0180] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for grading cancer stages from blood samples is provided. The pluripotent stem cell biomarkers are selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof. The blood sample is processed using a method comprising the steps of: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain a treated second mixture containing pluripotent stem cells, wherein the pluripotent stem cell biomarkers are analyzed from the treated second mixture obtained from the blood sample.

[0181] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for monitoring the progress of anticancer therapy from blood samples is provided, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets of the above, and combinations thereof.

[0182] In one embodiment of this disclosure, the use of pluripotent stem cell biomarkers for monitoring the progress of anticancer therapy from blood samples is provided. The pluripotent stem cell biomarkers are selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets of the above, and combinations thereof. The blood sample is processed using a method comprising the steps of: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) processing the second mixture to obtain a treated second mixture containing pluripotent stem cells, and wherein the pluripotent stem cell biomarkers are analyzed from the treated second mixture obtained from the blood sample.

[0183] In one embodiment of this disclosure, a method for treating cancer is provided, the method comprising: (a) obtaining a blood sample from a subject; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control detects cancer; and (f) administering an anticancer therapy to the subject for treating cancer.

[0184] In one embodiment of this disclosure, a method for treating cancer is provided, the method comprising: (a) obtaining a blood sample from a subject; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control detects cancer; (f) performing sequence-based determination of the nucleic acids and analyzing mutations in at least one cancer-related biomarker, wherein the presence of mutations in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker; and (g) administering an anticancer therapy to the subject for treating cancer.

[0185] In one embodiment of this disclosure, a method for treating cancer is provided, the method comprising: (a) obtaining a blood sample from a subject; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture of step (b); (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with a control, wherein an increase in the expression level of the at least one biomarker compared to the control detects cancer; and (f) performing sequence-based sequencing on the nucleic acids. (g) Identify and analyze mutations in at least one cancer-related biomarker, wherein the presence of a mutation in the at least one cancer-related biomarker indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker; and (g) administer anticancer therapy to the subject for the treatment of cancer, wherein the at least one biomarker of pluripotent stem cells is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset thereof, and a combination thereof, and wherein the cancer-related biomarker refers to any biomarker associated with cancer detection.In another embodiment, the cancer-related biomarker is selected from at least one of the following: ABL1, EVI1, MYC, APC, IL2, TNFAIP3, ABL2, EWSR1, MYCL1, ARHGEF12, JAK2, TP53, AKT1, FEV, MYCN, ATM, MAP2K4, TSC1, AKT2, FGFR1, NCOA4, BCL11B, MDM4, TSC2, ATF1, FGFR1OP, NFKB2, BLM, MEN1, VHL, BCL11A, FGFR 2. NRAS, BMPR1A, MLH1, WRN, BCL2, FUS, NTRK1, BRCA1, MSH2, WT1, BCL3, GOLGA5, NUP214, BRCA2, NF1, BCL6, GOPC, PAX8, CARS , NF2, BCR, HMGA1, PDGFB, CBFA2T3, NOTCH1, BRAF, HMGA2, PIK3CA, CDH1, NPM1, CARD11, HRAS, PIM1, CDH11, NR4A3, CBLB, IRF 4. PLAG1, CDK6, NUP98, CBLC, JUN, PPARG, CDKN2C, PALB2, CCND1, KIT, PTPN11, CEBPA, PML, CCND2, KRAS, RAF1, CHEK2, PTEN , CCND3, LCK, REL, CREB1, RB1, CDX2, LMO2, RET, CREBBP, RUNX1, CTNNB1, MAF, ROS1, CYLD, SDHB, DDB2, MAFB, SMO, DDX5, SDHD Combinations of DDIT3, MAML2, SS18, EXT1, SMARCA4, DDX6, MDM2, TCL1A, EXT2, SMARCB1, DEK, MET, TET2, FBXW7, SOCS1, EGFR, MITF, TFG, FH, STK11, ELK4, MLL, TLX1, FLT3, SUFU, ERBB2, MPL, TPR, FOXP1, SUZ12, ETV4, MYB, USP6, GPC3, SYK, ETV6, IDH1, TCF3 and above.

[0186] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein the cancer being treated is an early-stage cancer that is undetectable by conventional methods and therefore cannot be treated with conventional anticancer therapies. The treatment method described herein does not involve any invasive techniques such as biopsy.

[0187] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein nucleic acids are obtained from a mixture by means of any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkali extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0188] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein the analysis of the expression of at least one biomarker using nucleic acids is performed by a technique selected from quantitative PCR, flow cytometry, and next-generation sequencing (NGS).

[0189] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein a control is the expression level of at least one biomarker from pluripotent stem cells obtained from cancer-free subjects.

[0190] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer solution in a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution in a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells.

[0191] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein enriching pluripotent stem cells from a blood sample comprises: (a) contacting the blood sample with a neutral buffer at a ratio ranging from 1:1 to 1:20 to obtain a first mixture; (b) contacting the first mixture with at least one salt solution at a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and (c) treating the second mixture to obtain enriched pluripotent stem cells, wherein the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and combinations thereof.

[0192] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased at least 2-fold compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 3-fold compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased at least 5-fold compared to a control. In alternative embodiments, the expression level of at least one biomarker of pluripotent stem cells is increased by 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold.

[0193] In one embodiment of this disclosure, a method for treating cancer as described herein is provided, wherein the expression level of at least one biomarker of pluripotent stem cells is increased by 10-20 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 20-30 times compared to a control. In another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 30-40 times compared to a control. In yet another embodiment, the expression level of at least one biomarker of pluripotent stem cells is increased by 40 times or more compared to a control.

[0194] In one embodiment of this disclosure, a method for treating cancer is provided, the method comprising: (a) obtaining a blood sample from a subject at a time point following anticancer therapy; (b) enriching pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells; (c) obtaining nucleic acids from the mixture; (d) using the nucleic acids to determine the expression level of at least one biomarker of the pluripotent stem cells; (e) comparing the expression level of at least one biomarker of the pluripotent stem cells with the expression level of at least one biomarker of the pluripotent stem cells in the reference for monitoring the response to anticancer therapy, wherein an increase in the expression level of at least one biomarker of the pluripotent stem cells compared to the reference indicates a negative response to the anticancer therapy; and (f) administering an alternative anticancer therapy for treating cancer, wherein the alternative anticancer therapy is administered to treat the cancer, and wherein the reference is selected from at least one of: (i) a blood sample obtained prior to the administration of the anticancer therapy; (ii) a blood sample obtained at a previous time point compared to the time point mentioned in step (a); and (iii) a blood sample obtained from a cancer-free subject.

[0195] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing lateral flow assays.

[0196] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a chip-based assay.

[0197] In one embodiment of this disclosure, an in vitro method for detecting cancer as described herein is provided, wherein the method is optimized for performance in a chip-based assay.

[0198] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a lateral flow assay.

[0199] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a chip-based assay.

[0200] In one embodiment of this disclosure, an in vitro method for predicting cancer as described herein is provided, wherein the method is optimized for performance in a chip-based assay.

[0201] In one embodiment of this disclosure, an in vitro method for detecting metabolically altered cells as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a lateral flow assay.

[0202] In one embodiment of this disclosure, an in vitro method for detecting metabolically altered cells as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a chip-based assay.

[0203] In one embodiment of this disclosure, an in vitro method for detecting metabolically altered cells as described herein is provided, wherein the method is optimized for use in a chip-based assay.

[0204] In one embodiment of this disclosure, an in vitro method for detecting quiescent cells as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a lateral flow assay.

[0205] In one embodiment of this disclosure, an in vitro method for detecting quiescent cells as described herein is provided, wherein the determination of the expression of at least one biomarker for evaluating pluripotent stem cells using the nucleic acid is performed by employing a chip-based assay.

[0206] In one embodiment of this disclosure, an in vitro method for detecting quiescent cells as described herein is provided, wherein the method is optimized for use in a chip-based assay.

[0207] In one embodiment of this disclosure, a kit is provided that is optimized with relevant components for performing the in vitro methods described herein.

[0208] Although the subject matter has been described with reference to specific embodiments, this description is not intended to be limiting. Various modifications to the disclosed embodiments, as well as alternative embodiments of the subject matter, will become apparent to those skilled in the art upon reference to the description of the subject matter. Therefore, it is conceivable that such modifications may be made without departing from the spirit or scope of the inventive subject matter as defined.

[0209] This invention provides, but is not limited to, the following embodiments:

[0210] 1. An in vitro method for detecting the presence of cells with metabolic alterations, the method comprising:

[0211] a) Obtain a blood sample;

[0212] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0213] c) Obtain nucleic acids from the mixture of step (b);

[0214] d) Determining the expression level of at least one biomarker of pluripotent stem cells using the said nucleic acid; and

[0215] e) Compare the expression levels of at least one biomarker of the pluripotent stem cells in the sample with the expression levels of at least one biomarker in the control sample.

[0216] The increase in the expression level of the at least one biomarker in the sample, compared to the expression level of the control sample, detected the presence of cells with metabolic alterations.

[0217] 2. An in vitro method for detecting the presence of quiescent cells, the method comprising:

[0218] a) Obtain a blood sample;

[0219] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0220] c) Obtain nucleic acids from the mixture of step (b);

[0221] d) Determining the expression level of at least one biomarker of pluripotent stem cells using the said nucleic acid; and

[0222] e) Compare the expression levels of at least one biomarker of the pluripotent stem cells in the sample with the expression levels of at least one biomarker in the control sample.

[0223] The increase in the expression level of the at least one biomarker in the sample compared to the expression level of the control sample detected the presence of quiescent cells.

[0224] 3. An in vitro method for detecting cancer, the method comprising:

[0225] a) Obtain a blood sample;

[0226] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0227] c) Obtain nucleic acids from the mixture of step (b);

[0228] d) Determining the expression level of at least one biomarker of pluripotent stem cells using the said nucleic acid; and

[0229] e) Compare the expression levels of at least one biomarker of the pluripotent stem cells in the sample with the expression levels of at least one biomarker in the control sample.

[0230] The increase in the expression level of the at least one biomarker in the sample, compared to the expression level of the control sample, detected cancer.

[0231] 4. An in vitro method for predicting cancer, the method comprising:

[0232] a) Obtain a blood sample;

[0233] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0234] c) Obtain nucleic acids from the mixture of step (b);

[0235] d) Determining the expression level of at least one biomarker of pluripotent stem cells using the said nucleic acid; and

[0236] e) Compare the expression levels of at least one biomarker of the pluripotent stem cells in the sample with the expression levels of at least one biomarker in the control sample.

[0237] An increase in the expression level of at least one biomarker in the sample, compared to the expression level of the control sample, predicts cancer.

[0238] 5. The method of any one of embodiments 1-4, wherein the method further comprises analyzing the nucleic acid by performing sequence-based determination.

[0239] 6. The method as described in Embodiment 5, wherein the type of cancer is detected by analyzing the nucleic acid through sequence-based assays.

[0240] 7. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by at least 2 times compared with the control.

[0241] 8. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by at least 3 times compared with the control.

[0242] 9. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by at least 5-fold compared with the control.

[0243] 10. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by 10-20 times compared with the control.

[0244] 11. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by 20-30 times compared with the control.

[0245] 12. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by 30-40 times compared with the control.

[0246] 13. The method of any one of embodiments 1, 3 or 4, wherein the expression level of at least one biomarker of the pluripotent stem cells is increased by 40-50 times compared with the control.

[0247] 14. An in vitro method for monitoring the response to anticancer therapy, the method comprising:

[0248] a) Obtain a blood sample at one point during cancer therapy;

[0249] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0250] c) Obtain nucleic acids from the mixture of step (b);

[0251] d) Determining the expression level of at least one biomarker of pluripotent stem cells using the said nucleic acid; and

[0252] f) Compare the expression levels of at least one biomarker of the pluripotent stem cells in the sample with the expression levels of at least one biomarker of the pluripotent stem cells in a reference for monitoring the response to anticancer therapy.

[0253] 15. The method of embodiment 14, wherein the reference is selected from at least one of: (a) a blood sample obtained before the administration of anticancer therapy; (b) a blood sample obtained at a previous time point compared to the time point described in step (a) of embodiment 14; (c) a blood sample obtained at a subsequent time point compared to the time point described in step (a) of embodiment 14; and (d) a blood sample obtained from a cancer-free subject.

[0254] 16. The method of embodiment 14, wherein a decrease in the expression level of at least one biomarker of the pluripotent stem cells indicates a positive response to the anticancer therapy compared to the expression level in the reference, and wherein the reference is selected from at least one of: (a) a blood sample obtained before administration of the anticancer therapy; (b) a blood sample obtained at a previous time point compared to the time point described in step (a) of embodiment 14; and (c) a blood sample obtained from a cancer-free subject.

[0255] 17. An in vitro method for detecting a positive response to anticancer therapy, the method comprising:

[0256] a) Obtain a blood sample before administering anticancer therapy - I;

[0257] b) Obtaining blood samples after administration of anticancer treatment - II;

[0258] c) Enrich pluripotent stem cells from blood sample-I to obtain mixture-I containing said pluripotent stem cells;

[0259] d) Enrich pluripotent stem cells from blood sample-II to obtain mixture-II containing said pluripotent stem cells;

[0260] e) Obtain nucleic acid I from mixture I;

[0261] f) Obtaining nucleic acid II from mixture II;

[0262] g) Independently analyze the expression levels of at least one biomarker of pluripotent stem cells using said nucleic acid-I and said nucleic acid-II; and

[0263] h) Compare the expression levels of at least one biomarker from pluripotent stem cells of said nucleic acid-II with the expression levels of at least one biomarker from pluripotent stem cells of said nucleic acid-I.

[0264] A decrease in the expression level of at least one biomarker from pluripotent stem cells of nucleic acid-II, compared to the expression level of at least one biomarker from pluripotent stem cells of nucleic acid-I, detected a positive response to cancer treatment.

[0265] 18. An in vitro method for detecting cancer, the method comprising:

[0266] a) Obtain a blood sample;

[0267] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0268] c) Obtain nucleic acids from the mixture of step (b);

[0269] d) Use the nucleic acid to analyze the expression level of at least one biomarker of pluripotent stem cells;

[0270] e) Compare the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker in a control sample, wherein an increase in the expression level of at least one biomarker in the sample, compared to the expression level of at least one biomarker in the control sample, indicates the presence of cancer; and

[0271] f) Sequence-based determination of the nucleic acid and analysis of mutations in at least one cancer-related biomarker.

[0272] The presence of a mutation in at least one of the cancer-related biomarkers indicates the presence of a specific type of cancer based on the analyzed cancer-related biomarker.

[0273] 19. The method of any one of embodiments 1-4, 14, 17 or 18, wherein at least one biomarker of the pluripotent stem cell is selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset of the above and a combination thereof.

[0274] 20. The method of any one of embodiments 1-4, 14, 17 or 18, wherein the nucleic acid is obtained from the mixture by any of the following methods: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

[0275] 21. The method of any one of embodiments 1-4, 14, 17 or 18, wherein the determination of the expression of at least one biomarker by analyzing the nucleic acid is performed by a technique selected from quantitative PCR, flow cytometry and next-generation sequencing (NGS).

[0276] 22. The method of any one of embodiments 1-4, 14 or 18, wherein the control is the expression level of at least one biomarker of pluripotent stem cells obtained from cancer-free subjects.

[0277] 23. The method of any one of embodiments 1-4, 14, 17 or 18, wherein enriching pluripotent stem cells from the blood sample comprises:

[0278] a) Contact the blood sample with a neutral buffer solution at a ratio ranging from 1:1 to 1:20 to obtain a first mixture;

[0279] b) Contacting at least one salt solution with the first mixture in a ratio ranging from 1:2 to 1:10 to obtain a second mixture; and

[0280] c) Process the second mixture to obtain enriched pluripotent stem cells.

[0281] 24. The method of embodiment 23, wherein the treatment of the second mixture comprises at least one method selected from: (a) extraction; (b) washing; (c) centrifugation and a combination thereof.

[0282] 25. The method of embodiment 18, wherein the cancer-related biomarker is selected from well-known biomarkers identified as being associated with cancer.

[0283] 26. The method of any one of embodiments 1-4, 14, 17 or 18, wherein the method is independent of invasive techniques.

[0284] 27. Use of pluripotent stem cell biomarkers for the detection of cancer from blood samples, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset of the above, and combinations thereof.

[0285] 28. Use of pluripotent stem cell biomarkers for predicting cancer from blood samples, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset of the above, and combinations thereof.

[0286] 29. The use of pluripotent stem cell biomarkers for grading cancer stages from blood samples, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset of the above, and combinations thereof.

[0287] 30. Use of pluripotent stem cell biomarkers for monitoring the progress of anticancer therapy from blood samples, said pluripotent stem cell biomarkers being selected from Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, a subset of the above, and combinations thereof.

[0288] 31. A method for treating cancer, the method comprising:

[0289] a) Obtaining blood samples from the subject;

[0290] b) Enrich pluripotent stem cells from the blood sample to obtain a mixture containing the pluripotent stem cells;

[0291] c) Obtain nucleic acids from the mixture of step (b);

[0292] d) Use the nucleic acid to analyze the expression level of at least one biomarker of pluripotent stem cells;

[0293] e) Comparing the expression level of at least one biomarker of the pluripotent stem cells in the sample with the expression level of at least one biomarker in a control sample, wherein an increase in the expression level of at least one biomarker in the sample compared to the expression level of at least one biomarker in the control sample detects cancer; and

[0294] f) Administer anticancer therapy to the subject for the treatment of cancer.

[0295] Example

[0296] This disclosure will now be illustrated with working examples, which are intended to illustrate the work of this disclosure and are not intended to restrictively imply any limitation on the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods and materials can be used to implement the disclosed methods and compositions, exemplary methods, apparatus, and materials are described herein. It should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0297] The following paragraphs illustrate examples of how the methods described in this disclosure work.

[0298] Example 1

[0299] Research Design

[0300] This diagnostic human clinical study was prospectively registered with the Indian Council of Medical Research (ICMR) – Indian Clinical Trials Registry (CTRI) under the following number: CTRI / 2019 / 01 / 017166. This study was a double-blind study of blood samples from major centers in India. The study was conducted with 1000 samples, and the results showed that 500 of the 1000 samples were from healthy subjects.

[0301] Example 2

[0302] Detailed in vitro methods for studying the expression of at least one biomarker of pluripotent stem cells

[0303] Blood samples (5-10 ml) are obtained as part of the registration study. The samples are processed using known techniques, and nucleic acids are then extracted. The processed samples are subjected to a procedure for enriching pluripotent stem cells, followed by expression analysis of at least one biomarker for pluripotent stem cells. The procedures listed below are used to detect, predict, and monitor cancer in the subjects. Detailed procedures applied are described below.

[0304] 1. Blood samples (test samples) were obtained at one point in time as part of this study.

[0305] 2. Contact the blood sample with neutral buffer at a ratio ranging from 1:1 (blood sample: neutral buffer) to 1:20 to obtain a first mixture.

[0306] 3. Contact at least one salt solution with a first mixture at a ratio ranging from 1:2 (salt solution: first mixture) to 1:10 to obtain a second mixture.

[0307] 4. Process the second mixture to obtain a treated second mixture containing pluripotent stem cells.

[0308] 5. Obtaining nucleic acids from pluripotent stem cells using methods known in the art. For the purposes of this example, total mRNA was isolated from pluripotent stem cells using methods known in the art.

[0309] 6. Expression studies using mRNA samples, and quantitative PCR methods to investigate the expression of at least one biomarker from pluripotent stem cells selected from Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, NAD, RAS, ERC, erbB-2, ABL, subsets of the above, and combinations thereof. Expression studies shall be conducted using methods known in the art. Expression shall be investigated by NGS or qPCR.

[0310] 7. In cases where the research objective is detection or prediction, the expression levels obtained from previous steps are compared with a control, wherein the control represents the expression level of at least one biomarker of pluripotent stem cells obtained from blood samples isolated from non-cancer subjects.

[0311] 7A. In cases where the research objective is to monitor cancer, the obtained expression levels are compared with a reference level, wherein the reference level is selected from at least one of: (a) a blood sample obtained before the administration of anticancer therapy; (b) a blood sample obtained at a previous time point compared to the time point mentioned in the first step; (c) a blood sample obtained at a subsequent time point compared to the time point mentioned in the first step; and (d) a blood sample obtained from a cancer-free subject.

[0312] The methodologies and reagents used to implement the above methods are described below.

[0313] RNA isolation and expression analysis (steps 5 and 6)

[0314] Total RNA was isolated using RNA Plus (MP Biomedicals, Irvine, CA) according to the manufacturer's instructions. First-strand cDNA was synthesized using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Science, UK) according to the manufacturer's instructions. Briefly, 1 μg of total RNA was incubated with a mixture of 5x reaction buffer and reverse transcriptase. Reactions were performed in an Applied Biosystems GeneAmp® Thermal Cyclist 9700 (Applied Bio-systems, USA) according to the manufacturer's instructions. Gene transcript expression levels were estimated using the Thermo Scientific Maxima SYBR Green / ROX qPCRMaster Mix Kit (Thermo Science, UK) via a real-time PCR system ABI 7500 (Applied Bio-systems, USA). 18S rRNA was used as a housekeeping gene. The amplification conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 40 cycles of denaturation at 94°C for 10 seconds, annealing for 20 seconds, and extension at 72°C for 30 seconds, followed by melting curve analysis. Emitted fluorescence was collected during the extension step of each cycle. The homogeneity of the PCR amplicons was verified by running the products on a 2% agarose gel and also by studying the melting curves. All PCR amplifications were performed in triplicate. The average Ct value generated in each experiment using 7500 Manager software (Applied Biosystems, UK) was used to calculate mRNA expression levels. Fold change was calculated using the ΔΔCt method. The relative expression levels of each gene were compared to a baseline level. NGS technology was also used to analyze the expression and / or mutations of biomarkers or cancer-related biomarkers.

[0315] Treatment of the second mixture containing pluripotent stem cells (step 4)

[0316] The treatment of the second mixture includes a combination of extraction, washing, and centrifugation methods to obtain a treated second mixture containing the pluripotent stem cells.

[0317] For the purposes of this example, the process includes:

[0318] 1. Centrifuge the second mixture at a speed of 1000-6000 rpm for 5-20 minutes to obtain the supernatant and precipitate.

[0319] 2. Extract the supernatant or wash the precipitate to obtain a third mixture.

[0320] 3. Centrifuge the third mixture sequentially at varying speeds of 1000-10,000 rpm for 2-8 cycles to obtain a treated mixture containing the pluripotent stem cells.

[0321] Salt solution (step 3)

[0322] The salt solution used in step 3 is sodium chloride. It should be understood that any suitable salt solution may also be used.

[0323] Neutral buffer solution (step 2)

[0324] The neutral buffer used in step 2 is selected from ficoll hypaque. It should be understood that any suitable neutral buffer may also be used.

[0325] Example 3

[0326] Analysis of expression levels studied in Example 2

[0327] The in vitro method described in Example 2 can detect and predict cancer in samples. Furthermore, this method can monitor cancer progression to check whether the subject provides a positive response to anti-cancer treatment. Additionally, once treatment is completed, the method can monitor the performance of follow-up care to check for any chance of cancer recurrence. Different types of analyses that can be based on the methods of this disclosure are provided below.

[0328] Analysis of comparative expression levels:

[0329] 1. If the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample is increased by 5 to 50 times compared with the control, the sample is declared cancer-positive.

[0330] 2. If the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample increases less than 2-fold compared with the control, the sample is declared cancer-negative.

[0331] Classifying cancer into different stages: The methods disclosed in this disclosure can also be used to classify different stages of cancer. In ongoing studies, the different stages of cancer are determined as follows.

[0332] 1. When the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample increases by 6-10 times compared with the control – precancerous stage.

[0333] 2. Stage I cancer when the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample increases by 10-20 times compared with the control.

[0334] 3. Stage II cancer when the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample increases by 20-30 times compared with the control.

[0335] 4. Stage III cancer when the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample increases by 30-40 times compared with the control.

[0336] 5. Stage IV cancer when the expression level of at least one biomarker of pluripotent stem cells obtained from the test sample is increased by 40-fold or higher compared with the control.

[0337] The correlation between the fold increase in the expression of at least one biomarker of pluripotent stem cells and the results of the in vitro methods disclosed herein has been collected in Table 1 below.

[0338] Table 1:

[0339]

[0340] The terms “HrC range” or “fold change range” described in this disclosure are used interchangeably to refer to the fold change range of at least one biomarker of pluripotent stem cells. The terms “numerical value” or “HrC value” or “fold change” are used interchangeably to refer to a specific value within a specific range. Suggestions for analysis of expression levels of fold changes are herein provided. Ranges provided in the tables are referenced to “control” or “reference” as defined herein, as appropriate.

[0341] Analysis used to monitor cancer progression:

[0342] 1. A decrease in the expression level of at least one biomarker of pluripotent stem cells obtained from a test sample compared to a reference indicates a positive response to anticancer therapy, wherein the reference is selected from: (a) a blood sample obtained before administration of anticancer therapy; (b) a blood sample obtained at a previous time point compared to the time point of the test blood sample; and (c) a blood sample obtained from a cancer-free subject.

[0343] 2. An increase in the expression level of at least one biomarker of pluripotent stem cells obtained from a test sample compared to a reference indicates a negative response to anticancer therapy, wherein the reference is selected from at least one of: (a) a blood sample obtained before administration of anticancer therapy; (b) a blood sample obtained at a previous time point compared to the time point of the test blood sample; (c) a blood sample obtained at a subsequent time point compared to the time point of the test blood sample; and (d) a blood sample obtained from a cancer-free subject.

[0344] Example 4

[0345] Clinical research results obtained by evaluating the expression of the Sox2 biomarker

[0346] In samples obtained as part of the design of this study, the fold change in expression level of Sox2 (gene ID: 6657) in 20 samples derived from pluripotent cells obtained from corresponding blood samples was investigated. Figure 1 A graph depicting the fold change in Sox2 expression levels. From Figure 1 It was observed that samples 1 through 11 had fold changes in Sox2 expression of less than 20. Samples 2 and 4 had fold changes in expression of less than 6, and were therefore associated with samples without cancer, while all the other samples 1 through 11 had values ​​associated with their precancerous stage. Samples 13 through 20 had fold changes in expression of more than 50, which, according to this disclosure, are associated with stage IV cancer. The observations of this disclosure are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0347] Example 5

[0348] Clinical research results obtained by evaluating the expression of Nanog biomarkers

[0349] Twenty different samples (different from those selected in Example 4) were considered for studying the expression of Nanog (gene ID: 79923) as a biomarker. Figure 2 The fold changes in Nanog expression in 20 samples were described. It was observed that samples 1 through 11 had fold changes within 2, which, according to this document, are associated with samples as non-cancer samples and samples from cancer-free subjects. Samples 12 and 18 had fold changes in the range of 10-20 folds, and are therefore associated with stage I cancer, while samples 13-17, 19, and 20 had fold changes in the range of 20-30 folds compared to controls, and are therefore associated with stage II cancer. The observations in this embodiment are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0350] Example 6

[0351] Clinical research results obtained by evaluating the expression of the Oct-4a biomarker

[0352] This example considers 20 different samples (different from those mentioned in Examples 4 and 5) for studying Oct-4a (gene ID: ) as a biomarker. 642559 The expression of ). Figure 3A graph depicting the fold change values ​​of 20 samples is presented. It can be observed that the fold change values ​​of the samples are confirmed by observations of both cancer-free and cancer-present outcomes. Samples 1 to 4, 9, 10, and 15-17 have values ​​within 2 and are therefore correlated with cancer-free observations, while the other samples are correlated with stage I, II, III, or IV cancer based on their values. The observations in this embodiment are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the ability of the method of this disclosure to detect the presence of cancer.

[0353] Example 7

[0354] Clinical research results obtained by evaluating the expression of the Sirt-1 biomarker

[0355] In this embodiment, 20 different samples (different from those mentioned in Examples 4-6) were considered to study the expression of Sirt-1 (gene ID: 23411) as a biomarker.

[0356] Figure 4 A graph depicting the fold change values ​​of 20 samples is presented. Since none of the samples had a fold change value less than 2, none were associated with the absence of cancer. Samples 6, 9, and 13-20 had fold change values ​​greater than 50, thus being associated with the presence of stage IV cancer. The observations in this embodiment are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0357] Example 8

[0358] Clinical research results obtained by evaluating the expression of the Sirt6 biomarker

[0359] In samples obtained as part of this study design, the fold-wise variation of Sirt6 (gene ID: 51548) expression levels was investigated in 20 different samples (different from Examples 4-7), the Sirt6 cells derived from pluripotent cells obtained from the corresponding blood samples. The method for detecting the presence of cancer is as described in this disclosure and Example 2.

[0360] Figure 5 A graph depicting the fold change in Sirt6 expression levels. From Figure 5 It was observed that samples 5 to 9 had fold change values ​​within the range of 0-2, which are associated with healthy samples. Samples 11-20 had fold change values ​​within the carcinogenic range and were therefore labeled as cancer-positive samples. The observations of this disclosure are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0361] Example 9

[0362] Clinical research results obtained by evaluating the expression of NFκB biomarkers

[0363] Twenty different samples (different from those selected in Examples 4-8) were considered for studying the expression of NFκB (gene ID: 4791) as a biomarker. The method for detecting the presence of cancer is as described in this disclosure and Example 2.

[0364] Figure 6 The fold changes in NFκB expression in 20 samples were described. It was observed that samples 1 through 9 had fold change values ​​within the range specified for stage I or stage II cancer. Samples 10 through 20 had fold change values ​​within 2, and therefore correlated with non-cancer readings. The observations in this embodiment were correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0365] Example 10

[0366] Clinical research results obtained by evaluating the expression of Oct-4 biomarkers

[0367] This example considers 20 different samples (different from those mentioned in Examples 3-8) for studying Oct-4 (gene ID: ) as a biomarker. 642559 The expression is as described in this disclosure and Example 2. The method for detecting the presence of cancer is as described in this disclosure and Example 2.

[0368] Figure 7 A graph depicting the fold change values ​​of 20 samples is presented. It can be observed that samples 1 through 12 have fold change values ​​within 5 compared to the control, and are therefore associated with the absence of cancer. Samples 7, 8, 10, and 12, with values ​​greater than 2, are classified as having inflammation and are suspected of having some abnormality or condition causing inflammation in at least a part of the subject's body. Samples 13-19, with fold change values ​​in the range of 40-50 times, are associated with samples having stage IV cancer, while sample 20 is associated with the presence of stage II cancer because its fold change value is in the range of 20-30 times. The observations in this embodiment are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0369] Example 11

[0370] Clinical research results obtained by evaluating the expression of p53 biomarker

[0371] This embodiment considered 20 different samples (different from those mentioned in Examples 4-10) to study the expression of p53 (gene ID: 7157) as a biomarker. The method for detecting the presence of cancer is as described in this disclosure and Example 2.

[0372] Figure 8 A graph depicting the fold change values ​​of 20 samples is presented. Samples 5, 8, 9, 16, and 17 have fold change values ​​within 2 and are therefore cancer-free. Samples 2, 3, 6, 7, 15, 19, and 20 have fold change values ​​exceeding 50 and are therefore associated with advanced (stage IV) cancer. The observations in this embodiment are correlated with the medical history of the corresponding samples obtained as part of a double-blind study, thus demonstrating the capability of the method of this disclosure in detecting the presence of cancer.

[0373] Example 12

[0374] Results of all samples collected as part of this study design

[0375] This embodiment describes the results of a 1000-sample study conducted to determine the operation of the in vitro methods disclosed herein. Figures 9 to 18 The fold change (HrC value) of expression of at least one biomarker selected from pluripotent stem cells, including Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets of the above, and combinations thereof, is described. The method used to obtain the fold change value is performed according to the in vitro method of this disclosure as described in Example 2. Figures 9 to 18 The figures in the table depict data from 100 samples each. Green bars represent samples that are considered healthy according to the methods described in this disclosure, red bars represent cancer-positive samples, and yellow bars indicate samples that belong to a high-risk category for developing cancer.

[0376] Several case studies were conducted as part of the method for evaluating the contents of this disclosure. Some selected case studies are given below as part of this embodiment. The term "HrC test" refers to the in vitro procedure as disclosed in this disclosure.

[0377] Case Study 1

[0378] Subject details -- A blood sample was received from a 68-year-old male as part of this study, and no other details are known. The sample was tested for HrC.

[0379] The HrC test result on day 0 showed a reading of 9.78, indicating that the individual was a high-risk candidate. In addition to the high-risk category, due to... hoxb13A mutation was found in the gene, and the HrC test specifically detected the prostate as the organ at risk. At this stage, circulating tumor cells (CTCs) were absent. The results were shared with the individual, who underwent further analysis, which revealed elevated prostate-specific antigen (PSA) levels and prostate swelling (day 7). Following a consultation with an oncologist, the individual underwent a biopsy, which indicated… hoxb13 The mutation was detected on day 45, and the patient underwent a prostatectomy (day 65). On the day of surgery, the HrC value was 10.89, indicating that cancer was indeed developing. On day 75, gene expression results from the prostate tissue showed that the gene expression and mutation results obtained from the HrC test (from the blood sample) were almost identical to those obtained from the tissue. Therefore, the HrC test can provide the information that a biopsy can provide, and at an earlier stage, without using any invasive techniques. Six weeks after tumor resection (day 95), the HrC test showed a reading of 2.1, indicating that the individual was cancer-free.

[0380] The advantages provided by this disclosure This case study provides evidence that the HrC test has been successful in diagnosing and prognosing cancer. Gene expression and mutation outcomes are nearly identical in (cancer) tissue and blood. The HrC test was able to detect cancer from blood samples alone, even before CTCs were present.

[0381] Medical history – The subject’s medical history form disclosed the following details: hyperacidity, fatty liver, and diabetes.

[0382] Case Study 2

[0383] Subject Details – A blood sample was received from a 39-year-old woman as part of this study. The subject had been diagnosed with cervical adenocarcinoma and was scheduled for surgery to remove the tumor.

[0384] On the day of surgery (Day 0), the HrC value was 32.116 and CTCs were positive. Ten days post-surgery (Day 10), the HrC value decreased to 20.142 due to tumor removal. Post-surgery, she received 6 cycles of adjuvant chemotherapy (Days 20 to 160), and upon completion of chemotherapy, CTC testing was negative, while a PET scan showed no lesions. Based on imaging techniques, the patient was declared cancer-free, but an HrC value of 6.48 (high-risk category) (Day 167) was detected by the in vitro methods of this disclosure as proportionately indicating that the patient was in a high-risk category and at risk of recurrence. In addition to the inputs provided in this study, she is currently receiving an additional 3 cycles of chemotherapy.

[0385] This disclosure content Advantages--The HrC test has successfully assisted oncologists in monitoring disease progression and recurrence risk using non-invasive techniques. Therefore, the HrC test can detect cancer and recurrence risk before the presence of CTCs.

[0386] Case Study 3

[0387] Subject details -- Blood samples were received from women aged 65 years as part of this study.

[0388] Medical history – The subject was diagnosed with a tumor above the ovary.

[0389] The patient's HRC value was 41.28 (day 0), and cancer antigen 125 (CA125) was 198.8, accompanied by abdominal pain (day 3). Following diagnosis, the patient underwent surgery (day 7) to remove both ovaries, the uterus, and fallopian tubes. Immunohistochemical analysis of the excised tissue (day 10) indicated the primary site of cancer was in the stomach, as the tissue was positive for CK-20 and CDX2 / SATB2 markers. The primary site of cancer could not be detected by the physician, further impacting the treatment process (day 30). An HRC test accurately detected the primary site of cancer as the appendix (day 44).

[0390] The advantages provided by this disclosure The HrC test has been successful in diagnosing cancer and detecting the primary site of cancer (where conventional methods fail to detect the primary site). Therefore, the HrC test helps oncologists plan the treatment process in detail even when the primary site is undetectable, thereby increasing the patient's chances of survival.

[0391] Overview of the 1000-sample study: As part of this study, the types of cancer that were detected, predicted, and monitored are summarized in the table below.

[0392] Table 2:

[0393]

[0394] Table 2 suggests that, based on sample availability, the methods disclosed in this paper can effectively and accurately detect, predict, or monitor a wide range of cancers.

[0395] Advantages of this disclosure --The case studies mentioned in this embodiment clearly demonstrate that the in vitro methods disclosed in this disclosure offer more advantages and are more versatile and comprehensive than currently used techniques such as PET, CT, and liquid biopsy. These advantages are listed below.

[0396] Cancer detection--The methods disclosed in this disclosure enable the simple detection of cancer by analyzing blood samples, even before the advent of CTCs and even before PET or CT scans could be effective, thus increasing the survival chances of patients with cancer.

[0397] Predicting cancer --The method disclosed in this disclosure can easily predict the likelihood of cancer appearing in a subject by analyzing a blood sample. This method can accurately predict the type of cancer that will appear in a subject, which is currently unavailable in other technologies.

[0398] Monitoring cancer treatment --The method disclosed herein monitors cancer progression in subjects receiving anticancer therapy through a simple blood analysis. A specific advantage of this method is that it can be performed very frequently compared to PET scans (which can only be performed once every 6 months), thus saving valuable time in anticancer therapy. This method also checks for cancer recurrence and can guide oncologists in understanding the need for any available methods of cancer therapy without wasting time.

[0399] Detecting specific types of cancer without the need for a biopsy. The methods disclosed herein combine the analysis of expression levels of at least one biomarker of pluripotent stem cells with the study of mutations in cancer-related biomarkers to accurately detect specific types and stages of cancer from blood samples, thereby avoiding the use of any invasive techniques such as biopsies. Furthermore, since biopsies carry the risk of activating cancer-related activity in the subject, the methods disclosed herein have the particular advantage of not carrying any such risk.

[0400] Table 3 below describes the advantages of the in vitro methods of this disclosure compared to known techniques such as PET scans, CT scans, and conventional blood and tissue biopsy studies.

[0401] Table 3:

[0402]

[0403] This disclosure provides a simple, effective, safe, and sensitive method for detecting, predicting, and monitoring cancer and detecting cancer mutations in subjects from simple blood samples obtained from subjects. The method includes enriching pluripotent stem cells from the blood sample and analyzing the expression of pluripotent stem cell markers.

[0404] A significant advantage of the method disclosed in this disclosure is that it can predict and / or detect cancer in a subject without using any invasive techniques. Another significant advantage is that the method disclosed herein can analyze mutations in a set of genes, which can effectively infer the organs affected by cancer. Other methods known in the art cannot effectively infer organs infected by cancer from simple blood tests. The method of this disclosure effectively infers the organ where cancer is located solely from a blood sample. Conventional methods require the use of PET scans with radioactive isotopes to confirm the affected organ. The method of this disclosure involves a assay using only a blood sample from the subject, rather than the routinely performed tissue biopsy. Because this method does not use any radioactive isotopes, it can be performed frequently on subjects who have already received cancer treatment. Therefore, it can even be used to check whether the treatment provided is effective. The method is highly sensitive in such a way that it can predict the likelihood of a subject being diagnosed with cancer in the near future using a blood sample from the subject. The method disclosed herein can be used in combination with conventional techniques to provide an efficient combination procedure for detecting, predicting, or monitoring cancer in a subject. The methods disclosed in this disclosure are applicable to all types of cancer, and a non-limiting list has been disclosed in this disclosure.

Claims

1. Use of reagents for detecting Oct-4a in the preparation of a kit for analyzing blood samples from cancer patients, said detection comprising: (a) Enriching pluripotent stem cells from the blood sample of the subject to obtain a mixture containing the pluripotent stem cells; (b) Separating messenger ribonucleic acid (mRNA) from the mixture containing the pluripotent stem cells; (c) Using the isolated mRNA to determine the expression level of Oct-4a; and (d) Compare the expression level of Oct-4a in the blood sample with the expression level of Oct-4a in the control sample. The expression level of Oct-4a in the blood sample is increased by at least 10-fold compared to the expression level in the control sample, for the purpose of detecting the cancer in the subject.

2. The use as claimed in claim 1, wherein the assay for analyzing the isolated mRNA includes a sequence-based assay.

3. The use as claimed in claim 2, further comprising analyzing the isolated mRNA in response to a mutation in at least one cancer-associated biomarker, wherein the presence of the mutation in the at least one cancer-associated biomarker indicates the presence of a specific type of cancer.

4. The use as claimed in claim 1, wherein the assay for analyzing the isolated mRNA comprises quantitative polymerase chain reaction (PCR).

5. The use as claimed in claim 1, wherein the assay for analyzing the isolated mRNA comprises flow cytometry.

6. The use as claimed in claim 1, wherein the assay for analyzing the isolated mRNA comprises next-generation sequencing (NGS).

7. The use as claimed in claim 1, wherein the increase in the expression level of Oct-4a in the blood sample compared to the control sample is in the range of 10-20 times.

8. The use as claimed in claim 1, wherein the increase in the expression level of Oct-4a in the blood sample compared to the control sample is in the range of 20-30 times.

9. The use as claimed in claim 1, wherein the increase in the expression level of Oct-4a in the blood sample is in the range of 30-40 times compared to the control sample.

10. The use as claimed in claim 1, wherein the increase in the expression level of Oct-4a in the blood sample compared to the control sample is in the range of 40-50 times.

11. The use as claimed in claim 1, wherein the isolation of the mRNA comprises a method selected from the following: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

12. The use as claimed in claim 1, wherein the control sample is obtained from a cancer-free subject.

13. The use as claimed in claim 1, wherein the use is independent of invasive techniques.

14. Use of reagents for detecting Oct-4a in the preparation of a kit for analyzing blood samples from a subject, said detection comprising: (a) Enriching pluripotent stem cells from the blood sample of the subject to obtain a mixture containing the pluripotent stem cells; (b) Separating messenger ribonucleic acid (mRNA) from the mixture containing the pluripotent stem cells; (c) Using the isolated mRNA to determine the expression level of Oct-4a; and (d) Compare the expression level of Oct-4a in the blood sample with the expression level of Oct-4a in the control sample. An increase of up to twice the expression level of Oct-4a in the blood sample compared to the expression level in the control sample indicates that the subject does not have cancer.

15. The use as described in claim 14, wherein the assay for analyzing the isolated mRNA comprises a sequence-based assay.

16. The use as claimed in claim 15, further comprising analyzing the isolated mRNA in response to a mutation in at least one cancer-associated biomarker, wherein the presence of the mutation in the at least one cancer-associated biomarker indicates the presence of a specific type of cancer.

17. The use as claimed in claim 14, wherein the assay for analyzing the isolated mRNA comprises quantitative polymerase chain reaction (PCR).

18. The use as claimed in claim 14, wherein the assay for analyzing the isolated mRNA comprises flow cytometry.

19. The use as claimed in claim 14, wherein the assay for analyzing the isolated mRNA comprises next-generation sequencing (NGS).

20. The use as claimed in claim 14, wherein the isolation of the mRNA comprises a method selected from the following: (a) guanidine thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) hexadecyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid-phase nucleic acid extraction.

21. The use as claimed in claim 14, wherein the control sample is obtained from a cancer-free subject.

22. The use as described in claim 14, wherein the use is independent of invasive techniques.

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