Detection of tumor-derived nucleic acids using red blood cells

Abstract

Described herein are methods of diagnosing cancer in a subject. The method comprises: contacting a sample containing erythrocytes from the subject with an agent capable of detecting tumor-associated molecules in the sample; and diagnosing the subject with a tumor when the tumor-associated molecule is detected in the sample. In some embodiments, the suitable sample is less than 10 L.

Description

[0001] Cross-references 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 described.

[0002] Citations of materials submitted electronically are incorporated The contents of the electronic serial number (UPN-22-10081-PCT.xml; size: 45,266 bytes; and creation date: May 6, 2024) are incorporated herein by reference in their entirety. Background Technology

[0003] Lung cancer is a leading cause of cancer-related death worldwide, with a 5-year survival rate of only 23%. Analysis of circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping, monitoring disease burden, and early cancer detection. ctDNA consists of short DNA fragments released from tumor cells into the bloodstream. It can be analyzed using highly sensitive sequencing assays to detect tumor-specific mutations, structural variations, and copy number alterations in patients with lung cancer. ctDNA analysis is increasingly being used for non-invasive mutation detection to guide targeted therapy selection in patients with advanced disease. However, the short half-life of ctDNA in circulation (only 1–2 hours) and its typically small fraction of total circulating cell-free DNA significantly limit the sensitivity and clinical applicability of these assays.

[0004] There is a need in the art for improved methods for detecting tumor DNA in patient samples. Summary of the Invention

[0005] In a first aspect, this document provides a method for diagnosing cancer in a subject. The method includes: contacting a sample containing red blood cells from the subject with a reagent capable of detecting a tumor-related molecule in the sample; and diagnosing the subject with cancer when the tumor-related molecule is detected in the sample. In some embodiments, the reagent is specific for tumor-related gene mutations. In some embodiments, the reagent comprises multiple reagents, each capable of detecting a different specific tumor-related molecule. In some embodiments, the reagent contains primers capable of binding to tumor-related DNA or RNA. In some embodiments, tumor-related gene mutations are found in the EGFR, ALK, KRAS, ROS1, BRAF, NTRK, MET, RET, ERBB2, PIK3CA, BRCA1, BRCA2, FGFR2, FGFR3, EZH2, or PDGFRA genes.

[0006] In other respects, compositions and kits for performing the methods described herein are provided. Attached Figure Description

[0007] Figures 1A to 1H This illustrates how RBCs acquire tumor DNA from lung cancer cells. The lung cancer cells (A549 cells carrying the G12S mutation) were processed at a rate of 1x10... 6 Cells were seeded at a density of 10 cells / well in 6-well plates. Cells were grown to confluence and then 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 separated by centrifugation. The RBCs were then washed twice before freezing at -80°C. DNA was extracted from the frozen RBCs as previously described by our group and qPCR was performed using primers specific to G12 S (A and D), commercially available mutant multiplex primers (B & E), or universal KRAS primers. G: Summary of the 24-hour and 72-hour co-culture studies. H: DNA gels demonstrating the presence of G12S amplicons in A549 cells or RBCs treated with A549 cells, with an expected amplicon size of 98 bp. As shown in the figure above, the RBCs did not contain KRAS DNA under basal conditions but were obtained from tumor cells. Figures 1A to 1G The data are presented as cycle thresholds (Ct), where a lower Ct indicates more abundant DNA. Technological repetitions are presented in Figure 1.

[0008] Figures 2A to 2C This demonstrates that the allele-specific primers were specific and did not amplify in control cell lines. Lung cancer cells (A549 cells carrying the G12S mutation, H358 cells carrying the G12C mutation, or H1299 cells) were seeded in 6-well plates. The next day, the cells were treated with different doses of purified RBCs. The cell culture supernatant was then aspirated, and the RBCs were separated by centrifugation. Subsequently, the RBCs were washed twice before freezing at -80°C. DNA was extracted from the frozen RBCs, as 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. A summary heatmap of all experimental conditions is provided below the bar chart. As shown in the figure above, the RBCs did not contain KRAS DNA under basal conditions but were obtained from tumor cells. Data are presented as cycle thresholds (Ct), where a lower Ct indicates more abundant DNA.

[0009] Figures 3A to 3CRBCs were used to isolate tumor cell DNA. Different volumes of lung cancer cell lysates (A549, H358, and H1299) were incubated with different amounts of RBCs for 2 hours. The RBC pellet was then separated from the supernatant by sucrose density centrifugation (SN). The RBC pellet was separated from the supernatant (200 μL) or the RBC pellet (10 μL)... 7 DNA was extracted and qPCR was performed using allele-specific primers or universal KRAS primers. As shown in the figure, RBCs isolated tumor DNA. Data are presented as Ct and volume of tumor lysates, and a corresponding heatmap for each experiment is also shown.

[0010] Figure 4A and 4B The results showed that RNA content differed between healthy and patient RBCs. RBCs were magnetically purified using blood group glycoprotein A conjugated beads. RNA extracted from 10⁷ RBCs using the Zymo Quick DNA / RNA Microprep kit was analyzed via TapeStation. Figure 4A Electrophoresis of RNA from RBCs and ( Figure 4B The corresponding signal intensity distributions in the two samples. EL1(L): electron ladder, nt: nucleotide.

[0011] Figures 5A to 5D The results showed that RBCs from patients with early-stage lung cancer were positive for tumor DNA. Figure 5A and Figure 5B qPCR was performed on RBC samples from patients with lung cancer (stages 1-1A, 2-IIB) using mutation-specific primers targeting the KRAS G12C mutation. Amplification curves were observed at... Figure 5A and Figure 5B As shown in the image, both patient samples demonstrated the presence of KRAS G12C tumor DNA. Figure 5C The figure shows the cycling thresholds for RBC samples from patients, positive controls (from lysates of tumor cells from the H358 lineage carrying the G12 C mutation), and negative controls. The lower the Ct, the more DNA is present in the sample. Figure 5D : Gel containing PCR products. Lanes are as follows: Blank marker, 1-RBC from patient 1, 2-RBC sample from a patient with stage IIIA lung cancer, 3-RBC sample from another patient with stage IIIA lung cancer, 4-RBC from patient 2, 5-RBC from a stage 1A patient (unknown positive for G12C), 6-RBC from a stage 1A patient. 7-Buffer. 8-Positive control, H358 cell lysate.

[0012] Figure 6The comparison of wild-type KRAS amplification on plasma and RBCs is shown. DNA was extracted from 5 μL of RBCs or plasma, and qPCR was run on KRAS (using universal primers that do not detect mutant KRAS). As shown in the figure, RBCs amplified earlier than plasma, indicating that the DNA was isolated by RBCs.

[0013] Figure 7 A diagram is shown representing the experiment conducted in Example 6.

[0014] Figures 8A to 8B The amount of human papillomavirus (HPV) DNA that binds to RBCs is shown when incubated with different amounts of HPV CpG.

[0015] Figure 9 A diagram is shown representing the experiment conducted in Example 7.

[0016] Figure 10 The HPV16 qPCR amplification curve is shown.

[0017] Figure 11 The diagram shows a graphical representation and HPV16 qPCR gel, demonstrating that RBCs obtained HPV16 DNA from cervical cancer cell lines.

[0018] Figure 12 Copy number analysis of cervical cancer samples is shown.

[0019] Figures 13A to 13C This study demonstrates the acquisition of HPV DNA from healthy donor RBCs and the detectability of HPV DNA in RBCs from patients with locally advanced HPV 16+ cervical cancer. RBCs from four different healthy donors were incubated with a cancer cell line (CaSki cells, an HPV 16-positive cell line) for 24 hours. After incubation, DNA was extracted from CaSki cell supernatant, RBCs incubated with CaSki cells (1e7+ CaSki), or individual RBCs (1e7 cells alone). Filtered cell-free culture medium and water were used as positive and negative controls. DNA was also extracted from varying amounts of CaSki cells. Figure 13A As shown, isolated RBCs do not express HPV, but HPV is detectable on RBCs cultured with CaSki cells. Five independent experiments were performed on four healthy donors. The points on the graph represent the mean Ct from each experiment. **** p<0.0001, one-way ANOVA using the Sidak multiple comparison test. Figure 13B The presence of HPV DNA (Gyn 10 and Gyn 11) was tested in red blood cells from two patients with locally advanced cervical cancer. As shown in the figure, both patients' RBCs demonstrated detectable HPV DNA (2e7 and 10 uL volumes). Figure 13CThe corresponding DNA gel test showed that HPV DNA was present on the patient's RBCs, but not in the plasma.

[0020] Figure 14 RBV binding and CpG isolation were analyzed using TLR9. Detailed Implementation

[0021] The ability to optimize the detection of rare ctDNA fragments using erythrocytes (RBCs) is undergoing a paradigm shift and impacting early cancer detection, leading to more personalized treatment strategies. RBCs constitute the majority of circulating cells in mammals and are crucial for respiration. While non-gas exchange functions of RBCs, such as chemokine regulation, complement fixation, and pathogen fixation, have been described, the immune functions of RBCs are not fully understood. RBCs traverse all tissues and come into contact with circulating pathogens and autologous inflammatory mediators, thus positioning them as ideal messengers between distant organs. Recently, the immune role of RBCs as nucleic acid sensors has been revealed, and we have found that human RBCs can isolate DNA and RNA by expressing toll-like receptors (TLRs).

[0022] The discovery of RBCs as a nucleic acid library is undergoing a paradigm shift. Improved ctDNA testing could revolutionize diagnostic approaches to ctDNA analysis. Furthermore, this technology has the potential to have broad implications for managing patients with lung cancer, including facilitating early disease detection, detecting minimal residual disease, assessing prognosis, and developing personalized treatment strategies.

[0023] This article describes a method for detecting cancer. Our data demonstrate that RBCs are a rich source of tumor-derived nucleic acids. Furthermore, using RBC-based nucleic acid amplification, we can detect tumors with less than a drop of blood. We demonstrate that RBCs can be used to detect lung tumors.

[0024] As used herein, "patient" or "subject" refers to a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical research, including non-human primates, dogs, and mice. More specifically, the subject of these methods is a human. In one embodiment, the subject is suspected of having a tumor or its complications.

[0025] sample All of the compositions, kits, and methods described herein rely on the observation of RBC-containing samples for the detection, diagnosis, treatment, and prediction of various tumor outcomes.

[0026] As used herein, the term “sample” or “patient sample” refers to a biological sample containing red blood cells (RBCs) derived from a subject. RBCs, also known as erythrocytes, are the most common cell type found in blood, with 4–6 million cells per cubic millimeter of blood.

[0027] In some embodiments, the sample is substantially free of all other blood components except RBCs. Therefore, in one embodiment, a whole blood sample is obtained from the subject, and RBCs are isolated, concentrated, or purified. In one embodiment, the sample is filtered to remove non-RBCs, and RBCs are separated from the sample based on size. RBCs have a diameter of approximately 6-8 µM.

[0028] Another advantage of the compositions, methods, and kits described herein is the small sample volume required. In some embodiments, the sample is a drop of blood, which can be obtained from a fingertip at the point of care. Prior art methods require venous blood draws, which are more invasive, more expensive, and require 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 a drop of blood or less. 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 yet another embodiment, the sample volume is about 1 µL to about 20 µL. In another embodiment, the sample volume is from about 1 µL to about 10 µL. In another embodiment, the sample volume is from about 2 µL to about 5 µL. Each of these ranges includes the endpoints and all integers in between.

[0029] In another embodiment, the sample is less than about 1 mL. In another embodiment, the sample is about 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.

[0030] 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 contains approximately 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 approximately 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, or 100 million RBCs.

[0031] In some embodiments, the sample is obtained from the subject and processed to purify or enrich the RBCs in the sample. For example, the sample may be filtered to remove components smaller and / or larger than RBCs, with RBCs having a diameter of approximately 6-8 µM. The sample may also be sorted by the density of blood components, wherein RBC components are separated for use as described herein.

[0032] tumor In some embodiments, the compositions, methods, and kits described herein are used for the detection and identification of cancerous tumors. As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. In one embodiment, the term "cancer" refers to any cancer characterized by the presence of a solid tumor. In another embodiment, the cancer is a blood cancer. When mentioned herein, cancer includes, but is 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, gastric cancer, testicular cancer, thyroid cancer, vaginal cancer, lung cancer, lymphoma, myeloma, and neuroendocrine cancer. In some embodiments, the cancer is cervical cancer caused by human papillomavirus (HPV).

[0033] In some embodiments, the compositions, methods, and kits include one or more reagents capable of detecting mutations in tumor-related molecules, such as tumor-related genes. In some embodiments, gene mutations are found in one of the genes listed in Table 1.

[0034] Table 1 In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the EGFR gene. In another embodiment, the tumor-associated molecule originates from a tumor-associated gene mutation in the ALK gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the KRAS gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the ROS1 gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRAF gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the NTRK gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the MET gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the RET gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the ERBB2 gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRCA1 gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the BRCA2 gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the FGFR2 gene. In some embodiments, the tumor-associated molecule originates from a tumor-associated gene mutation in the FGFR3 gene. In some embodiments, tumor-associated molecules are derived from mutations in tumor-associated genes 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.

[0035] For the identified genes, one or more mutations from the naturally encoded and / or protein sequences are known to be associated with cancer. For example, KRAS mutations are present in approximately 25% of tumors, making it one of the most common gene mutations associated with cancer. Several KRAS mutations are known, including KRAS G12C, G12D, and G12R. Using the methods described herein, specific KRAS mutations can be detected without the need for tumor biopsy. Tables 2 and 3 provide known mutations in some of the tumor-associated genes described herein.

[0036] Table 2: KRAS mutations Table 3: Mutations in tumor-related genes Detection and / or diagnostic methods This document provides methods for detecting tumors in samples from a subject. These methods include contacting a sample containing red blood cells from the subject with a reagent capable of detecting tumor-related molecules in the sample. In one embodiment, the subject is diagnosed with a tumor when a tumor-related molecule is detected in the sample. In some embodiments, the method further includes treating the subject when a tumor-related molecule is detected. In some embodiments, the tumor is cervical cancer. In some embodiments, the tumor is cervical cancer and the tumor-related molecule is a nucleotide from human papillomavirus (HPV).

[0037] In one embodiment, the reagent is capable of detecting tumors by forming a complex with tumor-associated molecules in a sample. In one embodiment, a subject is diagnosed with a tumor when a complex consisting of a tumor-associated molecule and the reagent is detected in a sample. In yet another embodiment, the reagent is capable of amplifying tumor-associated molecules or their products. As used herein, the term "tumor-associated molecule" refers to any biomolecule that originates from or indicates the presence of a tumor. Such molecules include nucleic acids (e.g., DNA, RNA, mRNA, etc.) and proteins found within or on tumors, including surface proteins (e.g., glycoproteins, spike proteins, capsid proteins, F proteins, G proteins, etc.), tumor-induced antibodies, and mRNA transcripts. The presence of a tumor-associated molecule indicates the presence of a tumor in a subject.

[0038] Various methods and techniques are known for detecting, amplifying, or binding tumor-associated molecules in samples. These methods include nucleic acid-based methods (e.g., PCR-based methods) and protein-based methods (e.g., ELISA or flow cytometry). The reagents described herein are specific for tumor-associated gene mutations. Specificity for tumor-associated gene mutations means that the reagent binds to, recognizes, or amplifies a specific nucleic acid containing the gene mutation. For example, in one embodiment, for the KRAS G12C mutation, the reagent is able to detect a specific codon change corresponding to the G12C mutation found in the DNA or mRNA transcript in the sample. Reagents such as these are known in the art or can be designed by those skilled in the art considering the nucleic acid / amino acid sequence of the gene / protein. For example, see Guarnaccia M, Iemmolo R, San Biagio F, Alessi E, Cavallaro S. Genotyping of KRAS Mutational Status by the In-CheckLab-on-Chip Platform. Sensors (Basel). 5 Jan 2018; 18(1):131, which describes the use of PCR for KRAS mutation genotyping. This reference is incorporated into this paper by way of citation.

[0039] Alternatively, in another embodiment, the reagent is capable of detecting any mutation at one or more specific locations in the nucleic acid. These examples are not intended to limit the term "tumor-specific" reagent, which means to encompass any reagent that can distinguish between two or more tumors. In one embodiment, the reagents described herein comprise a plurality of 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 the method.

[0040] In one embodiment, when tumor-associated molecules are present in a sample, the reagent is capable of detecting, binding to, specifically complexing with, or measuring the level of tumor-associated molecules. In one embodiment, the reagent is one capable of detecting or measuring the amount or level of tumor using nucleic acids (e.g., DNA or RNA). Kits for performing the same operations 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 primers and site-specific primers ensure the amplification of mutant alleles, while oligonucleotide blockers inhibit the amplification of wild-type alleles. Exemplary mutations that can be detected include those listed in Tables 2 and 3.

[0041] Diagnostic reagents can be polynucleotides or genomic probes that hybridize with tumor DNA or RNA. Such polynucleotides can be about 20, about 22, about 25, or more nucleotides in length. In another embodiment, the diagnostic 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, microfluidic card, computer-readable chip, or chamber. Suitable assays using the said polynucleotide, genomic probe, or a pair of PCR primers may include, but are not limited to, PCR, reverse transcriptase PCR, quantitative PCR, Southern blotting analysis, dot blot hybridization, and fluorescence in situ hybridization (FISH). Given the tumor to be detected, conventional methods or tools can be used by those skilled in the art to design suitable polynucleotides, genomic probes, or a pair of PCR primers, as described.

[0042] Depending on the form of assay used, reagents can be traced or labeled with reagents capable of providing a detectable signal. Such labels can provide a detectable signal alone or in conjunction with other compositions or compounds. Ideally, the labeling is visually detectable (e.g., colorimetrically). Various enzyme systems operate to reveal colorimetric signals in assays; for example, glucose oxidase (which uses glucose as a substrate) releases peroxides as a product, which, in the presence of the peroxidase and a hydrogen donor such as tetramethylbenzidine (TMB), produces oxidized TMB, which appears blue. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP) and hexokinase bound to glucose-6-phosphate dehydrogenase, which reacts with ATP, glucose, and NAD+ to produce products such as NADH, which is detected as an increase in absorbance at a wavelength of 340 nm.

[0043] Other labeling systems that can be used in the methods described herein can be detected in other ways, such as colored latex particles (Bangs Laboratories, Indiana). Other labels include fluorescent compounds, radioactive compounds, or elements. Preferably, the reagent is associated with or bound to a fluorescently detectable dye, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), curryphosphine-O (CPO), or tandem dyes, PE-anthocyanin-5 (PC5), and PE-Texas Red (ECD). Commonly used fluorescent dyes include fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC), and also include tandem dyes, PE-anthocyanin-5 (PC5), PE-anthocyanin-7 (PC7), PE-anthocyanin-5.5, PE-Texas Red (ECD), rhodamine, PerCP, fluorescein isothiocyanate (FITC), and Alexa dyes. Depending on the assay method, combinations of such labels may be used, such as Texas Red and Rhodamine, FITC + PE, FITC + PECy5, and PE + PECy7. The detectable label used for attachment to the antibody that can be used in the methods described herein can be readily selected from a variety of compositions known and readily available to those skilled in the art of diagnostic assays.

[0044] In one aspect, this article provides a method for diagnosing cancer in a subject. The method includes: contacting a sample containing red blood cells from the subject (as described herein) with a reagent capable of detecting a tumor-related molecule in the sample; and diagnosing the subject with cancer when the tumor-related molecule is detected in the sample. In one embodiment, the sample is substantially free of all other blood components except RBCs (e.g., plasma, white blood cells, platelets, etc.). In one embodiment, the sample volume is from about 1 µL to about 10 µL.

[0045] In some embodiments, DNA is extracted from a sample containing RBCs. Methods for DNA extraction are known in the art. For example, the sample is centrifuged and the supernatant is discarded. A 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37°C. Lysis is performed for 30 minutes. Alternatively, the DNA extract can be obtained by column purification. This DNA extract can be used as a sample for PCR.

[0046] In some embodiments, the DNA is then amplified by PCR. Therefore, in some embodiments, reagents capable of detecting tumor-related molecules in a sample include PCR primers. Using the extracted DNA and primers, the targeted gene region is amplified by PCR using primers known in the art and as described herein.

[0047] Specifically, by using a primer set consisting of a forward primer and a reverse primer, for example, the region of the gene mutation to be expected can be amplified. As the PCR reaction solution, for example, a nucleic acid synthesis substrate, primer set, nucleic acid synthase, sample DNA, buffer solution, and a solution containing water as the remaining component can be appropriately used. Similar embodiments have been considered for other genes described herein. For example, to identify KRAS mutations, primers amplifying relevant portions of exon 2, exon 3, and / or exon 4, such as those described above by Guarnaccia et al., 2018, can be used.

[0048] Table 4: Exemplary KRAS Primers In some embodiments, reagents capable of detecting tumor-associated molecules in a sample include oligonucleotide probes. In hybridization-based methods, probe design is a critical step: efficiency depends on the strong affinity between the specific target and the short oligonucleotide probe. In formats such as gene chip arrays, multiple (up to thousands) of specific DNA probes are provided that can bind to mutated regions 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 associated with KRAS mutations are found in Table 5 below, as described in Guarnaccia et al., 2018, cited above. SEQ ID NO. 7-50 are provided in descending order.

[0049] Table 5: Exemplary KRAS probes The prepared DNA sample is then added to the gene chip, allowing it to hybridize or bind to the DNA probes on the chip. If the DNA in the sample contains mutations or variations in its sequence, it will not hybridize with the corresponding probes on the chip. When the presence or absence of PCR amplification products is determined by the DNA chip, a label is added to the amplification products. There are no particular limitations on the labeling method, but fluorescent labeling is preferred. When fluorescently labeling via PCR, fluorescently labeled primers can be used to generate amplification products with only the ends labeled. Alternatively, fluorescently labeled nucleic acid synthesis substrates can be used to generate amplification products containing the label. In any case, Cy5 or Cy3 can be suitable as fluorescent labeling components. Additionally, labels other than fluorescent ones, such as digoxigenin, biotin, and radioisotopes, can also be used as labels.

[0050] Alternatively, a general thermal cycler or similar device can be used as the apparatus for performing the PCR reaction. The PCR reaction conditions can be, for example, as follows: (A) 94°C, 2 minutes; (b) 94°C (DNA denaturation step) 30 seconds; (c) 60°C (annealing step) 30 seconds; (d) 72°C (DNA synthesis step) 60 seconds ((b) to (D) 35 cycles); (e) 72°C, 3 minutes. These conditions are provided as examples and are not intended to limit the invention.

[0051] In some embodiments, DNA chips are required to simultaneously and specifically identify multiple tumor-associated molecules within a single system. Preferably, DNA chips with sequences immobilized thereon complementary to the probe sequences are used. These probes each have specific sequences for each target molecule and can hybridize only with the amplification products of the corresponding gene regions, enabling simultaneous and specific detection of each target molecule.

[0052] DNA chips can be fabricated using the probes described above through existing general methods. For example, in fabricating immobilized DNA chips, probes can be fixed onto a glass substrate using a DNA spotter, forming spots corresponding to each probe. When fabricating synthetic DNA chips, single-stranded oligoDNAs with the aforementioned sequences can be synthesized on a glass substrate using photolithography. Furthermore, the substrate is not limited to glass; plastic substrates, silicon wafers, etc., can also be used. Moreover, the shape of the substrate is not limited to a flat plate shape and can be various three-dimensional shapes; substrates with functional groups introduced to allow for chemical reactions on the surface can also be used.

[0053] treat In some embodiments of the methods described herein, the subject receives cancer treatment after being diagnosed with cancer. In some embodiments, subsequent tests, including DNA sequencing, biopsy, MRI, CAT scan, PET scan, etc., may be performed before or after the methods described herein.

[0054] In some embodiments, the treatment includes administration of a chemotherapeutic agent. The chemotherapeutic agent is a compound exhibiting anticancer activity and / or being cellularly harmful (e.g., a toxin). Suitable chemotherapeutic agents for use in the methods disclosed herein include, but are not limited to: toxins (e.g., saponins, ricin, abrin, ethidium bromide, diphtheria toxin, Pseudomonas exotoxin, and other toxins listed above); alkylating agents (e.g., nitrogen mustard, such as chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustard, melphalan, and uracil mustard; aziridine, such as thiotepa; methanesulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, and streptozocin; platinum complexes, etc.). Examples of anti-inflammatory drugs include cisplatin and carboplatin; bioreductive alkylating agents such as mitomycin, procarbazine, dacarbazine, and hexamethylmelamine; DNA strand breaking agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., acridine, actinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., procainoxantrone); antimetabolites (e.g., folic acid antagonists such as methotrexate and trimethoprim; pyrimidine antagonists such as fluoride). Uracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and fluorouridine; purine antagonists, such as mercaptopurine, 6-thioguanine, fludarabine, and pentostatin; asparaginases; and ribonucleotide reductase inhibitors, such as hydroxyurea; microtubule interactors (e.g., vincristine, vinblastine, and paclitaxel); hormonal preparations (e.g., estrogens; conjugated estrogens; ethinylestradiol; diethylstilbestrol; chlorestradiol; idetriestrol; progestins, etc.). Examples of anti-steroids include hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; and androgens such as testosterone, testosterone propionate, flumethasone, and methyltestosterone; adrenocortical steroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); luteinizing hormone-releasing hormone (LH-R) or gonadotropin-releasing hormone (GnRH) antagonists (e.g., leuprolide acetate and goserelin acetate); and anti-hormone antigens (e.g., tamoxifen, anti-androgens such as flutamide, and anti-adrenergic agents such as mitotane and aminoglutethimide). In one embodiment, the chemotherapeutic agent is selected from the group consisting of: paclitaxel (paclitaxel®), cisplatin, docetaxel, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5-FU), gemcitabine, estradiol, carmustine, doxorubicin, etoposide, arsenic trioxide, irinotecan, and epormycin derivatives.

[0055] In some embodiments, the treatment for cancer optionally includes one or more of the following in addition to chemotherapy: Radiation therapy: This treatment involves using high-energy radiation to kill cancer cells. It can be applied topically or internally and is often used in conjunction with surgery or chemotherapy.

[0056] 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.

[0057] Targeted therapy: This type of treatment targets specific molecules or proteins involved in the growth and spread of cancer cells. Examples of targeted therapies include tyrosine kinase inhibitors and monoclonal antibodies.

[0058] Hormone therapy: This therapy is used for hormone-sensitive cancers, such as breast cancer and prostate cancer. It works by blocking or reducing the production of hormones that stimulate the growth of cancer cells.

[0059] Surgery: This treatment involves removing cancerous tumors or tissue from the body. It is often used in combination with other treatments, such as radiation therapy or chemotherapy.

[0060] Palliative care: This treatment focuses on managing symptoms and improving the quality of life for patients with advanced or terminal cancer. It includes pain management, emotional support, and other forms of supportive care.

[0061] Compositions and kits The compositions, kits, and methods described herein include reagents capable of detecting, binding to, specifically conjugating, or measuring the levels of tumor-associated molecules. Such reagents include those capable of detecting said molecules at the nucleic acid level or measuring the abundance of said molecules. Suitable reagents include those for detection by polymerase chain reaction (PCR). Suitable reagents are commercially available. Furthermore, suitable reagents can be designed by those skilled in the art based on publicly available sequences of the specific tumor of interest. In one embodiment, the reagent is a PCR primer and / or probe. Additionally, other suitable components are included to allow for the identification and / or quantification of the test tumor. Such components include, for example, enzymes, buffers and deoxynucleotides required for reverse transcription and / or PCR (preferably qualitative and / or quantitative RT-PCR), detectable probes, and / or internal controls.

[0062] Any combination of the reagents used to detect tumor-related molecules in a subject can be assembled in a 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-related gene mutations. In one embodiment, the kit includes reagents for 5 or 6 tumor-related gene mutations. In one embodiment, one or more of the reagents associate with or bind to a detectable marker or bind to a substrate.

[0063] For these reagents, the label can be selected from many known diagnostic labels, including those described above. Similarly, the substrate used for fixation can be any of common substrates, glass, plastic, microarrays, microfluidic cards, chips, or chambers.

[0064] It is anticipated that any of the compositions described herein may be a kit containing multiple reagents or one or more individual reagents. For example, one embodiment of the composition includes a substrate on which one or more of the reagents are immobilized. In another embodiment, the composition is a kit that also contains optional detectable markers, immobilized substrates, optional enzyme-labeled substrates, and other laboratory items. In one embodiment, the kit includes standards for use as controls.

[0065] The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the invention should in no way be construed as limited to these examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0066] Example Example 1: Obtaining tumor DNA from lung cancer cells using RBCs RBCs traverse all tissues, constantly coming into contact with host and pathogen-derived mediators, including highly vascularized tumors. We recently discovered that RBCs isolate mitochondrial DNA via cell surface expression of the receptor TLR9. Although we have not previously detected nuclear DNA bound to RBCs (Hotz AJRCCM, Supplement), we hypothesize that RBCs may acquire tumor DNA due to the highly vascular nature of tumors. We hypothesize that RBCs isolate tumor DNA and that tumor DNA could be detected in less than a drop of RBCs.

[0067] We first investigated whether RBCs acquire DNA from lung cancer cell lines carrying KRAS mutations. As shown in Figure 1, we were able to detect tumor DNA on RBCs co-cultured with tumor cells.

[0068] Example 2: Obtaining mutant DNA from cultured tumor cells using RBC Next, we examined other cell lines, including the H358 cell line carrying the KRAS G12C mutation and the H1299 cell line without any KRAS mutation. Similar to Figure 1, cells were seeded into 6-well plates before incubation with purified raw RBCs. However, the tumor cells did not grow to confluence several days before RBC treatment, but were only seeded overnight before the experiment. qPCR was performed on the extracted DNA against the G12S and G12C KRAS mutants. As controls, mutant multiplex and universal KRAS PCR were also run. As shown in Figure 2, mutant DNA was obtained from cultured tumor cells using RBCs.

[0069] Example 3: RBCs can detect and bind to extremely low amounts of tumor cell lysates. Next, we investigate whether RBCs can acquire tumor DNA at extremely low doses and whether they can isolate tumor DNA from plasma / SN. RBCs were incubated with varying volumes of tumor cell lysates. The lysates were produced by one million tumor cells. As shown below, RBCs were able to detect and bind to extremely low amounts of tumor cell lysates.

[0070] Example 4: RBC's tODN load From healthy donors (10 7 RBCs were incubated with fluorescently labeled tODN at different doses (0 nM / 1 million RBCs - 100 nM tODN / 1 million RBCs) at 37°C with gentle stirring at different time points (30 min, 60 min, 120 min). Sequences found in common somatic variants in lung cancer (KRAS G12C, EGFR L858R, and EGFR exon 19 deletion (synthesized by IDT)) were used. qPCR was used to compare the RBC-bound mutant ODN with the mutant ODN in the supernatant.

[0071] RBC-tODN binding will be determined using FACS, as we previously described for CpG ODN.

[0072] RBCs will be incubated with the unlabeled tODN described above. After incubation, RBCs will be separated from the supernatant (SN) by rotation over a 30% sucrose gradient. qPCR will be performed on the SN and RBC-associated DNA to quantify the binding and unbinding of tODN. If qPCR is not sensitive to detecting RBC-ODN binding, but we detect binding by flow cytometry, we will perform digital drop PCR on the RBCs and supernatant.

[0073] Red blood cells (RBCs) from patients with advanced NSCLC driven by known oncogenes and those with NSCLC without oncogene-driven tumors, as well as from healthy donors, will be isolated. Human red blood cells will be obtained from whole blood and isolated using glycoprotein A beads, as described in the literature. PCR for known somatic variants will be performed on RBCs and plasma from patients and control cancer patients (without driver mutations) and healthy controls. Fifteen patients / groups will be analyzed in this pilot study.

[0074] Example 5: RBCs isolate extracellular RNA and tumor RNA can be detected from RBCs.

[0075] We recently explored the possibility of obtaining RNA from RBCs derived from healthy donors and hospitalized patients in an inflammatory state. As shown in Figure 4, we detected significant amounts of RNA in RBCs during acute inflammatory syndrome (sepsis) compared to controls. Since RBCs do not synthesize new proteins or contain RNA, this RNA must have been acquired from other host or microbial sources. Given the reported increase in cell-free nucleic acids in cancer patients, we hypothesized that RBCs from cancer patients would contain substantial amounts of RNA, providing an opportunity to characterize circulating cell-free RNA patterns in lung cancer.

[0076] Example 6: Detection of HPV DNA on RBC Next, we will explore whether it is possible to... in vitro Human papillomavirus (HPV) DNA was detected on RBCs from healthy donors and patients that were incubated with HPV DNA. Figure 7 As shown, a FITC-tagged DNA motif from the E6 sequence of HPV16 was incubated with RBCs. RBC-HPV DNA binding was then assessed by flow cytometry. Figure 8A The binding of donor RBCs to HPV DNA after incubation with HPV CpG at doses of 0 nM, 5 nM, 25 nM, 50 nM, or 250 nM is shown. The amount of RBCs binding to HPV DNA increases with increasing HPV CpG dosage. Furthermore, in… Figure 8B In the figure, RBCs from four donors were incubated with a wider range of HPV CpG doses. The figure confirms that the percentage of cells binding to HPV DNA increases with increasing dose, with approximately 80% of cells being positive for HPV DNA after incubation with 2000 nM HPV CpG.

[0077] Example 7: Detection of HPV DNA from cancer cells on RBCs Next, we will explore whether RBCs can obtain HPV DNA from HPV-infected cervical cancer cell lines. For example... Figure 9As shown, RBCs were incubated with CaSki cells (a known cancer cell line containing HPV16) for 24 hours. DNA was then extracted from the RBCs, and HPV16 DNA was detected by qPCR. HPV16 qPCR amplification curves were generated from this experiment. Figure 10 The experiment was repeated in four independent studies using four unique healthy RBC donors. Data from these studies were... Figure 11 Provided in [the database]. In summary, these data demonstrate that RBCs obtain HPV16 DNA from cervical cancer cell lines.

[0078] The data presented above can be used clinically to diagnose HPV and cervical cancer in patients.

[0079] Example 8: Detection of HPV DNA in Cancer Cells on RBCs Next, we explored whether RBCs could obtain HPV DNA from other HPV-infected cervical cancer cell lines. First, we used PCR to detect the copy number of HPV16 DNA in various cell lines. Figure 12 Next, cells from patients with locally advanced HPV16+ cervical cancer were incubated with RBCs. HPV16 DNA was detected on 2e7 RBCs and 10 μL pRBCs in both patients. Figure 13B These results were confirmed using gel electrophoresis. Figure 13C ).

[0080] This process was then repeated in five independent experiments involving four healthy donors. Figure 13A This data confirms that HPV DNA was obtained from healthy donor RBCs.

[0081] Example 9: The mechanism of combining RBC and CpG Next, we analyzed the mechanism of RBC binding and CpG isolation. This mechanism was analyzed by comparing RBC DNA binding in WT mice and TLR9KO mice. Figure 14 RBCs from TLR9KO mice had significantly lower % CpG DNA than RBCs from wild-type mice. This is in TLR9 - and TLR + This was confirmed in human cells. The location of circulating DNA was also identified by comparing the amount of cfDNA in plasma with that in RBCs. Limited cfDNA was present in plasma in both mice and humans. This indicates that RBV preferentially isolates circulating DNA. Example

[0082] 1. A method for diagnosing cancer in a subject, the method comprising: The sample containing red blood cells from the subject was brought into contact with a reagent capable of detecting tumor-associated DNA molecules in the sample; and When the tumor-associated DNA molecule is detected in the sample, the subject is diagnosed with cancer.

[0083] 2. A method for diagnosing cancer in a subject, the method comprising: The sample containing red blood cells from the subject was brought into contact with a reagent capable of detecting tumor-related molecules in the sample; and When the tumor-associated molecule is detected in the sample, the subject is diagnosed with cancer.

[0084] 3. The method according to Example 1 or 2, wherein the reagent is specific for tumor-related gene mutations.

[0085] 4. The method according to any one of Examples 1 to 3, wherein the cancer is a cancer of the lung, cervix, breast, prostate, thyroid, colon, or pancreas.

[0086] 5. The method according to Example 1, wherein the reagent comprises a variety of reagents, each capable of detecting different specific tumor-associated molecules.

[0087] 6. The method according to any of the foregoing embodiments, wherein the reagent comprises primers capable of binding tumor-associated DNA or RNA.

[0088] 7. The method according to any of the foregoing embodiments, wherein specific tumor-related gene mutations found in the sample are identified.

[0089] 8. The method according to any of the foregoing embodiments, wherein tumor-related gene mutations occur in... EGFR, ALK, KRAS ROS1, BRAF, NTRK, MET, RET, ERBB2, PIK3CA, BRCA1, BRCA2, FGFR2, FGFR3, EZH2 or PDGFRA It was discovered in genes.

[0090] 9. The method according to Example 8, wherein the tumor-related gene mutation is in KRAS It was discovered in genes.

[0091] 10. The method according to any of the foregoing embodiments, wherein the method further includes treating the subject for cancer when diagnosed with cancer.

[0092] 11. The method according to Example 10, wherein the treatment is specific to the identified tumor-related gene mutation.

[0093] 12. The method according to any of the foregoing embodiments, wherein the diagnostic method is performed at the point of care.

[0094] 13. The method according to any of the foregoing embodiments further includes filtering the blood sample to remove non-RBC blood cells.

[0095] 14. The method according to any one of Examples 1 to 13, wherein the sample volume is 10 μL or less.

[0096] 15. The method according to Example 14, wherein the sample volume is about 1 μL to about 5 μL.

[0097] 16. The method according to any of the foregoing embodiments, wherein the sample contains at least 1 million RBCs.

[0098] 17. The method according to any of the foregoing embodiments, wherein the RBC is enriched from the sample.

[0099] 18. The method according to any of the foregoing embodiments, wherein the sample is substantially free of other blood components.

[0100] 19. The method according to any of the foregoing embodiments, wherein the subject is suspected of having cancer.

[0101] 20. A reagent capable of detecting tumor-associated DNA molecules in biological samples containing red blood cells for the purpose of diagnosing a subject with cancer when the tumor-associated DNA molecule is detected in the sample.

Claims

1. A method for diagnosing cancer in a subject, the method comprising: The sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-associated DNA molecules in the sample; as well as When the tumor-associated DNA molecule is detected in the sample, the subject is diagnosed with cancer.

2. A method for diagnosing cancer in a subject, the method comprising: The sample containing red blood cells from the subject is brought into contact with a reagent capable of detecting tumor-related molecules in the sample; as well as When the tumor-related molecules are detected in the sample, the subject is diagnosed with cancer.

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

4. The method of 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, each capable of detecting different specific tumor-associated molecules.

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

7. The method of claim 1, wherein the specific tumor-associated gene mutations found in the sample are identified.

8. The method of claim 1, wherein tumor-related gene mutations occur in... EGFR, ALK, KRAS, ROS1, BRAF, NTRK, MET, RET, ERBB2, PIK3CA, BRCA1, BRCA2, FGFR2, FGFR3, EZH2 or PDGFRA It was discovered in genes.

9. The method of claim 8, wherein the tumor-related gene mutation is... KRAS It was discovered in genes.

10. The method of claim 1, wherein the method further comprises treating the subject for cancer when the subject is diagnosed with cancer.

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

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

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

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

15. The method of claim 14, wherein the sample volume is about 1 μL to about 5 μL.

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

17. The method of claim 1, wherein the RBC is enriched from the sample.

18. The method of claim 1, wherein the sample is substantially free of other blood components.

19. The method of claim 1, wherein the subject is suspected of having cancer.

20. A reagent capable of detecting tumor-associated DNA molecules in a biological sample containing red blood cells for the purpose of diagnosing a subject with cancer when said tumor-associated DNA molecules are detected in said sample.

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