Detection of tumor-derived nucleic acids using red blood cells

Abstract

This specification describes a method for diagnosing cancer in a subject. The method involves contacting a red blood cell-containing sample from the subject with a reagent capable of detecting tumor-related molecules in the sample, and diagnosing the subject as having a tumor if tumor-related molecules are detected in the sample. In some embodiments, the preferred sample volume is less than 10 μL.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 500,136, filed May 4, 2023, and U.S. Provisional Application No. 63 / 636,309, filed April 19, 2024, the entire contents of each of which are incorporated herein by reference as if fully set forth herein.

[0002] Incorporation by Reference of Material Submitted in Electronic Form The contents of the electronic sequence listing (UPN - 22 - 10081 - PCT.xml, size: 45,266 bytes, and creation date: May 6, 2024) are incorporated herein by reference in their entirety.

Background Art

[0003] Lung cancer is the leading cause of cancer - related death worldwide, with a 5 - year survival rate of only 23%. Profiling of circulating tumor - free cell DNA (ctDNA) has emerged as a promising tool for tumor genotyping without the need for biopsies, monitoring disease burden, and early cancer detection. ctDNA consists of short DNA fragments released into the bloodstream by tumor cells. ctDNA can be analyzed using high - sensitivity sequencing assays, enabling the detection of tumor - specific mutations, structural variants, and copy - number abnormalities in lung cancer patients. Analysis of ctDNA is increasingly being used for non - invasive mutation detection to guide the selection of targeted therapies in patients with progressive disease. However, ctDNA has a short half - life of only 1 - 2 hours in circulation and generally accounts for only a small fraction of total circulating cell - free DNA, significantly limiting the sensitivity and clinical utility of these assays.

[0004] In the art, there is a need for improved methods for detecting tumor DNA in patient samples.

Summary of the Invention

[0005] In a first embodiment of this specification, a method for diagnosing cancer in a subject is provided. This method includes contacting a red blood cell-containing sample from the subject with a reagent capable of detecting tumor-associated molecules in the sample, and diagnosing the subject as having cancer if tumor-associated molecules are detected in the sample. In certain embodiments, the reagent is specific to tumor-associated gene mutations. In certain embodiments, the reagent comprises a plurality of reagents, each capable of detecting a different specific tumor-associated molecule. In certain embodiments, the reagent includes a primer capable of binding to tumor-associated DNA or tumor-associated RNA. In certain embodiments, the tumor-associated gene mutation is found in the EGFR gene, ALK gene, KRAS gene, ROS1 gene, BRAF gene, NTRK gene, MET gene, RET gene, ERBB2 gene, PIK3CA gene, BRCA1 gene, BRCA2 gene, FGFR2 gene, FGFR3 gene, EZH2 gene, or PDGFRA gene.

[0006] In other embodiments, compositions and kits for carrying out the methods described herein are provided. [Brief explanation of the drawing]

[0007] [Figure 1A]This shows that RBCs obtained tumor DNA from lung cancer cells. Lung cancer cells (A549 cells with the G12S mutation) were seeded at a density of 1 × 10⁶ cells / well in 6-well plates. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to a method previously described by our group, and qPCR was performed using G12S-specific primers or universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the ground state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. A technical replica is shown in Figure 1. [Figure 1B] This shows that RBCs obtained tumor DNA from lung cancer cells. Lung cancer cells (A549 cells with the G12S mutation) were seeded at a density of 1 × 10⁶ cells / well in 6-well plates. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to a method previously described by our group, and qPCR was performed using commercially available mutant multiplex primers or universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the basal state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. A technical replica is shown in Figure 1. [Figure 1C]This shows that RBCs obtained tumor DNA from lung cancer cells. Lung cancer cells (A549 cells with the G12S mutation) were seeded at a density of 1 × 10⁶ cells / well in 6-well plates. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to a method previously described by our group, and qPCR was performed using universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the ground state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. A technical replica is shown in Figure 1. [Figure 1D] This shows that RBCs obtained tumor DNA from lung cancer cells. Lung cancer cells (A549 cells with the G12S mutation) were seeded at a density of 1 × 10⁶ cells / well in 6-well plates. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to a method previously described by our group, and qPCR was performed using G12S-specific primers or universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the ground state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. A technical replica is shown in Figure 1. [Figure 1E]Lung cancer cells (A549 cells with the G12S mutation) were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated, and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to a method previously described by our group, and qPCR was performed using commercially available mutant multiplex primers or universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the basal state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. Technical replicates are shown in Figure 1. [Figure 1F] Lung cancer cells (A549 cells with the G12S mutation) were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated, and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using universal KRAS primers. As seen in the figure above, RBCs do not contain KRAS DNA in the ground state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. Technical replicates are shown in Figure 1. [Figure 1G]Lung cancer cells (A549 cells with the G12S mutation) were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated, and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using universal KRAS primers. This figure summarizes the 24-hour and 72-hour co-culture tests. As seen in the figure above, RBCs do not contain KRAS DNA in the basal state, but DNA is obtained from tumor cells. This data is presented by cycle threshold (Ct), where a lower Ct indicates a richer DNA content. Technical replicates are shown in Figure 1. [Figure 1H] Lung cancer cells (A549 cells with the G12S mutation) were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. After growing the cells to confluence, they were incubated with a specified dose of highly purified RBCs for 24 or 72 hours. The cell culture supernatant was then aspirated, and the RBCs were isolated by centrifugation. The RBCs were then washed twice and cryopreserved at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using universal KRAS primers. This figure shows a DNA gel showing the G12S amplicon present in A549 cells or RBCs treated with A549 cells, with an expected amplicon size of 98 bp. As seen in the figure above, RBCs do not contain KRAS DNA in the basal state, but DNA is obtained from tumor cells. This data is presented with a cycle threshold (Ct), where a lower Ct indicates a richer DNA content. A technical replica is shown in Figure 1. [Figure 2A]This demonstrates that allele-specific primers are specific and do not amplify in control cell lines. Lung cancer cells (A549 cells with the G12S mutation, H358 cells with the G12C mutation, or H1299 cells) were seeded in 6-well plates. The following day, the cells were treated with various doses of purified RBCs. The RBCs were then washed twice and frozen at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using allele-specific primers (G12S or G12C), commercially available mutant multiplex primers, or universal KRAS primers. Below the bar graph, a heatmap summary of all experimental conditions is provided. As seen in the figure above, RBCs do not contain KRAS DNA under basal conditions, but DNA is obtained from tumor cells. The data are shown as cycle threshold (Ct), with lower Ct indicating a higher amount of DNA. [Figure 2B] This demonstrates that allele-specific primers are specific and do not amplify in control cell lines. Lung cancer cells (A549 cells with the G12S mutation, H358 cells with the G12C mutation, or H1299 cells) were seeded in 6-well plates. The following day, the cells were treated with various doses of purified RBCs. The RBCs were then washed twice and frozen at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using allele-specific primers (G12S or G12C), commercially available mutant multiplex primers, or universal KRAS primers. Below the bar graph, a heatmap summary of all experimental conditions is provided. As seen in the figure above, RBCs do not contain KRAS DNA under basal conditions, but DNA is obtained from tumor cells. The data are shown as cycle threshold (Ct), with lower Ct indicating a higher amount of DNA. [Figure 2C]This demonstrates that allele-specific primers are specific and do not amplify in control cell lines. Lung cancer cells (A549 cells with the G12S mutation, H358 cells with the G12C mutation, or H1299 cells) were seeded in 6-well plates. The following day, the cells were treated with various doses of purified RBCs. The RBCs were then washed twice and frozen at -80°C. DNA was extracted from the frozen RBCs according to the method previously described by our group, and qPCR was performed using allele-specific primers (G12S or G12C), commercially available mutant multiplex primers, or universal KRAS primers. Below the bar graph, a heatmap summary of all experimental conditions is provided. As seen in the figure above, RBCs do not contain KRAS DNA under basal conditions, but DNA is obtained from tumor cells. The data are shown as cycle threshold (Ct), with lower Ct indicating a higher amount of DNA. [Figure 3A] Figures A–C demonstrate that RBCs (reactive cytoplasmic bacteria) take up and retain tumor cell DNA. Different amounts of lung cancer cell lysates (A549, H358, and H1299) were incubated with varying amounts of RBCs for 2 hours. RBC pellets were then isolated from the supernatant (SN) via sucrose density centrifugation. DNA was extracted from the supernatant (200 μL) or RBC pellet (10⁷ cells) and qPCR was performed using allele-specific primers or universal KRAS primers. As shown in the figure, RBCs take up and retain tumor DNA. Data are presented as Ct against the amount of tumor lysate, and heatmaps corresponding to each experiment are also shown. [Figure 3B]Figures A–C demonstrate that RBCs (reactive cytoplasmic bacteria) take up and retain tumor cell DNA. Different amounts of lung cancer cell lysates (A549, H358, and H1299) were incubated with varying amounts of RBCs for 2 hours. RBC pellets were then isolated from the supernatant (SN) via sucrose density centrifugation. DNA was extracted from the supernatant (200 μL) or RBC pellet (10⁷ cells) and qPCR was performed using allele-specific primers or universal KRAS primers. As shown in the figure, RBCs take up and retain tumor DNA. Data are presented as Ct against the amount of tumor lysate, and heatmaps corresponding to each experiment are also shown. [Figure 3C] Figures A–C demonstrate that RBCs (reactive cytoplasmic bacteria) take up and retain tumor cell DNA. Different amounts of lung cancer cell lysates (A549, H358, and H1299) were incubated with varying amounts of RBCs for 2 hours. RBC pellets were then isolated from the supernatant (SN) via sucrose density centrifugation. DNA was extracted from the supernatant (200 μL) or RBC pellet (10⁷ cells) and qPCR was performed using allele-specific primers or universal KRAS primers. As shown in the figure, RBCs take up and retain tumor DNA. Data are presented as Ct against the amount of tumor lysate, and heatmaps corresponding to each experiment are also shown. [Figure 4A] This shows that RNA content differs between healthy RBCs and patient RBCs. RBCs were purified magnetically using glycophorin A-binding beads. RNA extracted from 107 RBCs was analyzed using TapeStation with the Zymo Quick DNA / RNA Micropreparation Kit. This figure shows the electrophoresis of RNA from RBCs. EL1(L): electron ladder, nt: nucleotide(s). [Figure 4B]This shows that RNA content differs between RBCs from healthy individuals and those from patients. RBCs were purified magnetically using glycophorin A-binding beads. RNA extracted from 107 RBCs was analyzed using TapeStation with the Zymo Quick DNA / RNA Micropreparation Kit. This figure shows the corresponding signal intensity profiles of the two samples. EL1(L): electron ladder, nt: nucleotide(s). [Figure 5A] This shows that RBCs from early-stage lung cancer patients are positive for tumor DNA. qPCR for mutant KRAS G12C was performed on RBC samples from lung cancer patients (Patient 1 - Stage 1A, Patient 2 - Stage IIB) using mutant-specific primers. This figure shows the tumor amplification curve. This patient sample indicates the presence of KRAS G12C tumor DNA. [Figure 5B] This shows that RBCs from early-stage lung cancer patients are positive for tumor DNA. qPCR for mutant KRAS G12C was performed on RBC samples from lung cancer patients (Patient 1 - Stage 1A, Patient 2 - Stage IIB) using mutant-specific primers. This figure shows the tumor growth curve. This patient sample shows the presence of KRAS G12C tumor DNA. [Figure 5C] This shows that RBCs from early-stage lung cancer patients are positive for tumor DNA. The cycle thresholds are shown for RBC samples from patients, positive controls (tumor cell lysates from the H358 strain with the G12C mutation), and negative controls. A lower Ct indicates a higher concentration of DNA in the sample. [Figure 5D] This study shows that RBCs from early-stage lung cancer patients are positive for tumor DNA. Gel electrophoresis was performed on the PCR products. The lanes were as follows: blank-marker, 1-RBC from patient 1, 2-RBC sample from a stage IIIA lung cancer patient, 3-RBC sample from another stage IIIA lung cancer patient, 4-RBC from patient 2, 5-RBC from a stage 1A patient (not known to be G12C positive), 6-RBC from a stage 1A patient, 7-buffer, 8-positive control, H358 cell lysate. [Figure 6] This study compares wild-type KRAS amplification in plasma and RBCs. DNA was extracted from 5 μL of RBCs or plasma, and qPCR was performed for KRAS (using universal primers that do not detect mutant KRAS). As shown in the figure, amplification occurred faster in RBCs than in plasma, suggesting that DNA is incorporated and retained by RBCs. [Figure 7] This diagram shows a schematic representation of the experiment conducted in Example 6. [Figure 8A] This shows the amount of human papillomavirus (HPV) DNA that binds to RBCs when incubated with various amounts of HPV CpG. [Figure 8B] This shows the amount of human papillomavirus (HPV) DNA that binds to RBCs when incubated with various amounts of HPV CpG. [Figure 9] This diagram shows a schematic representation of the experiment conducted in Example 7. [Figure 10] This shows the qPCR amplification curve for HPV16. [Figure 11] The graph and HPV16qPCR gel show that RBCs acquire HPV16 DNA from cervical cancer cell lines. [Figure 12] This shows the copy number analysis of cervical cancer samples. [Figure 13A]RBCs from healthy donors acquire HPV DNA, indicating that HPV DNA is detectable in RBCs from locally advanced HPV 16+ cervical cancer patients. RBCs from four different healthy donors were incubated with a cancer cell line (CaSki cells, an HPV16-positive cell line) for 24 hours. After incubation, DNA was extracted from the supernatant of CaSki cells, RBCs incubated with Caski cells (1e7+CaSki), or RBCs alone (1e7 alone). Filtered cell-free culture medium and water were used as positive and negative controls. DNA was also extracted from different amounts of CaSki cells. As seen in the figure, RBCs alone do not express HPV, but HPV is detectable in RBCs cultured with CaSki cells. This is five independent experiments with four healthy donors. The points on the graph represent the mean Ct from individual experiments. ****p<0.0001, one-way ANOVA with Sidak's multiple comparison test. [Figure 13B] RBCs from healthy donors acquire HPV DNA, indicating that HPV DNA is detectable in RBCs from locally advanced HPV16+ cervical cancer patients. RBCs from four different healthy donors were incubated with a cancer cell line (CaSki cells, an HPV16-positive cell line) for 24 hours. After incubation, DNA was extracted from the supernatant of CaSki cells, RBCs incubated with Caski cells (1e7+CaSki), or RBCs alone (1e7 alone). Filtered cell-free culture medium and water were used as positive and negative controls. DNA was also extracted from different amounts of CaSki cells. RBCs from two patients with locally advanced cervical cancer were tested for the presence of HPV DNA (Gyn10 and Gyn11). As seen in the graph, RBCs from both patients showed detectable HPV DNA (amounts of 2e7 and 10 uL) [Figure 13C]RBCs from healthy donors acquire HPV DNA, indicating that HPV DNA is detectable in RBCs from locally advanced HPV16+ cervical cancer patients. RBCs from four different healthy donors were incubated with a cancer cell line (CaSki cells, an HPV16-positive cell line) for 24 hours. After incubation, DNA was extracted from the supernatant of CaSki cells, RBCs incubated with CaSki cells (1e7+CaSki), or RBCs alone (1e7 alone). Filtered cell-free culture medium and water were used as positive and negative controls. DNA was also extracted from different amounts of CaSki cells. This figure is the corresponding DNA gel showing the presence of HPV DNA on the patient's RBCs but not in the plasma. [Figure 14] Analysis of CpG binding and uptake retention of RBV via TLR9.

Mode for Carrying Out the Invention

[0008] The ability to employ red blood cells (RBCs) to optimize the detection of rare ctDNA fragments is a paradigm shift, influencing early cancer detection and enabling more individualized treatment strategies. RBCs make up the majority of mammalian circulating cells and are essential for respiration. Functions of RBCs other than gas exchange, such as chemokine regulation, complement binding, and pathogen immobilization, have been reported, but the immunological functions of RBCs are not fully understood. RBCs pass through all tissues, are in contact with pathogens and self-derived inflammatory mediators in circulation, and are positioned as ideal messengers between distant organs. Recently, the immunological role of RBCs as nucleic acid sensors has been revealed, and we discovered that human RBCs can take up and retain DNA and RNA through the expression of toll-like receptors (TLRs).

[0009] The discovery that RBCs are a reservoir of nucleic acids is a paradigm shift. Improved ctDNA detection has the potential to revolutionize the diagnostic approach to ctDNA analysis. In addition, this technology has broad implications for the management of patients with lung cancer, including accelerating early disease detection, detecting minimal residual disease, assessing prognosis, and developing personalized treatment strategies.

[0010] This specification describes a method for detecting cancer. Our data show that RBCs are a rich source of tumor-derived nucleic acids. Furthermore, using RBC-based nucleic acid amplification methods, we can detect tumors using less than a drop of blood. We demonstrate that lung tumors can be detected using RBCs.

[0011] As used herein, the terms “patient” or “subject” mean mammals, including humans, veterinary or agricultural animals, domestic or companion animals, and animals commonly used in clinical research, such as non-human primates, dogs, and mice. More specifically, the subjects of these methods are humans. In one embodiment, the subject is suspected to have a tumor or a complication thereof.

[0012] sample All compositions, kits, and methods described herein are based on the knowledge that samples containing RBCs can be used to assist in the detection, diagnosis, treatment, and prediction of outcomes for various tumors.

[0013] As used herein, the terms “sample” or “patient sample” refer to a biological sample derived from an subject containing red blood cells (RBCs). RBCs, also known as red blood cells, are the most common type of cell found in blood, with approximately 4 to 6 million cells per cubic millimeter of blood.

[0014] In certain embodiments, the sample substantially contains no blood components other than RBCs. Therefore, in one embodiment, a whole blood sample is obtained from the subject, and the RBCs are isolated, concentrated, or purified. In one embodiment, the sample is filtered to remove non-RBCs, and the RBCs are isolated from the sample based on size. The RBCs have a diameter of about 6-8 μM.

[0015] Another advantage of the compositions, methods, and kits described herein is the small sample volume required. In some embodiments, the sample is a single drop of blood, which can be collected by point-of-care fingertip puncture. Prior art methods require blood collection from a vein, which is more invasive, more expensive, and requires specialized equipment and training. In contrast, in some embodiments, the compositions, methods, and kits described herein require only about 1 μL to about 10 μL of blood, or less than one drop of blood. In one embodiment, the sample is about 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, or 10 μL. In another embodiment, the sample is about 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, or 20 μL. In yet another embodiment, the sample is about 10 μL or less. In another embodiment, the sample volume is approximately 1 μL to approximately 20 μL. In yet another embodiment, the sample volume is approximately 1 μL to approximately 10 μL. In yet another embodiment, the sample volume is approximately 2 μL to approximately 5 μL. Each of these ranges includes the endpoint and all integers between them.

[0016] In another embodiment, the sample volume is less than approximately 1 mL. In yet another embodiment, the sample volume is approximately 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, or 1 mL (including all integers in between).

[0017] The sample must contain a sufficient number of red blood cells. In one embodiment, the sample contains at least 1 million RBCs. In another embodiment, the sample contains at least 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, or 10 million RBCs. In yet another embodiment, the sample contains at least 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, or 100 million RBCs. In another embodiment, the sample contains up to 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, or 10 million RBCs. In yet another embodiment, the sample contains up to 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, or 100 million RBCs. In another embodiment, the sample consists of approximately 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, and 5.5 million samples. In yet another embodiment, the sample contains 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, or 10 million RBCs.

[0018] In some embodiments, a sample is taken from the subject, and the sample is processed to purify or concentrate RBCs. For example, the sample may be filtered to remove components smaller than and / or larger than RBCs with a diameter of approximately 6–8 μM. The sample may also be sorted by the density of blood components to isolate the RBC components for use as described herein.

[0019] tumor In some embodiments, the compositions, methods, and kits described herein are used to detect and identify cancerous tumors. As used herein, the term “cancer” typically refers to or describes a physiological condition in mammals characterized by uncontrolled cell proliferation. In one embodiment, the term “cancer” means any cancer characterized by the presence of a solid tumor. In another embodiment, cancer is a blood cancer. Cancers referred to herein include, but are not limited to, melanoma, cervical cancer, breast cancer, brain cancer, colorectal cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, endometrial cancer, esophageal cancer, eye cancer, kidney cancer, laryngeal cancer, liver cancer, head and neck cancer, nasopharyngeal cancer, osteosarcoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rhabdomyosarcoma, salivary gland cancer, stomach cancer, testicular cancer, thyroid cancer, vaginal cancer, lung cancer, lymphoma, myeloma, and neuroendocrine cancer. In certain embodiments, cancer is cervical cancer caused by the human papillomavirus.

[0020] In certain embodiments, the composition, method, and kit include one reagent capable of detecting tumor-related molecules, such as tumor-related gene mutations. In certain embodiments, the gene mutation is found in one of the genes listed in Table 1. [Table 1] In certain embodiments, tumor-related molecules are involved in tumor-related inheritance in the EGFR gene. It originates from a sub-mutation. In another embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the ALK gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the KRAS gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the ROS1 gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRAF gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the NTRK gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the MET gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the RET gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the ERBB2 gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRCA1 gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRCA2 gene. In a particular embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the FGFR2 gene. In certain embodiments, the tumor-associated molecule is derived from a tumor-associated gene mutation in the gene FGFR3. In certain embodiments, the tumor-associated molecule is derived from a tumor-associated gene mutation in the gene PDGFRA. The nucleic acid and amino acid sequences of the genes in Table 1 are known in the art and are incorporated herein by reference.

[0021] For the identified genes, one or more mutations from the original coding sequence and / or protein sequence are known to be associated with cancer. For example, KRAS mutations are present in approximately 25% of tumors and are one of the most common gene mutations associated with cancer. Several KRAS mutations are known, including KRAS G12C, G12D, and G12R. The methods described herein make it possible to detect specific KRAS mutations without requiring tumor biopsy. Tables 2 and 3 provide known mutations for some of the tumor-associated genes described herein. [Table 2] [Table 3]

[0022] Methods of detection and / or diagnosis Provided herein is a method for detecting tumors in a sample from a subject. The method involves contacting a red blood cell-containing sample from the subject with a reagent capable of detecting tumor-associated molecules in the sample. In one embodiment, if tumor-associated molecules are detected in the sample, the subject is diagnosed with a tumor. In a particular embodiment, the method further includes treating the subject if tumor-associated molecules are detected. In a particular embodiment, the tumor originates from cervical cancer. In a particular embodiment, the tumor originates from cervical cancer, and the tumor-associated molecule is a nucleotide derived from human papillomavirus (HPV).

[0023] In one embodiment, the reagent can detect tumors by forming a complex with tumor-related molecules in a sample. In another embodiment, if a complex consisting of tumor-related molecules and the reagent is detected in the sample, the subject is diagnosed as having a tumor. In yet another embodiment, the reagent can amplify tumor-related molecules or their products. As used herein, the term “tumor-associated molecules” refers to any biological molecule that originates from or indicates the presence of a tumor. Such molecules include nucleic acids (e.g., DNA, RNA, mRNA, etc.) and proteins found in or on tumors, namely surface proteins (e.g., glycoproteins, spike proteins, capsid proteins, F proteins, G proteins, etc.), as well as tumor-induced antibodies and mRNA transcripts. The presence of tumor-associated molecules indicates the presence of a tumor in the subject.

[0024] Various methods and techniques are known for detecting, amplifying, or binding tumor-related molecules in samples. Such methods include nucleic acid-based methods (e.g., PCR-based methods), flow cytometry, and protein-based methods (e.g., ELISA or flow cytometry). The reagents described herein are specific to tumor-related gene mutations. Specific to tumor-related gene mutations means that the reagent binds to a specific nucleic acid containing the gene mutation, identifying or amplifying it. For example, in one embodiment, for the KRAS G12C mutation, the reagent can detect a specific codon change in the DNA or mRNA transcript found in the sample that corresponds to the G12C mutation. Such reagents are known in the art or can be designed by those skilled in the art, in view of the nucleic acid / amino acid sequences of genes / proteins. For example, Guarnaccia M, Iemmolo R, San Biagio See F, Alessi E, Cavallaro S. Genotyping of KRAS Mutational Status by the In-Check Lab-on-Chip Platform. Sensors (Basel). 2018 Jan 5;18(1):131. This paper describes genotyping of KRAS mutations using PCR. This reference is incorporated herein by reference.

[0025] Alternatively, in another embodiment, the reagent can detect any mutation at specific locations (may be multiple) of nucleic acids. These examples are not intended to limit the term "tumor-specific" reagent, but rather to encompass any reagent capable of identifying two or more tumors. In one embodiment, the reagents described herein include multiple reagents, each capable of detecting a different specific tumor. In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more different reagents are included in the composition or used in this method.

[0026] In one embodiment, the reagent can detect, bind to, specifically complex with, or measure the level of tumor-related molecules when present in a sample. In another embodiment, the reagent can detect or measure the amount or level of tumor using nucleic acids, such as DNA or RNA. Kits for carrying this out are commercially available, including, but not limited to, Competitive Allele-Specific TaqMan® polymerase chain reaction (CastPCR) (Applied Biosystems). CastPCR has high sensitivity and specificity. Its allele-specific and locus-specific primers ensure amplification of mutant alleles, while oligonucleotide blockers suppress amplification of wild-type alleles. Exemplary mutations that can be detected include those listed in Tables 2 and 3.

[0027] The diagnostic reagent may be a polynucleotide or genomic probe that hybridizes to tumor DNA or RNA. Such polynucleotides may have a nucleotide length of about 20, about 22, about 25, or more. In another embodiment, the diagnostic The reagent is a PCR primer-probe set that amplifies and detects the polynucleotide sequence of a suspected tumor. In one embodiment, the reagent is immobilized on a substrate. In another embodiment, the diagnostic reagent includes a microarray, a microfluidic card, a computer-readable chip, or a chamber. Suitable assays utilizing the described polynucleotides, genomic probes, or PCR primer pairs include, but are not limited to, PCR, reverse transcriptase PCR, quantitative PCR, Southern blot analysis, dot blot hybridization, and fluorescence in situ hybridization (FISH). Those skilled in the art can utilize conventional methods or tools in designing suitable polynucleotides, genomic probes, or PCR primer pairs, such as those described, taking into account the tumor to be detected.

[0028] Reagents may be tagged or labeled with reagents capable of providing a detectable signal, depending on the assay format employed. Such labels can provide a detectable signal alone or in cooperation with other compositions or compounds. Most preferably, the labels are detectable visually, for example, colorimetrically. A variety of enzyme systems function to produce colorimetric signals in assays. For example, glucose oxidase (using glucose as a substrate) releases a peroxide as a product that produces oxidized TMB, which is seen as blue, in the presence of peroxidase and a hydrogen donor such as tetramethylbenzidine (TMB). Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase conjugated with glucose-6-phosphate dehydrogenase. Hexokinase reacts with ATP, glucose, and NAD+ to produce NADH, which is detected as an increase in absorbance at a wavelength of 340 nm, among other products.

[0029] Other labeling systems that may be used in the methods described herein are detectable by other means, for example, colored latex microparticles (Bangs Laboratories, Indiana). Further labels include fluorescent compounds, radioactive compounds, or radioactive elements. Preferably, the reagent is associated with or conjugated to a fluorescently detectable fluorescent dye, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO), or a tandem dye, PE-cyanine-5 (PC5), and PE-Texas Red (ECD). Commonly used fluorescent dyes include fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC), as well as tandem dyes, PE-cyanine-5 (PC5), PE-cyanine-7 (PC7), PE-cyanine-5.5, PE-Texas Red (ECD), rhodamine, PerCP, fluorescein isothiocyanate (FITC), and Alexa dye. Combinations of such labels, such as Texas Red and rhodamine, FITC+PE, FITC+PECy5, and PE+PECy7, may be used depending on the assay method. Detectable labels for attachment to antibodies useful in the methods described herein can be readily selected from a large number of compositions known and readily available to those skilled in the art of diagnostic assays.

[0030] Provided herein, in one embodiment, is a method for diagnosing cancer in a subject. The method includes contacting a red blood cell-containing sample (as described herein) from the subject with a reagent capable of detecting tumor-related molecules in the sample, and diagnosing the subject with cancer if tumor-related molecules are detected in the sample. In one embodiment, the sample substantially contains no other blood components other than RBCs (e.g., plasma, leukocytes, platelets, etc.). In one embodiment, the sample volume is about 1 μL to about 10 μL.

[0031] In some embodiments, DNA is extracted from RBC-containing samples. Methods for this are well known in the art. For example, the sample is centrifuged and the supernatant is discarded. To the resulting precipitate, 20 mg / mL of lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added at 37°C. Dissolution is carried out for 30 minutes. Furthermore, a DNA extract can be obtained by column purification. This DNA extract can be used as a sample for PCR.

[0032] In some embodiments, this DNA is then subjected to amplification by PCR. Therefore, in certain embodiments, the reagent capable of detecting tumor-related molecules in the sample includes PCR primers. Using the extracted DNA and primers, the gene region to be amplified is amplified by PCR using primers known in the art and as described herein.

[0033] Specifically, for example, by using a primer set consisting of forward primers and reverse primers, the region in which a gene mutation is expected can be amplified. The PCR reaction mixture can preferably be a solution containing a nucleic acid synthesis substrate, a primer set, a nucleic acid synthesis enzyme, sample DNA, a buffer, and water as the remaining component. Similar embodiments are conceivable for other genes as described herein. For example, to identify KRAS mutations, primers that amplify relevant portions of exon 2, exon 3, and / or exon 4, such as those described by Guarnaccia et al, 2018, cited above, may be used.

[0034] [Table 4]

[0035] In certain embodiments, reagents capable of detecting tumor-related molecules in a sample include oligonucleotide probes. In hybridization-based methods, probe design is a critical step, and its efficiency depends on the strong affinity between the specific target and the short oligonucleotide probe. For example, in the gene chip format, multiple (up to several thousand) specific DNA probes are provided that can bind to the mutated region of the DNA sequence being analyzed. Each probe is a short DNA sequence complementary to a specific region of the DNA being analyzed. Exemplary probes for KRAS mutations are found in Table 5 below, as described by Guarnaccia et al, 2018, cited above. Sequence IDs 7-50 are listed in descending order. [Table 5]

[0036] Next, the prepared DNA sample is added to a gene chip, allowing it to hybridize or bind to the DNA probe on the chip. If the DNA in the sample contains mutations or sequence variations, this DNA will not hybridize with the corresponding probe on the chip. When the presence or absence of a PCR amplification product is identified by the DNA chip, the amplification product is labeled. The labeling method is not particularly limited, but fluorescent labeling is preferably used. When fluorescent labeling is performed by PCR, a fluorescently labeled primer can be used to generate an amplification product in which only the ends are labeled. In addition, a fluorescently labeled nucleic acid synthesis substrate can be used to generate an amplification product that contains the label internally. In either case, Cy5 or Cy3 can preferably be used as the fluorescent labeling component. Furthermore, non-fluorescent labels such as digoxigenin, biotin, and radioisotopes can also be used as labels.

[0037] Furthermore, a general thermal cycler or similar device can be used to carry out the PCR reaction. The PCR reaction conditions can be carried out as follows, for example: (A) 94°C for 2 minutes, (b) 94°C for 30 seconds (DNA denaturation step), (c) 60°C for 30 seconds (annealing step), (d) 72°C for 60 seconds (DNA synthesis step) ((b) to (D) for 35 cycles), (e) 72°C for 3 minutes. These conditions are illustrative and are not intended to limit the present invention.

[0038] In certain embodiments, it is desirable to use DNA chips to simultaneously and specifically identify multiple tumor-associated molecules in a single system. It is preferable to use DNA chips immobilized with sequences complementary to the probe sequences. Each of these probes has a specific sequence for each target molecule and can hybridize only with amplification products of the corresponding gene regions, thus enabling the specific and simultaneous detection of each target molecule being tested.

[0039] DNA chips can be fabricated using existing common methods with the probes described above. For example, when fabricating a fixed DNA chip, probes can be immobilized on a glass substrate using a DNA spotter to form spots corresponding to each probe. When fabricating a synthetic DNA chip, single-stranded oligoDNA having the above sequence can be synthesized on a glass substrate using photolithography techniques. Furthermore, the substrate is not limited to glass; plastic substrates or silicon wafers can also be used. Moreover, the shape of the substrate is not limited to a flat plate; it can have various three-dimensional shapes, and substrates with functional groups introduced to allow chemical reactions to occur on the surface can be used.

[0040] treatment In some embodiments of the methods described herein, the subject receives treatment after being diagnosed with cancer. In certain embodiments, follow-up examinations, including DNA sequencing, biopsy, MRI, CAT scan, PET scan, etc., may be performed before or after the implementation of the methods described herein.

[0041] In certain embodiments, treatment involves the administration of chemotherapeutic agents. Chemotherapeutic agents are compounds that exhibit anticancer activity and / or compounds harmful to cells (e.g., toxins). Suitable chemotherapeutic agents for use in the methods disclosed herein include toxins (e.g., saporin, lysine, abrin, ethidium bromide, diphtheria toxin, Pseudomonas exotoxin, and others listed above), alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, ifosfamide, mechloretamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulfonic acid esters such as busulfan; nitrosoureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin; bioreducing alkylating agents such as mitomycin, procarbazine, dacarbazine, and altoretamine), DNA strand cleavage agents (e.g., bleomycin), topoisomerase II inhibitors (e.g., A (Musacrin, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide), DNA minor groove binders (e.g., plicamycin), antimetabolites (e.g., folate antagonists such as methotrexate and trimethrexate, pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and phloxuridine, purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, and pentostatin, ribonucleotide reductase inhibitors such as asparaginase and hydroxyurea), tubulin interaction agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol)), hormones (e.g., estrogen, conjugated estrogen, ethinyl Examples of such substances include, but are not limited to, progestins such as lutestradiol, diethylstilbestrol, chlortrianicene, dienestrol, hydroxyprogesterone caproate, medroxyprogesterone, and megestrol, as well as androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone; corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); luteinizing hormone-releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and antihormone antigens (e.g., antiandrogens such as tamoxifen and flutamide, and antiadrenal agents such as mitotane and aminoglutethimide). In one embodiment, the chemotherapeutic agent is selected from the group consisting of paclitaxel (Taxol®), cisplatin, docetaxel, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, and epothyron derivatives.

[0042] In certain embodiments, cancer treatment may optionally include one or more of the following in addition to chemotherapy:

[0043] Radiation therapy: This treatment involves using high-energy radiation to kill cancer cells. It can be administered externally or internally and is often used in combination with surgery or chemotherapy.

[0044] Immunotherapy: This treatment uses the body's immune system to fight cancer. It works by stimulating the immune system to recognize and attack cancer cells. Some examples of immunotherapy include checkpoint inhibitors, CAR-T cell therapy, and cancer vaccines.

[0045] Targeted therapy: This type of treatment targets specific molecules or proteins involved in the proliferation and metastasis of cancer cells. Examples of targeted therapies include tyrosine kinase inhibitors and monoclonal antibodies.

[0046] Hormone therapy: This treatment is used for hormone-sensitive cancers, such as breast cancer and prostate cancer. It works by inhibiting or reducing the production of hormones that stimulate the growth of cancer cells.

[0047] Surgical intervention: This treatment involves removing cancerous tumors or cancerous tissue from the body. This is often used in combination with other treatments such as radiation therapy or chemotherapy.

[0048] Palliative care: This treatment focuses on managing symptoms and improving the quality of life for patients with advanced or terminal cancer. This includes pain management, psychological support, and other forms of supportive therapy.

[0049] Compositions and kits The compositions, kits, and methods described herein include reagents capable of detecting, binding to, specifically encombining with, or measuring the levels of tumor-related molecules. Such reagents include those capable of detecting the molecules at the nucleic acid level or measuring their abundance. Preferred reagents include those for detection by polymerase chain reaction (PCR). Suitable reagents are commercially available.

[0050] In addition, suitable reagents may be designed by those skilled in the art based on the published sequences of the specific tumors of interest. In one embodiment, the reagents are PCR primers and / or probes. In addition, other suitable components are included to enable the identification and / or quantification of the tumors of interest. Such components include, for example, enzymes, buffers, and deoxyribonucleotides required for reverse transcription and / or PCR, preferably qualitative and / or quantitative RT-PCR, as well as detectable probes and / or internal controls.

[0051] Any combination of the described reagents for detecting the target tumor-associated molecule can be incorporated into the diagnostic kit. For example, one embodiment of the diagnostic kit includes reagents for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 tumor-associated gene mutations. In one embodiment, the kit includes reagents for 5 or 6 tumor-associated gene mutations. In one embodiment, one or more reagents are associated with or bound to a detectable label or bound to a substrate.

[0052] For these reagents, the label can be selected from a wide range of known diagnostic labels, including those described above. Similarly, the substrate for immobilization may be a general substrate, glass, plastic, microarray, microfluidic card, chip, or chamber.

[0053] Any of the compositions described herein is intended to be a kit containing multiple reagents or one or more individual reagents. For example, one embodiment of a composition includes a substrate on which one or more reagents are immobilized. In another embodiment, the composition is a kit that includes other laboratory equipment in addition to an optional detectable label, an immobilized substrate, and an optional substrate for enzyme labeling. In one embodiment, the kit includes a standard substance for use as a control.

[0054] The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the present invention to these examples, but rather as encompassing any variations that become apparent as a result of the teachings provided herein. [Examples]

[0055] Example 1: RBCs obtain tumor DNA from lung cancer cells RBCs (reactive protein cells) traverse all tissues and are constantly in contact with host and pathogen-derived mediators, including highly vascularized tumors. We recently discovered that RBCs take up and retain mitochondrial DNA through the cell surface expression of the receptor TLR9. Although we have never previously detected nuclear DNA bound to RBCs (Hotz AJRCCM, appendix), we hypothesized that RBCs could acquire tumor DNA due to the highly vascularized nature of tumors. We hypothesized that RBCs take up and retain tumor DNA, and that tumor DNA could be detected in less than one drop of RBC.

[0056] We first investigated whether RBCs (reactive bone cell lines) could acquire DNA from lung cancer cell lines containing KRAS mutations. As shown in Figure 1, we were able to detect tumor DNA on RBCs co-cultured with tumor cells.

[0057] Example 2: RBCs obtain mutant DNA from cultured tumor cells Next, we examined additional cell lines, including the H358 cell line with the KRAS G12C mutation and the H1299 cell line without any KRAS mutations. Similar to Figure 1, Cells were seeded in 6-well plates before incubation with prepared naive RBCs. However, tumor cells were seeded only overnight before the experiment, rather than being grown to confluence for several days before RBC treatment. G12S and G12C were used against the extracted DNA. qPCR was performed against KRAS variants. Mutant multiplex PCR and universal KRAS PCR were also performed as control experiments. As shown in Figure 2, RBCs obtained mutant DNA from cultured tumor cells.

[0058] Example 3: RBCs can detect and bind to minute amounts of tumor cell lysates. We then investigated whether RBCs acquire tumor DNA with extremely low doses of DNA and whether RBCs take up and retain tumor DNA from plasma / SN. RBCs were incubated with varying amounts of tumor cell lysate. This lysate was generated from 1 million tumor cells. As seen below, RBCs can detect and bind to ultra-low volumes of tumor cell lysate.

[0059] Example 4: RBC loads tODN RBCs from healthy donors (10 7 RBCs (RBCs) are incubated with synthetic tODN fluorescently labeled at various doses (0 nM / 1 million RBCs to 100 nM tODN / 1 million RBCs) at 37°C for various time points (30 minutes, 60 minutes, 120 minutes) with gentle stirring. We use sequences commonly found in somatic variants of lung cancer (KRAS G12C, EGFR L858R, and EGFR exon 19 deletion (synthesized by IDT)). RBC-bound mutant ODN and supernatant mutant ODN are compared using qPCR.

[0060] As we previously described for CpG ODNs, we measure RBC-tODN binding using FACS.

[0061] RBCs are incubated with unlabeled tODN as described above. After incubation, RBCs are separated from the supernatant (SN) by centrifugation under a 30% sucrose gradient. qPCR is performed on DNA associated with SN and RBCs to quantify bound and unbound tODN. Although qPCR is insensitive to the detection of RBC-ODN binding, if we detect binding by flow cytometry, we perform digital drop PCR on the RBCs and supernatant.

[0062] RBCs will be isolated from patients with known oncogene-driven progressive NSCLC, NSCLC patients without oncogene-driven tumors, and healthy donors. Human red blood cells will be collected from whole blood and isolated using glycophorin A beads as described in the literature. PCR for known somatic variants will be performed on RBCs and plasma from patients, control cancer patients (without driver mutations), and healthy controls. In this preliminary study, 15 patients per group will be analyzed.

[0063] Example 5: RBCs take up and retain extracellular RNA, and tumor RNA can be detected from RBCs. We recently investigated whether RNA could be obtained from RBCs (reactive bone cell) from healthy donors and from RBCs from patients hospitalized with inflammation. As shown in Figure 4, we detected a significant amount of RNA on RBCs in patients with acute inflammatory syndrome (sepsis), which was considerably different from the control group. Since RBCs do not synthesize new proteins and do not contain RNA, this RNA must have been obtained from another host-derived source or a microbial source. Because it has been reported that extracellular nucleic acids are increased in cancer patients, we hypothesized that RBCs from cancer patients contain a significant amount of RNA and would offer an opportunity to characterize the circulating free RNA pattern in lung cancer.

[0064] Example 6: Detection of HPV DNA on RBCs Next, we investigated whether human papillomavirus (HPV) DNA could be detected on RBCs from healthy donors and patients incubated with HPV DNA in vitro. As shown in Figure 7, a FITC-labeled DNA motif derived from the E6 sequence of HPV16 was incubated with RBCs. RBC-HPV DNA binding was then evaluated by flow cytometry. Figure 8A shows the binding of donor RBCs to HPV DNA after incubation with 0 nM, 5 nM, 25 nM, 50 nM, and 250 nM HPV CpG. As the dose of HPV CpG increased, HPV The amount of RBCs that bind to DNA also increases. Furthermore, in Figure 8B, RBCs from four donors were incubated with a wider range of HPV CpG doses. From this figure, HPV The percentage of cells that bind to DNA increases with increasing dose, and after incubation with 2000 nM HPV CpG, approximately 80% of cells were found to be positive for HPV DNA.

[0065] Example 7: Detection of HPV DNA from cancer cells on RBCs Next, we investigated whether RBCs can acquire HPV DNA from HPV-infected cervical cancer cell lines. As shown in Figure 9, RBCs were incubated with CaSki cells, a cancer cell line known to contain HPV16, for 24 hours. DNA was then extracted from the RBCs, and HPV16 DNA was detected by qPCR. An HPV16 qPCR amplification curve was created from this experiment (Figure 10). This experiment was repeated in four independent experiments using four unique healthy RBC donors. The data obtained from these experiments are shown in Figure 11. Taken together, these data indicate that RBCs acquire HPV16 DNA from cervical cancer cell lines.

[0066] The data presented above in this specification can be clinically applied to patients as diagnostic tests for HPV and cervical cancer.

[0067] Example 8: Detection of cancer cell HPV DNA on RBCs Next, we investigated whether RBCs could obtain HPV DNA from other HPV-infected cervical cancer cell lines. First, we detected the copy number of HPV16 DNA in various cell lines using PCR (Figure 12). Then, we incubated cells from patients with locally advanced HPV16+ cervical cancer with RBCs. In both patients, HPV16 DNA was detected on 2e7 RBCs and 10 μL of pRBCs (Figure 13B). These results were confirmed using gel electrophoresis (Figure 13C).

[0068] Subsequently, this process was repeated in five independent experiments with four healthy donors (Figure 13A). This data confirmed that RBCs from healthy donors could acquire HPV DNA.

[0069] Example 9: Mechanism of RBC binding to CpG Next, we analyzed the mechanism by which RBCs bind to, take up, and retain CpG. This mechanism was analyzed by comparing DNA binding by RBCs in WT mice and TLR9KO mice (Figure 14). RBCs from TLR9KO mice had a significantly lower percentage of CpG DNA than RBCs from wild-type mice. This was confirmed in TLR9-human cells and TLR+ human cells. We also confirmed the location of circulating DNA by comparing the amount of cfDNA in plasma with the amount of cfDNA in RBCs. In both mice and humans, the amount of cfDNA present in plasma was limited. This indicates that RBVs preferentially take up and retain circulating DNA.

[0070] Embodiment 1. A method for diagnosing cancer in a subject, wherein the method is A sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-related DNA molecules in the sample. A method comprising: diagnosing a subject with cancer if the tumor-related DNA molecule is detected in the sample. 2. A method for diagnosing the cancer in the subject, wherein the method is A sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-related molecules in the sample. A method comprising: diagnosing a subject with cancer if the tumor-related molecule is detected in the sample. 3. The method according to Embodiment 1 or 2, wherein the reagent is specific to tumor-associated gene mutations. 4. The method according to any one of Embodiments 1 to 3, wherein the cancer is a cancer of the lung, cervix, breast, prostate, thyroid, colon, or pancreas. 5. The method according to Embodiment 1, wherein the reagent comprises a plurality of reagents, each capable of detecting a different specific tumor-related molecule. 6. The method according to any of the embodiments, wherein the reagent comprises a primer capable of binding to tumor-associated DNA or RNA. 7. The method according to any of the embodiments, wherein a specific tumor-associated gene mutation found in the sample is identified. 8. The method according to any of the embodiments, wherein the tumor-associated gene mutation is found in the EGFR, ALK, KRAS, ROS1, BRAF, NTRK, MET, RET, ERBB2, PIK3CA, BRCA1, BRCA2, FGFR2, FGFR3, EZH2, or PDGFRA gene. 9. The method according to Embodiment 8, wherein the tumor-associated gene mutation is found in the KRAS gene. 10. The method according to any of the embodiments, further comprising treating the target cancer when the person has been diagnosed with cancer. 11. The method according to Embodiment 10, wherein the treatment is specific to the identified tumor-associated gene mutation. 12. The method according to any of the embodiments, wherein the diagnostic method is performed at a point of care. 13. The method according to any of the embodiments, further comprising filtering the blood sample to remove non-RBC blood cells. 14. The method according to any one of Embodiments 1 to 13, wherein the amount of the sample is 10 uL or less. 15. The method according to Embodiment 14, wherein the amount of the sample is approximately 1 uL to approximately 5 uL. 16. The method according to any of the embodiments, wherein the sample contains at least 1 million RBCs. 17. The method according to any of the embodiments, wherein the RBC is concentrated from the sample. 18. The method according to any of the embodiments, wherein the sample substantially does not contain other blood components. 19. The method according to any of the embodiments described above, wherein the subject is suspected of having cancer. 20. Use of a reagent capable of detecting tumor-related DNA molecules in a biological sample containing red blood cells, for diagnosing the subject as having the cancer if the tumor-related DNA molecules are detected in the sample.

Claims

1. A method for diagnosing cancer in a subject, wherein the method is A sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-related DNA molecules in the sample. A method comprising: diagnosing a subject with cancer if the tumor-related DNA molecule is detected in the sample.

2. A method for diagnosing the cancer in the subject, wherein the method is A sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-related molecules in the sample. A method comprising: diagnosing a subject with cancer if the tumor-related molecule is detected in the sample.

3. The method according to claim 1, wherein the reagent is specific to tumor-associated gene mutations.

4. The method according to claim 1, wherein the cancer is a cancer of the lung, cervix, breast, prostate, thyroid, colon, or pancreas.

5. The method according to claim 1, wherein the reagent comprises a plurality of reagents, and each reagent is capable of detecting different specific tumor-related molecules.

6. The method according to claim 1, wherein the reagent comprises a primer capable of binding to tumor-associated DNA or RNA.

7. The method according to claim 1, wherein specific tumor-related gene mutations found in the sample are identified.

8. The method according to claim 1, wherein the tumor-associated gene mutation is found in the EGFR gene, ALK gene, KRAS gene, ROS1 gene, BRAF gene, NTRK gene, MET gene, RET gene, ERBB2 gene, PIK3CA gene, BRCA1 gene, BRCA2 gene, FFFR2 gene, FFFR3 gene, EZH2 gene, or PDGFRA gene.

9. The method according to claim 8, wherein the tumor-associated gene mutation is found in the KRAS gene.

10. The method according to claim 1, further comprising the method treating the cancer of the subject if the subject is diagnosed with the cancer.

11. The method according to claim 10, wherein the treatment is specific to the identified tumor-associated gene mutation.

12. The method according to claim 1, wherein the diagnostic method is performed at a point of care.

13. The method according to claim 1, further comprising filtering the blood sample to remove non-RBC blood cells.

14. The method according to claim 1, wherein the sample volume is 10 μL or less.

15. The method according to claim 14, wherein the amount of the sample is about 1 uL to about 5 uL.

16. The method according to claim 1, wherein the sample contains at least 1 million RBCs.

17. The method according to claim 1, wherein the RBC is concentrated from the sample.

18. The method according to claim 1, wherein the sample substantially does not contain other blood components.

19. The method according to claim 1, wherein the subject is suspected to have the cancer.

20. The use of a reagent capable of detecting tumor-related DNA molecules in a biological sample containing red blood cells, for diagnosing the subject as having the cancer if tumor-related DNA molecules are detected in the sample.

Images