A method for detecting cancer using circulating cell-free DNA

Abstract

This specification provides a method for measuring the concentration of circulating cell-free DNA (cfDNA) from a sample of interest for the purpose of cancer or tumor detection and / or characterization.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority from U.S. Provisional Patent Application No. 63 / 500,478, filed May 5, 2023, the content of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods for detecting or characterizing cancer or tumors in a subject by optimizing cfDNA concentration thresholds. Specifically, the methods provided herein relate to evaluating the performance of thresholds for determining the presence or absence of cancer.

Background Art

[0003] As veterinary medicine continues to improve, companion animals such as dogs and cats are enjoying longer lives. However, this increased lifespan has led to a higher incidence of cancer among companion animals. According to some estimates, over 50% of dogs over 10 years old will die from cancer - related health problems. Cats are also prone to various cancers. Therefore, veterinary oncology is needed to provide early and effective cancer detection. Among the most common cancers in these animals are lymphoma, hemangiosarcoma (cancer in blood vessels), histiocytic sarcoma, squamous cell carcinoma (skin cancer), breast cancer, mast cell tumor, oral tumors, fibrosarcoma (soft tissue cancer), osteosarcoma (bone cancer), lung cancer, intestinal adenocarcinoma, pancreatic adenocarcinoma, liver cancer, anal sac adenocarcinoma, and urinary tract cancer.

[0004] Companion animals, including dogs of various breeds, are susceptible to certain cancers (Rafalko, 2023). For example, larger dogs are more prone to developing osteosarcoma. German Shepherds, Golden Retrievers, Labrador Retrievers, Pointers, Boxers, English Settlers, Great Danes, Poodles, and Siberian Huskies are susceptible to hemangiosarcoma (HSA). HSA tends to affect larger breeds of animals more frequently than smaller breeds. Unfortunately, there are no good tumor biomarkers for cancer, and its detection and diagnosis are often difficult, typically requiring invasive surgery and biopsy to make a diagnosis.

[0005] Current methods for diagnosing cancer in companion animals include imaging, microneedle aspiration cytology, and biopsy. Liquid biopsy, including blood tests for detecting cancer, provides diagnostic information that is otherwise only accessible through invasive and risky biopsies. The initial applications of liquid biopsy are based on the detection of genetic markers such as sex differences, genetic polymorphisms, or mutations.

[0006] Cancer fluid biopsies can also provide information about circulating cell-free DNA (cfDNA), which is extracellular DNA released into the bloodstream primarily as a result of apoptosis, necrosis, and secretion. Elevated levels of cfDNA have been associated with physiological conditions, including the presence of cancer or tumors. Several studies have demonstrated that increased plasma cfDNA concentrations are associated with dogs having immune-mediated hemolytic anemia, cancer, sepsis, gastric dilatatum volvulus syndrome, and trauma. However, compared to the human literature, there is relatively little literature on the relationship between cfDNA concentration and cancer in veterinary medicine. Currently available techniques involving the determination of cfDNA in veterinary medicine are risky and expensive. There is also a lack of blood-based non-invasive tests that provide a relatively inexpensive and simple method for accurately performing cancer detection. [Overview of the project]

[0007] This specification describes a method for measuring and optimizing cfDNA concentration thresholds from samples of subjects. In some embodiments, this method is used to improve cancer detection, diagnosis, and screening in subjects.

[0008] Several embodiments provided herein relate to methods for detecting cancer or tumors in a subject. In some embodiments, the method includes isolating circulating cell-free DNA (cfDNA) from a sample from the subject, extracting cfDNA from the sample, determining the concentration of cfDNA from the sample, generating a first experimental model from one or more cfDNA concentration distributions of subjects with cancer, generating a second experimental model from one or more cfDNA concentration distributions of subjects without cancer, and / or determining the presence of cancer or tumors based on a comparison of the first and second models with the cfDNA concentrations of the subject. In some embodiments, the sample is blood, plasma, urine, saliva, exudate, or cerebrospinal fluid. In some embodiments, the determination of cfDNA concentration is performed using an electrophoretic solution. In some embodiments, the determination of cfDNA concentration is performed using quantitative PCR (qPCR), digital PCR, or fluorescence assay. In some embodiments, one or more optimized thresholds are obtained from the first and second experimental models. In some embodiments, one or more optimized thresholds classify the cfDNA concentration into low, medium, and high categories. In some embodiments, the method further includes performing a genomic cancer screening assay on subjects classified as [specific type]. In some embodiments, one or more optimized thresholds differ for a given demographic variable. In some embodiments, the demographic variables are the sex of the subject, the size of the subject, the age of the subject, the breed of the subject, and / or whether the subject is spayed or neutered. In some embodiments, one or more optimized thresholds is a single threshold. In some embodiments, a cfDNA concentration above the threshold is a prediction of cancer. In some embodiments, the subjects are mammals. In some embodiments, the subjects are dogs, cats, horses, or humans. In some embodiments, the cancers are lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, mammary gland cancer, anal sac adenocarcinoma, and / or malignant melanoma.

[0009] Several embodiments provided herein relate to methods for detecting cancer or tumors in a subject. In some embodiments, the method includes obtaining a biological sample containing circulating cell-free DNA (cfDNA) from the subject, determining the concentration of cfDNA directly from the sample, generating a first experimental model from one or more cfDNA concentration distributions of subjects with cancer, generating a second experimental model from one or more cfDNA concentration distributions of subjects without cancer, and / or determining the presence of cancer or tumors based on a comparison of the first and second models with the cfDNA concentrations of the subject. In some embodiments, the sample is blood, plasma, urine, saliva, exudate, or cerebrospinal fluid. In some embodiments, the determination of cfDNA concentration is performed using an electrophoretic solution. In some embodiments, the determination of cfDNA concentration is performed using quantitative PCR (qPCR), digital PCR, or fluorescence assay. In some embodiments, one or more optimized thresholds are obtained from the first and second experimental models. In some embodiments, one or more optimized thresholds classify cfDNA concentrations into low, medium, and high categories. In some embodiments, the method further includes performing a genomic cancer screening assay on subjects classified as [specific type]. In some embodiments, one or more optimized thresholds differ for a given demographic variable. In some embodiments, the demographic variables are the sex of the subject, the size of the subject, the age of the subject, the breed of the subject, and / or whether the subject is spayed or neutered. In some embodiments, one or more optimized thresholds is a single threshold. In some embodiments, a cfDNA concentration above the threshold is a prediction of cancer. In some embodiments, the subjects are mammals. In some embodiments, the subjects are dogs, cats, horses, or humans. In some embodiments, the cancers are lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, mammary gland cancer, anal sac adenocarcinoma, and / or malignant melanoma.

[0010] In some embodiments of the methods described herein, the analysis is a pan-cancer analysis.

[0011] To illustrate how the above and other advantages and features of the embodiments described herein can be obtained, a more specific description will be given by reference to the specific embodiments illustrated in the accompanying drawings. With the understanding that these drawings depict only typical embodiments and are therefore not intended to limit the scope, this disclosure will be described and explained more specifically and in detail through the use of the accompanying drawings. [Brief explanation of the drawing]

[0012] [Figure 1A] An illustrative flowchart illustrating threshold analysis through the first analysis is shown. [Figure 1B] An exemplary flowchart illustrating threshold analysis through the second analysis is shown. [Figure 2A] Exemplary ROC curves demonstrating the performance of Concept 1 (Figure 1A) and Concept 2 (Figure 1B) as functions of demographic variables, including sex, are shown. The lighter colored lines represent the performance of male subjects, and the darker colored lines represent the performance of female subjects. [Figure 2B] Exemplary ROC curves demonstrating the performance of Concept 1 (Figure 1A) and Concept 2 (Figure 1B) as functions of demographic variables, including size, are shown. The lighter colored lines represent subjects above the median size, and the darker colored lines represent subjects below the median size. [Figure 2C] Exemplary ROC curves demonstrating the performance of Concept 1 (Figure 1A) and Concept 2 (Figure 1B) as functions of demographic variables, including age, are shown. The lighter colored lines represent those above the median age of the subjects, and the darker colored lines represent those below the median age of the subjects. [Figure 3] For a set of difficult-to-diagnose (D2D) cancers, an exemplary bar graph is shown, representing the estimated proportion of cancers to be diagnosed, assigned to each probability tier, known as the "cancer probability index" (low, medium, high). [Modes for carrying out the invention]

[0013] In the modes of carrying out the invention described herein, references are made to the accompanying drawings which form part of this specification. In the drawings, similar symbols typically identify similar components unless the context indicates otherwise. The exemplary embodiments described in the modes of carrying out the invention, the drawings, and the claims are not intended to be limiting. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that the aspects of this disclosure generally described herein and illustrated in the drawings can all be arranged, substituted, combined, separated, and designed in a wide variety of different configurations expressly contemplated herein. All references cited herein are expressly incorporated herein in their entirety by reference for the specific disclosures referenced herein.

[0014] Previous studies, such as Tagawa (2019) and Kim (2021), have demonstrated that elevated plasma cfDNA concentrations compared to healthy subjects tend to be associated with larger tumor size and malignancy. These studies argued that cfDNA concentration is a good screening tool for detecting distant metastasis and may be useful when used in combination with existing diagnostic tools. However, the cfDNA concentrations reported by Tagawa and Kim are much higher than the mean concentrations observed in other studies with larger datasets. Therefore, cfDNA concentration measurements are likely to be overestimated and may be contributed to by cfDNA and genomic DNA found in cells. Consequently, the disclosure in Tagawa is not feasible because successful measurement of cfDNA concentration was not achieved to draw conclusions. In addition, the small sample size (N<100) with limited representation of breed and other demographics, used in each analysis to detect differences between different groups, may have been influenced by multiple confounding factors. In contrast, the results presented in this application are generated from a much larger dataset (spanning thousands of dogs representing diverse demographics) using quantification methods specifically developed to target cfDNA-specific physical properties (e.g., taking into account fragment size profiles), and therefore can more accurately reflect the association between cfDNA concentration and the probability of cancer.

[0015] Additional studies have demonstrated that determining plasma nucleosome concentrations by the Nu.Q assay, an ELISA-based test for detecting nucleosome concentrations in cfDNA, is a good indicator of cancer progression, primarily for systemic cancers (higher metastasis rates). Wilson-Robles, 2022. The Nu.Q assay was only able to correctly detect half of the cancers tested, and many additional assays were needed to improve the sensitivity of liquid biopsy techniques in both human and veterinary subjects.

[0016] Having a large number of subjects with different types of cancer, and by using the screening analysis described herein, the methods disclosed herein may have the advantage of having a significantly more sensitive and optimized cfDNA concentration threshold for correctly detecting or characterizing cancer or tumors in the subjects.

[0017] Embodiments relate to methods for screening subjects for the possibility of having cancer or tumors. In some embodiments, cancer or tumors are screened by isolating circulating cell-free DNA (cfDNA) from a biological sample from a subject such as a dog, determining the concentration of cfDNA in the sample, creating a model or summary statistics of the cfDNA concentration, comparing the model of the cfDNA concentration of the subject to a second model derived from at least one healthy subject, and determining the presence or absence of cancer or tumors based on the comparison of the two models. Determining the concentration of cfDNA can be done through any method recognized by those skilled in the art, such as quantitative polymerase chain reaction (qPCR). Other non-limiting examples include methods using digital PCR, electrophoresis, or fluorescence assay methods.

[0018] In some embodiments, cancer or tumors are screened by comparing models. In some embodiments, these models are mixed models. These models are derived from the concentration of cfDNA. As disclosed herein, the screening method can be performed on subjects suspected of having cancer or tumors, as well as on models of one or more healthy subjects, specifically on subjects with difficult-to-diagnose cancers such as lymphoma, osteosarcoma, angiosarcoma, histiocytic sarcoma, leukemia, lung malignancies, and bladder / urethral cancers. These models may also be specific to the sex, size, and age of the subjects. Non-limiting examples of detectable differences include susceptibility, specificity, and distribution between the two models.

[0019] Various methods exist for determining the concentration of cfDNA within a subject. In one embodiment, a blood sample is taken from the subject. Circulating cell-free DNA (cfDNA) is obtained from the blood. cfDNA is isolated from blood cells in the sample. In some embodiments, cfDNA is measured directly from plasma without isolation or extraction.

[0020] The methods provided herein improve the detection, diagnosis, staging, screening, treatment, and management of cancer in subjects including dogs. In some embodiments, the methods include measuring the concentration of cfDNA directly from a biological sample.

[0021] Biological Samples: Some embodiments of the embodiments provided herein measure cfDNA present in biological samples. Biological samples as used herein include, for example, cell culture media, as well as tissues and fluids obtained from subjects. Samples obtained from subjects may include any tissue or fluid from subjects that may contain cfDNA. In some embodiments, the biological sample is whole blood, plasma, serum, lymph, vitreous fluid, cochlear fluid, tears, peripheral blood, serum, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's gland fluid or preejaculatory fluid, female vaginal fluid, sweat, fecal matter, hair, cystic fluid, pleural fluid and ascites, pericardial fluid, erosion, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, or other lavage fluid. The biological sample may also include blastocyst cavity, umbilical cord blood, or maternal circulation, which may be of fetal or maternal origin. The biological sample may also be a tissue sample or biopsy from which cfDNA can be obtained.

[0022] As used herein, "detecting" with respect to measuring cancer or a tumor includes the use of an instrument used to observe and record the level of cancer or a signal corresponding to the measurement, or the materials required to generate such a signal. In various embodiments, detecting includes any suitable method, including amplification, sequencing, arrays, fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.

[0023] It should be appreciated that the assays described herein can be part of a larger diagnostic panel used to determine the overall health of a subject. For example, the analysis of thresholds for determining the concentration of cfDNA in a subject can be used simultaneously or sequentially with other methods for the detection, diagnosis, staging, screening, monitoring, treatment, and management of cancer, including additional genetic variance analysis. These procedures can be useful for detecting various cancers, including lymphoma, leukemia, squamous cell carcinoma, feline mammary carcinoma, mastocytoma, bladder cancer, osteosarcoma, angiosarcoma, melanoma, or various other cancers afflicting the subject.

[0024] In some embodiments, the method includes obtaining or having obtained a biological sample from a subject suspected of having cancer. In some embodiments, the sample is a liquid biopsy sample such as a blood sample. In some embodiments, the sample contains cfDNA. In some embodiments, the sample is provided in an amount less than 10 mL, such as less than 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 500 μL, 250 μL, 100 μL, 50 μL, 25 μL, or 10 μL, or an amount within or less than the range defined by any two of the foregoing values. In some embodiments, the method includes extracting cfDNA from a plasma sample. In some embodiments, the method includes determining the concentration of cfDNA in a plasma sample. Determining the concentration of cfDNA can be achieved using techniques including, for example, quantitative PCR (qPCR), digital PCR, fluorescence assays, automated electrophoresis solutions, or commercially available kits for cfDNA quantification. In some embodiments, the method can be used to predict whether a subject has cancer based on the cfDNA concentration.

[0025] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety unless otherwise specified. In the event of multiple definitions for terms herein, the definitions in this section shall control unless otherwise specified.

[0026] As used herein, "a" or "an" can mean one or more than one.

[0027] Where used herein, the terms “about” or “approximately” have their ordinary meaning as understood by those skilled in the art, and thus indicate that the value refers to a range of numbers (e.g., ±5% to 10% of the enumerated value) that those skilled in the art would generally consider equivalent to (e.g., having the same function or result as) the enumerated value, including variations in the inherent error of the method employed to determine the value, or variations that exist between multiple determinations.

[0028] The dimensions and values ​​disclosed herein are not to be understood as strictly limited to the exact numerical values ​​listed. Rather, unless otherwise specified, each such dimension is intended to mean both the listed value and the functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "approximately 20 mm".

[0029] Throughout this specification, unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of the described step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. “Consisting of” means including and being limited to everything that precedes the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are necessary or essential, and that any other elements may not be present. “Consisting essentially of” means including any elements enumerated before this phrase, and being limited to other elements that do not interfere with or contribute to the activity or action identified in this disclosure for the enumerated elements. Therefore, the phrase "consisting essentially of" indicates that the enumerated elements are necessary or essential, while the other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0030] As used herein, the terms “function” and “functional” have their plain and ordinary meanings as understood in light of this specification, and refer to biological, enzymatic, or therapeutic functions.

[0031] As used herein, the term “yield” for any given substance, compound, or material has its plain and ordinary meaning as understood in light of this specification and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material is about, at least, at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less, or approximately less than or equal to, the expected total amount, including all decimal points in between. The yield may be affected by the efficiency of the reaction or process, unwanted side effects, decomposition, the quality of the input substance, compound, or material, or the loss of the desired substance, compound, or material at any step of production.

[0032] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of this specification and means (1) separated from at least some of the components associated with the original production (in the natural and / or experimental environment), and / or (2) produced, prepared, and / or manufactured by human hands. Isolated substances and / or entities may be separated from an amount equal to 10%, about 10%, at least 10%, at least about 10%, 10% or less, or about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% (or to the extent that includes and / or extends to the aforementioned values). In some embodiments, the isolated active ingredient is 80%, about 80%, at least 80%, at least about 80%, 80% or less, about 80% or less, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% (or to the extent that includes and / or extends the aforementioned values) pure. As used herein, the “isolated” substance may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multicellular organism or tissue.

[0033] As used herein, the term “circulation” has its plain and ordinary meaning as understood in light of this specification, and means (1) matter and / or entities that move or are moved continuously or freely through a closed system or area, and / or (2) matter and / or entities that pass or are passed from place to place or from person to person.

[0034] As used herein, the term “extract” has its plain and ordinary meaning as understood in light of this specification and refers to a substance and / or entity that is (1) transferred or removed by effort or force, or (2) obtained from something by a special method. Extracting circulating cell-free DNA from a plasma sample means removing cfDNA from plasma after centrifugation using different extraction kits such as CNA kits, RSC kits, and ME kits.

[0035] As used herein, the term “determine” has its ordinary meaning as understood by those skilled in the art, and therefore means (1) to cause or have something happen in a particular manner, or (2) to find or make a decision by investigation, reasoning or calculation.

[0036] As used herein, the term “to produce” has its ordinary meaning as understood by those skilled in the art, and therefore indicates (1) to bring something into existence or occur, or (2) to produce a set or sequence by performing a specified mathematical or logical operation on an initial set.

[0037] As used herein, the term “concentration” has its ordinary meaning as understood in light of this specification and refers to (1) the amount of dissolved substance in a unit volume, or (2) the ratio of solute in a solution to either the solvent or the total solution. Concentration is usually expressed in terms of mass per unit volume. Example of concentration units: g / cm³ 3 , kg / l, M, m, N, kg / L. A concentrated solution refers to a chemical solution containing a large amount of solute. A diluted solution refers to a chemical solution containing a small amount of solute dissolved in it. DNA concentration (DNA) can be measured in several different ways, such as ultraviolet (UV) absorbance, fluorescence, and the diphenylamine reaction.

[0038] As used herein, “nucleic acid,” “nucleic acid molecule,” or “nucleotide” refers to polynucleotides and / or oligonucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, exonuclease action, and synthetic synthesis. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications may include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar may be functionalized as an ether or ester. Furthermore, the entire sugar moiety may be replaced with a sterically and electronically similar structure, such as aza sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, and phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded.

[0039] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their plain and ordinary meanings as understood in light of this specification, and refer to macromolecules consisting of amino acids linked by peptide bonds. Numerous functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structural, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, though not always, produced biologically by ribosome complexes using nucleic acid templates, although chemosynthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations can be performed, such as substitution, deletion, cleavage, addition, replication, or fusion of one or more peptides, polypeptides, or proteins. These fusions of one or more peptides, polypeptides, or proteins can be joined adjacent to each other within the same molecule or with any extra amino acids between them, such as a linker, repeat, epitope, or tag, or any other sequence of any length within the range defined by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases, or any other sequence of any length within the range defined by any two of the aforementioned lengths, approximately that length, at least that length, at least approximately that length, less than or equal to that length, or approximately less than or equal to that length. The term “downstream” on a polypeptide, as used herein, has its plain and ordinary meaning as understood in light of this specification and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term “upstream” on a polypeptide has its plain and ordinary meaning as understood in light of this specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0040] The terms “cancer” and “cancerous” have their usual meanings as understood in light of this specification and refer to or describe a physiological condition in animals typically characterized by unregulated cell proliferation. A “tumor” comprises one or more cancerous cells. In some embodiments, a tumor is a solid tumor. Several major types of cancer exist. Carcinomas are cancers that originate from epithelial cells, e.g., skin cells or the intestinal lining. Sarcomas are cancers that originate from mesenchymal cells, e.g., bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is a cancer that originates from hematopoietic cells, such as those in the bone marrow, causing them to produce a large number of abnormal blood cells that enter the bloodstream. Lymphomas and multiple myelomas are cancers that originate from lymphoid cells in the lymph nodes. Central nervous system cancers are cancers that originate from the central nervous system and spinal cord. In some embodiments, cancers may be specific to companion animals, such as dogs. Examples of cancers include lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, breast cancer, anal sac adenocarcinoma, and malignant melanoma. In some embodiments, the study is a pan-cancer analysis, which is an analysis across a variety of tumor types.

[0041] In some embodiments, the methods provided herein assign subjects to a probability hierarchy referred to herein as a cancer probability index. As used herein, the term “cancer probability index” refers to the probability of having cancer at the time of performing the methods described herein and may include low, medium, or high probabilities, or probabilities within a range of these terms. For example, a low probability may include the probability of having cancer with a reduced likelihood compared to the incidence rate of cancer. The reduction in the likelihood of having cancer may be twofold or threefold. A medium probability may include the probability of having cancer with a slight increase compared to the incidence rate of cancer, for example, 10%, 20%, 30%, 40%, 50%, or 60%, or within a range defined by any two of the aforementioned values. A high probability may include the possibility of having cancer that is significantly higher than the cancer incidence rate, such as 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, or more, or within the range defined by any two of the aforementioned values.

[0042] As used herein, the term “amplification” has its usual meaning as understood in light of this specification and refers to any method known in the art for copying a target nucleic acid and thereby increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. The target nucleic acid can be either DNA or RNA. Typically, the sequence amplified in this way forms an “amplicon.” Amplification can be achieved in a variety of ways, including but not limited to polymerase chain reaction (“PCR”), transcription-based amplification, isothermal amplification, and rolling circle amplification. Amplification can be carried out with relatively similar amounts of each primer in a primer pair to produce a double-stranded amplicon. However, asymmetric PCR can be used, as is well known in the art, primarily or exclusively, to amplify single-stranded products (e.g., Poddar et al. Molec. And Cell. Probes 14:25-32 (2000)). This can be achieved using each pair of primers by significantly reducing the concentration of one primer in the pair relative to the other (e.g., a 100-fold difference). Amplification by asymmetric PCR is generally linear. Those skilled in the art will understand that different amplification methods can be used together.

[0043] As used herein, the terms “individual,” “subject,” “host,” or “patient” have their ordinary meanings as understood by those skilled in the art, and therefore include humans and non-human mammals. The term “mammal” is used in its ordinary biological sense. Therefore, it specifically includes, but is not limited to, primates, including true monkeys (chimpanzees, apes, and monkeys), humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0044] As used herein, the term “liquid biopsy” has its ordinary meaning as understood in light of this specification, and refers to the collection and testing of a sample, which is a non-solid biological tissue such as blood.

[0045] As used herein, the term "cfDNA" has its ordinary meaning as understood by those skilled in the art and refers to circulating cell-free DNA containing DNA fragments released into plasma. cfDNA may include circulating tumor deoxyribonucleic acid (ctDNA).

[0046] As used herein, the term “plasma” has its usual meaning as understood in light herein, and refers to the clear, yellowish liquid portion of blood, which is composed of 90–92% water. 55 percent of blood is plasma, and the remaining 45 percent consists of red blood cells, white blood cells, and platelets. Plasma acts as a transport medium for delivering nutrients and proteins to the cells of various organs of the body, and for transporting waste products derived from cellular metabolism to the kidneys, liver, and lungs for excretion. It is also the transport system for blood cells. Plasma helps distribute heat throughout the body, as well as maintain homeostasis, or biological stability, including acid-base balance in the blood and within the body. Plasma also contains 6–8% protein. One important group is coagulation proteins and their inhibitors, which are primarily synthesized in the liver.

[0047] As used herein, the term “susceptibility” has its ordinary meaning as understood by those skilled in the art, and refers to the true positive rate, which is the probability of a positive test result, provided that the subject is truly positive.

[0048] As used herein, the term “specificity” has its ordinary meaning as understood in light herein, and refers to the true negative rate, which is the probability of a negative test result given that the individual is truly negative.

[0049] As used herein, the term “Screening Concept 1” refers to a population in which only cfDNA concentration is measured.

[0050] As used herein, the term “Screening Concept 2” refers to a population of subjects with moderately high cfDNA concentrations that are further evaluated using the OncoK9 genomic test. OncoK9 is a cell-free DNA-based, non-invasive genomic cancer screening test for canine cancer detection, driven by next-generation sequencing (NGS) and using bioinformatics analysis. OncoK9 is capable of detecting a variety of cancers, including, for example, lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, mammary gland cancer, anal sac adenocarcinoma, and / or malignant melanoma. In some embodiments, the method involves performing a genomic cancer screening assay on subjects classified as intermediate. In some embodiments, the genomic cancer screening assay is the OncoK9 assay.

[0051] As used herein, the term “distribution” has its ordinary meaning as understood in light of this text, and refers to the frequency of a position, arrangement, or occurrence (as a member of a group) across a region or throughout a spatial or temporal unit. In statistics, a distribution is a mathematical function that describes the relationship of observations at different heights. A distribution is simply a set of data or scores about a variable. It describes the probability that a system takes a specific value or set of values. The highest point on a curve usually represents the most common value or mode, which is close to the mean of the population. As used herein, the term “cfDNA distribution” refers to the frequency of occurrence and may include the distribution of cfDNA across different cancers and / or across different subjects.

[0052] The above description discloses several methods and materials. This disclosure is susceptible to modifications of methods and materials, as well as alterations of manufacturing methods and equipment. Such modifications will be apparent to those skilled in the art from consideration of the disclosures disclosed herein. Accordingly, the embodiments described herein are not intended to be limited to the specific embodiments disclosed herein, but rather to cover all modifications and alternatives that fall within the true scope and spirit of this disclosure.

[0053] All references cited herein, including but not limited to published and unpublished applications, patents, and references to documents, are incorporated herein by reference in their entirety and thus become part of this Specified. To the extent that any published and unpublished applications or patent applications incorporated by reference conflict with the disclosures contained herein, this Specified is intended to supersede and / or take precedence over any such conflicting material.

[0054] In another embodiment of the methods described herein, any of the methods described herein may be used alone, or any of the methods described herein may be used in combination with any other or more of the methods described herein. [Examples]

[0055] Embodiments of the present invention are further defined by the following examples. It should be understood that these examples are given for illustrative purposes only. From the above considerations and these examples, those skilled in the art can confirm the essential characteristics of the present invention and make various changes and modifications to the embodiments of the invention to adapt them to various uses and conditions without departing from the spirit and scope thereof. Therefore, various modifications to the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosures of each reference cited herein are incorporated herein in their entirety by reference for the disclosures referenced herein.

[0056] Example 1: Extraction of cfDNA from the target Embodiments of cfDNA isolation described herein were carried out using a series of extractions. Blood samples were collected from canine subjects into anticoagulant blood collection tubes (BCTs) containing circulating cell-free DNA stabilizing components. Not limited examples of usable collection tubes include Roche cell-free DNA collection tubes, as well as Streck, Biomatrica, MagMax, or Norgen collection tubes. The BCTs were then centrifuged to separate the plasma fraction from the erythrocytes and leukocytes. The cell-free plasma layer was removed from the BCTs and either stored or incorporated directly into the circulating cell-free DNA (cfDNA) extraction.

[0057] cfDNA was extracted from 2–8 mL of plasma using a commercially available magnetic bead-based extraction kit (MagMax Cell-Free DNA Isolation Kit). Other equivalent extraction methods / kits, including column-based solid-phase and precipitation-based methods, could potentially be used in this process. cfDNA was eluted, and the cfDNA concentration was quantified by automated electrophoresis (e.g., Agilent TapeStation). The selection of the type of assay performed on the electrophoresis platform and the specific parameters used for quantification were optimized to target the specific fragment size profile of cell-free DNA. Other methods for quantifying cfDNA concentration include quantitative PCR (qPCR) or digital PCR, which can be performed by direct measurement from plasma samples without DNA extraction.

[0058] A whole-genome library was prepared from cfDNA by contacting the cfDNA sample with random primers configured to amplify the entire genome for sequencing. However, it will be understood by those skilled in the art that any method suitable for sequence amplification, such as next-generation sequencing, can be utilized. In one embodiment, library preparation may include incorporating unique molecular identifiers and unique sample-specific barcodes to enable multiplexing of samples from different subjects.

[0059] Example 2: Demographic analysis of individuals with and without cancer An embodiment of cfDNA analysis of subjects was performed by comparing sex, age, and weight of dogs with all types of cancer and dogs with difficult-to-diagnose (D2D) cancers compared with dogs without cancer. The dataset consisted of 754 cancer-diagnosed subjects and 1204 cancer-free subjects. Of the 754 cancer-diagnosed subjects, 324 were diagnosed with relevant D2D cancers. Table 1 shows the demographics of cancer-diagnosed subjects (all cancers and D2D cancers separately) and cancer-free subjects.

[0060] The samples were divided into training and testing sets in a 2:1 ratio based on cancer status. Specifically, two-thirds of the cancer-diagnostic samples were randomly assigned to training, and the remainder to testing, with the same ratio applied to the non-cancer samples. After this division, the training set consisted of 502 cancer-diagnostic samples (229 of which were D2D cancers) and 802 non-cancer samples, while the testing set consisted of 252 cancer-diagnostic samples (95 of which were D2D cancers) and 402 non-cancer samples.

[0061] Table 1: Population statistics for cancer diagnosis (all cancers and D2D cancers separately) and cancer-free populations JPEG2026516048000002.jpg67157

[0062] Example 3: Analysis to optimize susceptibility and specificity across different cancer cohorts The following examples demonstrate how data analysis can be performed on cfDNA to determine whether different cancer sets, as a whole, produce more robust data compared to cancer.

[0063] Cancers were divided into three distinct groups: all cancers, Nu.Q® cancers (as included in Nu.Q® marketing materials: lymphoma, osteosarcoma, angiosarcoma, soft tissue sarcoma, histiocytic sarcoma, mast cell tumor, malignant melanoma), Nu.Q+® cancers (as included in Nu.Q® marketing materials, plus additional cancer types for which the commercially available OncoK9 assay performs well in the International Clinical Cancer Detection (CANDiD) study in dogs: lymphoma, osteosarcoma, angiosarcoma, soft tissue sarcoma, histiocytic sarcoma, mast cell tumor, malignant melanoma, leukemia, mammary gland cancer, lung cancer), and D2D cancers (including lymphoma, osteosarcoma, angiosarcoma, histiocytic sarcoma, leukemia, lung malignancies, and bladder / urethral cancers).

[0064] The combination of lower and upper thresholds for cfDNA concentration was manually selected to optimize the overall sensitivity and specificity of the data analysis.

[0065] Susceptibility and specificity were calculated using the following formula. ● Susceptibility (Concept 1) = {Number of cancers with a high probability of diagnosis} / {Number of cancers with a high or low probability of diagnosis} ●Specificity (Concept 1) = {Number of cancer-free subjects with a low probability} / {Number of cancer-free subjects with a high or low probability}

[0066] In the analysis of Screening Concept 1, the proportion of subjects with intermediate probability results is excluded from the calculation, whereas in the analysis of Screening Concept 2, all intermediate probability results are automatically tested by OncoK9 / OKR before reporting, so these samples are included.

[0067] In Screening Concept 1, these calculations exclude the proportion of subjects with intermediate probability results, whereas in Screening Concept 2, all intermediate probability results are automatically tested by OncoK9 before reporting, as shown in Figures 1A and 1B, and these samples are included. In the figures, "OKR" refers to the OncoK9 genome test, "CSND" refers to no detected cancer signal, "CSD" refers to detected cancer signal, and "OKL" refers to Concept 1 or 2.

[0068] Receiver operating characteristic (ROC) curves were generated for different demographic variables (sex, weight, age). As shown in Figures 2A–2C, no significant differences in performance were observed in the ROC curves as a function of demographic variables. However, the optimal points on the curves did not correspond to the same cfDNA concentration threshold. Instead, sex-specific thresholds performed better than a single threshold.

[0069] The highest threshold may be selected to optimize sensitivity and specificity in the Nu.Q+(trademark) cancer set while minimizing the number of samples classified into the intermediate probability group. The final upper and lower thresholds for different sexes are listed in Table 2 along with the grid search results.

[0070] Table 2: Final cfDNA concentration threshold based on reported sex. JPEG2026516048000003.jpg21155

[0071] Example 4: Analysis of susceptibility across different cancer groups The following examples summarize the comparison of Concepts 1 and 2 in different cancer groups by measuring susceptibility and specificity as well as moderate cancer probability index results.

[0072] The following tables show the overall performance of screening concepts 1 and 2 for both training (Table 3) and trial (Table 4) sets when applied to D2D cancers.

[0073] Table 3: Performance of Screening Concepts 1 and 2 in the Training Set JPEG2026516048000004.jpg26145

[0074] Table 4: Performance of screening concepts 1 and 2 in the test set. JPEG2026516048000005.jpg36147

[0075] Example 5: Comparison of susceptibility between different cancer groups The following examples summarize the comparison of different cancer susceptibility across different cancer groups.

[0076] Susceptibility across both training and test sets was performed for each cancer type within the D2D cancer set and is shown in Table 5. Subjects with multiple diagnoses were included in Table 5 if at least one of those diagnoses was a given cancer. A single subject may be selected to be included in the performance estimates for both cancers if diagnosed with two D2D cancers.

[0077] Table 5: Training + test set performance for each cancer type within the D2D set. JPEG2026516048000006.jpg88166

[0078] With regard to the use of plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or singular to plural as appropriate to the context and / or use. Various singular / plural substitutions may be expressly shown herein for clarity.

[0079] In general, it will be understood by those skilled in the art that the terms used herein, and especially in the appended claims (e.g., the text of the appended claims), are generally intended to be “non-restrictive” terms (for example, “including” should be interpreted as “including but not limited to,” “having” as “having at least,” and “includes” as “includes but is not limited to,” etc.). It will further be understood by those skilled in the art that where a specific number of claims introduced is intended, such intention is explicitly stated in the claim, and where such a statement is not present, such intention is not present. For example, to aid understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, the use of such phrases should not be interpreted as implying that the introduction of a claim description by the indefinite article "a" or "an" implies limiting any particular claim containing such introduced description to embodiments containing only one such description, even if the same claim includes an introductory phrase such as "one or more" or "at least one" and an indefinite article such as "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"). The same is true for the use of indefinite articles used to introduce a claim description. In addition, even if a specific number of introduced claims is explicitly stated, a person skilled in the art will recognize that such a description should be interpreted as meaning at least that stated number (for example, the bare description "two descriptions" without other modifying phrases means at least two descriptions, or two or more descriptions).Furthermore, in cases where a convention similar to "at least one of A, B, and C, etc." is used, such a structure is generally intended to be understood by those skilled in the art (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any disjunct word and / or phrase presenting two or more alternative terms should be understood as construing the possibility of including one of those terms, either or both of those terms, whether or not they appear in the specification, claims, or drawings. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0080] In addition, if any feature or aspect of the present disclosure is described in terms of the Markush group, a person skilled in the art will recognize that the present disclosure is also described in terms of any individual member or subgroup of a member of the Markush group.

[0081] Any feature of the embodiments of the first to second embodiments is applicable to all embodiments and forms identified herein. Furthermore, any feature of the embodiments of the first to third embodiments can be independently combined in any way, in part or in whole, with other embodiments described herein, for example, one, two, or three or more embodiments may be combined in whole or in part. Furthermore, any feature of the embodiments of the first to third embodiments may be optionally selected from other embodiments or forms.

Claims

1. A method for detecting cancer or tumors in a subject, To isolate circulating cell-free DNA (cfDNA) from the sample from the aforementioned subject, Extracting the cf DNA from the sample, The concentration of the cfDNA from the sample is determined, To generate a first experimental model from the cfDNA concentration distribution of one or more subjects with cancer, To generate a second experimental model from the cfDNA concentration distribution of one or more subjects without cancer, The method comprising determining the presence of cancer or a tumor based on a comparison between the first and second models and the concentration of the target cfDNA.

2. The method according to claim 1, wherein the sample is blood, plasma, urine, saliva, exudate, or cerebrospinal fluid.

3. The method according to claim 1, wherein the determination of the concentration of the cfDNA is performed using an electrophoretic solution.

4. The method according to claim 1, wherein the determination of the concentration of the cfDNA is performed using quantitative PCR (qPCR), digital PCR, or fluorescence assay.

5. The method according to claim 1, wherein one or more optimized thresholds are obtained from the first and second experimental models.

6. The method according to claim 5, wherein the one or more optimized thresholds classify the concentration of the cfDNA into low, medium, and high categories.

7. The method according to claim 6, further comprising performing a genomic cancer screening assay on subjects classified as [specific category].

8. The method according to claim 5, wherein the one or more optimized thresholds differ with respect to a given demographic variable.

9. The method according to claim 8, wherein the demographic variables are the sex of the subject, the size of the subject, the age of the subject, the breed of the subject, and / or whether the subject is to be sterilized or castrated.

10. The method according to claim 5, wherein the one or more optimized thresholds is a single threshold, and the concentration of the cfDNA above the threshold is a prediction of cancer.

11. The method according to claim 1, wherein the subject is a mammal.

12. The method according to claim 11, wherein the subject is a dog, a cat, a horse, or a human.

13. The method according to claim 1, wherein the cancer is lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, breast cancer, anal sac adenocarcinoma, and / or malignant melanoma.

14. A method for detecting cancer or tumors in a subject, Obtain a biological sample containing circulating cell-free DNA (cfDNA) from the aforementioned subject, Determining the concentration of the cfDNA directly from the aforementioned sample, To generate a first experimental model from the cfDNA concentration distribution of one or more subjects with cancer, To generate a second experimental model from the cfDNA concentration distribution of one or more subjects without cancer, The method comprising determining the presence of the cancer or tumor based on a comparison between the first and second models and the concentration of the target cfDNA.

15. The method according to claim 14, wherein the sample is blood, plasma, urine, saliva, exudate, or cerebrospinal fluid.

16. The method according to claim 14, wherein the determination of the concentration of the cfDNA is performed using an electrophoretic solution.

17. The method according to claim 14, wherein the determination of the concentration of the cfDNA is performed using quantitative PCR (qPCR), digital PCR, or fluorescence assay.

18. The method according to claim 14, wherein one or more optimized thresholds are obtained from the first and second experimental models.

19. The method according to claim 18, wherein the one or more optimized thresholds classify the concentration of the cfDNA into low, medium, and high categories.

20. The method according to claim 19, further comprising performing a genomic cancer screening assay on subjects classified as intermediate.

21. The method according to claim 18, wherein the one or more optimized thresholds differ with respect to a given demographic variable.

22. The method according to claim 21, wherein the demographic variables are the sex of the subject, the size of the subject, the age of the subject, the breed of the subject, and / or whether the subject is to be spayed or neutered.

23. The method according to claim 18, wherein the one or more optimized thresholds is a single threshold, and the concentration of the cfDNA above the threshold is a prediction of cancer.

24. The method according to claim 14, wherein the subject is a mammal.

25. The method according to claim 24, wherein the subject is a dog, a cat, a horse, or a human.

26. The method according to claim 14, wherein the cancer is lymphoma, angiosarcoma, soft tissue sarcoma, mast cell tumor, osteosarcoma, breast cancer, anal sac adenocarcinoma, and / or malignant melanoma.

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