Means and methods for diagnosing cancer and / or acute inflammatory diseases

Abstract

The present invention relates to a method for diagnosing cancer and / or acute inflammatory disease in a subject. Furthermore, the present invention relates to a method for evaluating the response of cancer and / or acute inflammatory disease to a candidate treatment in a subject. Furthermore, the present invention relates to a method for evaluating the malignancy of cancer in a subject.

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing cancer and / or acute inflammatory disease in a subject. The present invention further relates to a method for evaluating the response of cancer and / or acute inflammatory disease to a candidate treatment in a subject. Furthermore, the present invention relates to a method for evaluating the malignancy of cancer in a subject. [Background technology]

[0002] This specification references numerous documents, including patent applications and manufacturer's manuals. While the disclosure of these documents is not considered relevant to the patentability of the present invention, they are incorporated by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated as being incorporated by reference.

[0003] Cancer is one of the leading causes of death worldwide, affecting an estimated 12.7 million people globally, with an equal number of men and women affected. This number is even projected to increase to 21 million by 2030 (Vinay DS. Semin Cancer Biol. (2015);35 Suppl:S185-S198). Timely detection of malignant tumors through early diagnosis and cancer screening provides patients with the opportunity to benefit effectively from cancer treatment, because the improvement in treatment response and the resulting survival rates is overwhelmingly more pronounced in the early stages of cancer (Siegel RL et al., Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30). The decline in mortality rates for some cancers, such as colorectal cancer and breast cancer, is at least in part due to the establishment of corresponding cancer screening tests (Byers T et al., The American Cancer Society; 2016;66(5):359-69).

[0004] However, the reality is that the majority of cancer patients are diagnosed only after symptoms appear, at a stage when the tumor has already progressed and treatment options are limited. Diagnostic screening tools exist for only some cancers, including microscopic analysis combined with DNA testing (cervical cancer (Tsikouras P et al. Journal of BUON: official journal of the Balkan Union of Oncology. 2016;21(2):320-5)), skin examination (Wolff T et al., Annals of internal medicine. 2009;150(3):194-8), imaging techniques (mammography (van den Biggelaar FJ et al., Breast (Edinburgh, Scotland). 2008;17(1):85-90.5), computed tomography (CT) colonography and colonoscopy (Pickhardt PJ et al., Radiology. 2011;259(2):393-405), and low-dose computed tomography of the chest (Hoffman RM et al., The Medical clinics of North America. These include blood-based tests (e.g., prostate-specific antigen (Brawer MK, Seminars in surgical oncology. 2000;18(1):3-9), carcinoembryonic antigen (Young GP et al., Cancer medicine. 2016;5(10):2763-72)), and liquid biopsy (detection of circulating tumor cells and tumor-derived DNA (Li J et al. Cancers (Basel). 2020;12(10);Liu MC et al., Ann Oncol. 2020;31(6):745-59)). However, many of these tests have limitations, with sensitivity and specificity being quite low, around 70%-80% and 60%-70%, respectively (Schiffman JD et al., American Society of Clinical Oncology Educational Book. 2015(35):57-65).Furthermore, sensitivity depends on tumor size, patient age, medical history, and histological composition (Humphrey et al., Annals of internal medicine. 2013;159(6):411-20; Ohuchi N et al., Lancet (London, England) 2016;387(10016):341-8; Sprague BL et al. Radiology. 2017;283(1):59-69).

[0005] Most cancer screening strategies currently available focus on detecting specific tumors, contradicting the “paradox” of cancer epidemiology: while the risk of any cancer is generally high throughout a person's life, the risk of a particular cancer at a particular time is rather low (Schiffman JD et al. American Society of Clinical Oncology Educational Book. 2015(35):57-65). A major drawback of current screening tests is that they can only detect specific cancers for which reliable markers are known and which are testable. Expanding the diagnosis to a wider range of cancers would require analyzing multiple biomarkers in parallel, resulting in increased laboratory workload, healthcare system costs, and a risk of false results.

[0006] Much research effort has been made toward the development of more universal cancer screening tests (so-called "multi-cancer" or "pan-cancer" screening tests) that are highly sensitive, highly specific, and highly accurate, enabling early diagnosis of the most common tumors, and possessing sufficient clinical applicability and usability (Shapley M et al. Br J Gen Pract. 2010;60(578):e366-77). For example, the development of approaches based on the detection of circulating cell-free DNA, including DNA methylation abnormalities, has made recent progress. However, the reported overall performance, particularly in terms of sensitivity, remains unsatisfactory, at least due to the inherently small amount of circulating tumor-derived DNA (Locke WJ et al. Front Genet. (2019) 14;10:1150;Srivastava S, Hanash S. Pan-Cancer Early Detection: Hype or Hope? Cancer Cell. 2020;38(1):23-24).

[0007] In this context, inflammatory diseases pose a significant burden on global health. Inflammation is the body's response to protect itself from invading pathogens and tissue damage. Acute inflammation is an immediate immune response that induces cytokines and chemokines, promoting the migration of immune cells to the site of infection or injury to eliminate pathogens and regenerate tissue. If the initial acute inflammatory response fails to eliminate the pathogens or the cause of tissue and cell damage, chronic inflammation can develop, potentially leading to further complications, including metabolic diseases such as coronary heart disease, type 2 diabetes, and rheumatoid arthritis. While inflammation's role in tissue regeneration and host defense has long been recognized, it has recently been implicated in the development and progression of cancer (Greten FR, Grivennikov SI. Inflammation and Cancer: Triggers, Mechanisms, and Consequences. Immunity. 2019;51(1):27-41). Early detection of inflammation, especially in the acute phase, is thought to be useful in assessing a person's predisposition to developing cancer in the future. Therefore, it would be ideal to have a laboratory screening test that can detect not only a wide range of cancers but also acute inflammatory diseases with high specificity, sensitivity, and reliability. [Overview of the project]

[0008] Therefore, there is an unmet need in the art for means and methods for diagnosing the presence of cancer and / or acute inflammatory disease in a subject, overcoming the limitations of currently available approaches. The present invention addresses this need and other needs and provides similarly related advantages.

[0009] Therefore, in a first embodiment, the present invention relates to a method for diagnosing cancer and / or acute inflammatory disease in a subject, the method comprising: (a) Subjecting the sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample in relation to the following: (bi) Levels of precipitated (poly)peptides in at least one correspondingly treated reference sample obtained from one or more reference subjects known to be positive for cancer and / or acute inflammatory disease; (b-ii) Levels of precipitated (poly)peptides in at least one corresponding processed reference sample obtained from one or more reference subjects known to be negative for cancer and acute inflammatory disease; and / or A predetermined standard determined based on (b-iii)(bi) and / or (b-ii); Here, the subjects are diagnosed with a positive result for cancer and / or acute inflammatory disease in the following cases: The level of precipitated (poly)peptide in the sample is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the level of precipitated (poly)peptide in the reference sample (bi), or compared to a predetermined standard; and / or The level of precipitated (poly)peptides in the sample is higher than the level of precipitated (poly)peptides in reference samples (b-ii), or is at least 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, and preferably at least 30% higher than the respective specified standards.

[0010] At least one corresponding processed reference sample is at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, and 100 corresponding processed reference samples, with larger numbers being preferable. Similarly, and independently of the number of reference samples, one or more reference subjects are obtained from at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, and 100 reference subjects, with larger numbers being preferable. In this regard, it should be understood that each reference sample may be obtained from one reference subject or from two or more reference subjects. In the latter case, the reference sample may be a “pooled” reference sample obtained from multiple reference subjects. It is also conceivable, and is particularly intended herein, that reference samples may be obtained from different body regions / tissues and / or different bodily fluids of one or two or more reference subjects.

[0011] The phrase "processed to correspond" means that the conditions for inducing the precipitation of one or more (poly)peptides are the same as those used in step (a).

[0012] The level of precipitated (poly)peptide is preferably defined as the weight of the precipitated (poly)peptide and / or the percentage of precipitated (poly)peptide compared to the total amount of (poly)peptides in the sample.

[0013] In this invention, the term "cancer" refers to any malignant abnormal proliferation of cells. Examples, but not limited to, include breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colorectal cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, stomach cancer, colorectal cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, and adrenocortical carcinoma. Examples include malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, pilocytic cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin lymphoma, soft tissue sarcoma, osteosarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of neoplastic carcinomas.

[0014] In a preferred embodiment, the cancer is selected from the group of solid cancers. In other embodiments, the cancer is selected from the group of liquid cancers. As used in the present invention, the term "solid cancer" refers to a cancer in which multiple malignant cells are related to each other, that is, contiguously localized within a limited site. As used in the present invention, the term "solid cancer" includes, but is not limited to, "carcinoma," "adenocarcinoma," and "sarcoma." "Sarcoma" is a cancer of connective tissue, cartilage, bone, and / or muscle. "Carcinoma" is a cancer of epithelial (endometrial) cells. "Adenocarcinoma" refers to a cancer originating from glandular cells. "Solid cancer" is in contrast to "liquid cancer" (also called "liquid carcinoma" or "hematogenous carcinoma"), in which malignant cells arise primarily as unbound cells or individual cells. Liquid cancer is a cancer that arises in the blood, bone marrow, or lymph nodes, and includes leukemia, lymphoma, myeloma, etc., and refers to the specific pathological conditions described above.

[0015] As is evident from the experimental results disclosed herein (see Examples 1 and 2, and in particular Figure 8), the assay of the present invention has been proven effective in distinguishing samples from patients suffering from a wide variety of cancers, including bile duct cancer, gallbladder cancer, bladder cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors (GIST), head and neck cancer, kidney cancer, laryngeal cancer, lung cancer, lymphoma, skin cancer, neuroendocrine tumors, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, rectal cancer, sarcoma, squamous cell carcinoma, testicular cancer, and uterine cancer. In light of this disclosed evidence, in a particularly preferred embodiment, cancer can be selected from any of these specific cancer species.

[0016] However, since there is no indication that certain types of cancer cannot be detected by the method of the present invention, it is possible to detect all types of cancer with this assay, and therefore, it is expected that this assay can be suitably adopted as a "pan-cancer" screening test (as mentioned at the beginning).

[0017] As used in this invention, the terms "inflammation" and "inflammatory" refer to a biological response involving the upregulation of the immune system, and include increased expression and / or activity of (poly)peptides related to inflammation or immune responses (e.g., inflammatory markers such as chemokines and cytokines, production of plasma haptoglobin), as well as symptoms of inflammation (e.g., pain, heat, redness, and / or edema). Generally, inflammation may be acute or chronic.

[0018] The term "inflammatory disease" as used in this invention has a general meaning in the art and refers to all diseases and conditions related to acute inflammation, chronic inflammation, or both. This term includes (1) inflammatory or allergic diseases such as systemic anaphylaxis or hypersensitivity reactions, drug allergies, and insect bite allergies; inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, and enteritis; vaginitis; inflammatory skin diseases such as psoriasis and dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, and urticaria; vasculitis; spondyloarthritis; scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, and hypersensitivity lung disease; (2) arthritis (rheumatic and psoriatic), osteoarthritis, multiple sclerosis, and systemic lupus erythematosus. (3) Graft rejection (including allograft rejection and graft-versus-host disease (GvHD)), and (4) other diseases in which undesirable inflammatory responses may occur (e.g., atherosclerosis, myositis, inflammatory CNS disorders, such as stroke and closed head injury, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis, and Bechet syndrome), but are not limited to these. In a particularly preferred embodiment, “inflammatory disease” is sepsis.

[0019] As used in this invention, the term "sepsis" has a general meaning in the art and refers to a serious medical condition characterized by a systemic inflammatory state. In addition to symptoms related to the induced infection, sepsis is characterized by the presence of acute inflammation throughout the body. In particular, sepsis is defined as an abnormal immune response to infection that leads to life-threatening organ dysfunction, and is therefore sometimes referred to as a fulminant systemic inflammatory response to infection. As used in this invention, the term "sepsis" also encompasses specific forms and complications such as "severe sepsis" and "septic shock."

[0020] An “inflammatory disease” that can be diagnosed according to the methods of the first and / or second aspects of the present invention is an “acute inflammatory disease,” characterized by an ongoing inflammatory process currently in progress (i.e., at the time the sample is obtained from the subject), particularly indicated by the presence of elevated levels of, for example, one or more inflammatory markers in the subject’s blood. Various inflammatory markers, such as C-reactive protein (CRP) and / or procalcitonin (PCT), are well known in the art and their measurement is now routinely performed. Particularly preferred inflammatory markers and their corresponding levels that are generally considered to indicate acute inflammation are described herein in the following preferred embodiments.

[0021] It will be understood that the methods and applications disclosed in this invention are useful in the fields of human medicine and veterinary medicine. Accordingly, the terms “subject” or “patient” used interchangeably in this invention refer to any vertebrate, but are not limited to humans and other primates (e.g., chimpanzees and other apes and monkey species), livestock (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, rabbits, guinea pigs and hamsters), laboratory animals (e.g., rodents such as mice, rats, rabbits, guinea pigs and hamsters), and birds (e.g., chickens, turkeys and other gosseous birds, domestic birds such as ducks and geese, wild birds and game birds). In a preferred embodiment, the subject is a mammal. In a more preferred embodiment, the subject is a human.

[0022] As used in the present invention, the terms "sample" or "body sample" generally refer to any biological sample that can be obtained from the body in question, such as body fluids (e.g., blood such as whole blood, plasma, or serum) or body tissues (e.g., tissue samples obtained by biopsy). The term "sample" may refer to a pure biological sample or a processed form thereof. For example, a "sample" may contain or consist of a fraction of body fluids (e.g., plasma or serum obtained from whole blood) or body tissue, and / or may contain further components such as anticoagulants and / or stabilizers, such as protease inhibitors. Particularly preferred samples, and further components that may preferably be contained therein or added thereto, are defined below in the present invention.

[0023] From the perspective that the method disclosed in the present invention is based on the evaluation of precipitated (poly)peptides resulting from the supply of precipitation-inducing conditions, it is understood that the sample used in the method disclosed in the present invention preferably contains substantially no precipitate, and in particular substantially no precipitated (poly)peptides. Therefore, the sample before use in the method disclosed in the present invention may preferably be subjected to treatment to remove any potentially present precipitate (e.g., centrifugation or filtration).

[0024] In this invention, the term "(poly)peptide" refers to a chain polymer of amino acid residues linked by peptide bonds in a specific sequence, and encompasses both the "polypeptide" group and the "peptide" group. In this invention, the "polypeptide" group, used interchangeably with the term "protein," consists of molecules with 30 or more amino acids and is distinguished from the "peptide" group, which consists of molecules with up to 30 amino acids. "Peptide" also refers to protein fragments with 30 or fewer amino acids. A (poly)peptide may further form dimers, trimers, and oligomers, i.e., structures consisting of two or more (poly)peptide molecules. The (poly)peptide molecules forming such dimers, trimers, etc., may be identical or non-identical. The corresponding higher-order structures are consequently expressed as homodimers or heterodimers, homotrimers or heterotrimers, etc. Homodimers or heterodimers, etc., also fall under the definition of "(poly)peptide." The term "(poly)peptide" also refers to chemically modified or post-translationally modified peptides and polypeptides.

[0025] The term "precipitation," as in its general sense, refers to a phase change from a colloidal dispersion to a solid mass when a colloidal dispersion is subjected to perturbation. More specifically, "precipitation" refers to the form of an insoluble solid mass resulting from a reaction occurring in a solution. For example, precipitation occurs when a suitable precipitating agent is added to a solution. When precipitation occurs, the resulting solid is called a "precipitate." The precipitate can be collected or separated from the remaining solution by various methods such as filtration, decantation, and centrifugation. As used in the present invention in relation to the disclosed methods, "precipitation" more specifically refers to the process by which one or more (poly)peptides initially present in a dissolved (i.e., soluble) form in a sample undergo a phase change and become insoluble as a result of being exposed to environmental stimuli (i.e., precipitation-inducing conditions described below).

[0026] When used in this invention, the terms “condition inducing precipitation” or “precipitation-inducing condition” are intended to refer to any physical and / or chemical conditions that can cause one or more (poly)peptides contained in a sample to precipitate. Typically, in the context of this invention, precipitation occurs as a result of supplying precipitation-inducing conditions to the sample, such as the addition of a precipitating agent (e.g., an acid), as described below.

[0027] The inventors have surprisingly discovered that (poly)peptides contained in body samples (e.g., plasma) of subjects suffering from cancer and / or acute inflammatory diseases exhibit different sensitivities to specific precipitation-inducing conditions compared to (poly)peptides contained in samples of healthy subjects (i.e., those not suffering from cancer and / or acute inflammatory diseases). In particular, when body samples of subjects with cancer and / or acute inflammatory diseases are exposed to such precipitation-inducing conditions, the (poly)peptides contained in these body samples are found to produce significantly more pronounced precipitation compared to (poly)peptides contained in correspondingly treated body samples from healthy subjects. Although the exact molecular details underlying this phenomenon remain unclear, further investigations conducted by the inventors, as disclosed herein, have revealed that this "differential precipitation behavior" is consistently and reproducibly observed in body samples from a wide variety of subjects suffering from cancer and / or acute inflammatory conditions. In this invention, it has been demonstrated that this mechanism can be effectively used as a diagnostic means to distinguish with high accuracy whether a body sample originates from a healthy subject or from a subject suffering from cancer and / or acute inflammatory disease.

[0028] Furthermore, further investigations conducted by the inventors revealed that this differential precipitation is most pronounced, and therefore, the distinction between samples derived from cancer / acute inflammation subjects and samples derived from healthy subjects is best achieved when the precipitation induction conditions employed are rather mild, i.e., conditions sufficient to initiate the precipitation process, rather than harsh precipitation induction conditions that result in the immediate precipitation and / or aggregation (or hydrolysis) of almost or substantially all (poly)peptides in both samples derived from cancer / acute inflammation subjects and samples derived from healthy subjects, thereby hindering the identification based on the effects disclosed herein.

[0029] Therefore, in a preferred embodiment, the precipitation induction conditions are such that at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% of the total amount or mass of (poly)peptides contained in the sample remain soluble, more preferably at least 95%, but preferably at least 1% of the total amount or mass of (poly)peptides contained in the sample precipitates.

[0030] As used in the present invention, the phrase "evaluate the level of precipitated (poly)peptides" (i.e., relative to a reference sample and / or a specified standard) is intended to refer to a direct or indirect evaluation technique that enables the detection of a quantitative signal or other measurement parameter correlated with the formation of precipitated (poly)peptides, and thus enables a comparison between the level of precipitated (poly)peptides in the sample of the subject under test and a corresponding processed reference sample derived from a known health condition (i.e., a subject known to have cancer and / or acute inflammatory disease, or a subject known not to have either of the reference conditions) and / or a specified standard subject.

[0031] Exemplary methods suitable for such evaluation include, in particular, separating the precipitated (poly)peptide from the sample (e.g., by centrifugation or filtration), determining the weight or volume of the precipitate, and comparing it to the weight or volume of the precipitated (poly)peptide obtained from a correspondingly processed reference sample. Alternatively, the separated precipitated (poly)peptide can be subjected to gel electrophoresis (e.g., polyacrylamide gel electrophoresis (PAGE)), e.g., SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and optionally Coomassie blue staining (or other staining approaches, e.g., silver staining) or Western blotting, and the resulting band intensity can be quantified (e.g., by densitometry) and compared to the band intensity from a correspondingly processed reference sample. The corresponding evaluation by SDS-PAGE (and staining such as Coomassie blue staining) and subsequent densitometry quantification of band intensity are carried out in Example 4 and corresponding Figure 13, and are particularly preferred. Other well-known methods for quantifying (poly)peptides include bicinchoninic acid (BCA) assays or other copper-based assays, Bradford assays, Lowry assays, and chromatographic approaches such as reverse-phase high-performance liquid chromatography (RP-HPLC). Further methods that can quantitatively assess (poly)peptide levels include mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), matrix-assisted laser desorption / ionization (MALDI; particularly MLDI-MS and MALDI-TOF-MS), light scattering (particularly dynamic light scattering (DLS)), and nuclear magnetic resonance (NMR) spectroscopy (e.g., solution-state NMR spectroscopy).

[0032] Those skilled in the art will understand that the level of precipitated (poly)peptides may alternatively be assessed indirectly or conversely from the remaining soluble fractions of the sample and reference sample, for example, by (i) first determining the total amount / mass of (poly)peptides contained in the sample or reference sample, and (ii) after providing precipitation-inducing conditions, preferably after removing the precipitate (e.g., by centrifugation and / or filtration), determining the amount / mass of soluble (poly)peptides contained in the remaining soluble fractions of the sample and reference sample. Any of the quantitative methods mentioned above can be employed by analogy for that purpose.

[0033] One further exemplary approach for that purpose is to measure the UV absorbance (A) at a wavelength of 280 nm of the starting sample (i.e., before providing precipitation induction conditions) and the remaining soluble (poly)peptide fraction (i.e., after providing precipitation induction conditions, preferably after removing the precipitated (poly)peptide). 280nm The method involves measuring the extinction coefficient of (poly)peptides and thereby determining the fraction of precipitated (poly)peptides from the total amount of (poly)peptides contained in the sample; and comparing the said fraction with the respective determined fractions of precipitated (poly)peptides in a correspondingly processed reference sample. Those skilled in the art will understand that, even without knowledge of the extinction coefficient of complex mixtures of (poly)peptides contained in the sample, each evaluation allows for a relative evaluation to compare the level of precipitated (poly)peptides in the sample with the level of precipitated (poly)peptides in the reference sample.

[0034] A more preferred approach for evaluating the level of one or more precipitated (poly)peptides in a sample, in particular a further absorbance-based approach, is defined in various embodiments of this specification below.

[0035] The phrase "diagnose a subject as positive for cancer and / or acute inflammatory disease" as used in the present invention does not mean to provide absolute (i.e., 100%) certainty of the presence of cancer and / or acute inflammatory disease in the subject, but rather to provide strong signs / suspicions that the subject has cancer and / or acute inflammatory disease, and if such a positive diagnosis is made by the method of the present invention, further evaluation (i.e., follow-up examination) may be performed for further verification, either to confirm or disprove the initial diagnosis; in the former case, this may also be to collect further information about the type of disease, for example, in the case of cancer, the type of cancer and / or its location in the subject's body. Such follow-up examinations may be performed, for example, by physical examination, blood and / or urine tests for evaluation of known biomarkers, ultrasound (sonography), mammography, and / or other imaging techniques such as X-ray, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Accordingly, the method objective "for diagnosing cancer and / or acute inflammatory disease" as referred to in the present invention in relation to the method of the first aspect of the present invention (and as referred to hereafter in relation to the method of the second aspect of the present invention) can be alternatively and interchangeably formulated as "for assessing whether a subject has an increased risk of having cancer and / or acute inflammatory disease," or "for screening a subject for the presence of cancer and / or acute inflammatory disease," or "for identifying a subject who has or is suspected of having cancer and / or acute inflammatory disease."

[0036] Furthermore, due to the observed differences in precipitation sensitivity, the inventors have found it advantageous that the distinction between whether a sample originates from a subject suffering from cancer and / or acute inflammatory disease, or from a healthy subject (a subject not suffering from either reference condition), can be reliably established alternatively, solely from the evaluation of the sample being tested (i.e., without the need for comparison with any reference sample and / or a given standard), that is, by determining the fraction of precipitated (poly)peptides from the total amount of (poly)peptides contained in the sample. As described in the invention (see, for example, Figures 1A-E and 10-13), when exposed to precipitation-inducing conditions, the proportion of (poly)peptides in the sample that precipitates relative to the total amount of (poly)peptides in the sample is characteristic of whether the sample originates from a pathological or non-pathological subject; however, the degree of the observable difference depends on the individual precipitation-inducing conditions applied. For example, as shown in Figure 1A, when precipitation was induced by adding 0.4% (v / v) acetic acid to a final concentration of approximately 0.15% (v / v) in the sample, the proportion of precipitated (poly)peptides in the pathological sample was approximately 2%, while the proportion of precipitated (poly)peptides in the non-pathological sample was only 1.5% of the total amount of (poly)peptides in the sample. Similarly, as shown in Figure 1B, when exposed to heat (70°C / 1 min), 7% of the (poly)peptides precipitated in the pathological sample, compared to 4% in the non-pathological sample. Therefore, this difference in precipitation, when evaluated based on the determination of the proportion of precipitated (poly)peptides relative to the total amount / mass of protein in the sample, becomes a highly discriminative determinant for intended diagnostic purposes. It is well known that IR induces protein precipitation.

[0037] Accordingly, in a second related embodiment, the present invention provides a method for diagnosing cancer and / or acute inflammatory disease in a subject, the method comprising: (a) Subjecting a sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample, wherein the conditions that induce precipitation are provided as follows: (i) Adjust the pH of the sample to a value in the range of pH 4.2 ± 0.05, which is preferable to a higher value, and most preferably to a lower value, between pH 2 and pH 6, pH 3 and pH 5.5, pH 3.4 and pH 5, pH 3.6 and pH 4.8, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, and pH 4.1 and pH 4.3; Preferably, this is done by adding an acid or an aqueous solution containing an acid, where the acid has a pKa in the range of preferably 3.0 and 7.0, preferably 3.5 and 6.5, preferably 3.8 and 5.8, preferably 4.1 and 5.5, and most preferably 4.75 ± 0.05; where preferably, the acid is an organic acid, where the organic acid is preferably a carboxylic acid, more preferably a monocarboxylic acid, and most preferably acetic acid; (ii) Increase in sample temperature; (iii) Addition of at least one salting agent; (iv) Addition of one or more aliphatic alcohols, preferably alkanols, most preferably ethanol; and / or (vi) Addition of a ketone, preferably acetone; and (b) Determine the fraction of precipitated (poly)peptides from the total amount of (poly)peptides in the sample by the following: - If precipitation is induced by a decrease in the pH of the sample, the determined fraction of the precipitated (poly)peptide is preferably greater than 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the total amount of (poly)peptides in the sample, and more preferably greater than 2.1%; - If precipitation is induced by an increase in sample temperature, the determined fraction of precipitated (poly)peptides is more preferably 5%, 5.5%, 6%, or 6.5% of the total amount of (poly)peptides in the sample, and more preferably more than 7%; - If precipitation is induced by the addition of at least one salting-out agent, the determined fraction of the precipitated (poly)peptide is preferably greater than 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the total amount of (poly)peptides in the sample, and more preferably greater than 2.1%; - If precipitation is induced by the addition of ethanol and / or other aliphatic alcohols, the determined fraction of the precipitated (poly)peptide is more preferably 4%, 6%, 8%, 10%, 11%, and more preferably 12% of the total amount of (poly)peptides in the sample; and / or - If precipitation is induced by the addition of ketones, the determined fraction of the precipitated (poly)peptide is more preferably 1%, 2%, 3%, or 4%, and more preferably more than 5%, of the total amount of (poly)peptides in the sample; The subjects are those diagnosed with cancer and / or acute inflammatory disease.

[0038] In relation to the first aspect of the present invention, the definitions and preferred embodiments described herein apply to the second aspect of the present invention. For example, further details and preferred embodiments of increasing the sample temperature (e.g., temperature and / or time) and adding at least one salting-out agent (e.g., examples and amounts of salting-out agents) are described below, meaning that these also apply to the second aspect of the present invention.

[0039] In this invention, the term "precipitated (poly)peptide fraction" in a sample refers to the proportion of precipitated (poly)peptides to the total amount of (poly)peptides contained in the sample, and is expressed as a percentage or decimal.

[0040] As demonstrated by the experimental evidence disclosed in this invention (see Examples 1 and 2, and Figures 1A-E), the precipitated (poly)peptide fraction was consistently higher in samples derived from pathological subjects compared to the precipitated (poly)peptide fraction measured for correspondingly treated non-pathological reference samples, regardless of the specific conditions employed to induce precipitation. However, the individually determined precipitated (poly)peptide percentage was found to depend on the specific precipitation induction conditions applied.

[0041] For example, when precipitation was induced by adding acid (particularly 0.4% (v / v) acetic acid to a final concentration of approximately 0.15% (v / v)), the proportion of precipitated (poly)peptides in the pathological sample was approximately 2% of the total amount of (poly)peptides present, compared to only about 1.5% in the non-pathological sample; see Figure 1A.

[0042] When the sample temperature was raised to 70°C and allowed to precipitate for 1 minute, the proportion of precipitated (poly)peptides in the pathological sample was approximately 7% of the total amount of (poly)peptides, compared to only about 5% in the non-pathological sample; see Figure 1B.

[0043] When a salting-out agent was added to precipitate instead, the proportion of precipitated (poly)peptides (in particular, 35% (NH4)2SO4 to a final concentration of 20.6%) was approximately 13.5% of the total amount of (poly)peptides in the pathological sample, compared to only about 10% in the non-pathological sample; see Figure 1C.

[0044] When precipitation was instead carried out by the addition of ketones or alcohols (in particular, 50% ice-cold acetone or 50% EtOH to a final concentration of 18.5% (v / v)), the proportion of precipitated (poly)peptides was approximately 6.2% and 28% of the total amount of (poly)peptides in the pathological samples. In contrast, the fraction of precipitated (poly)peptides in the non-pathological samples was only about 5% and 23%, respectively; see Figures 1C and D, respectively.

[0045] In the present invention, the term "ketone" refers to a ketone having 3 to 6 carbon atoms, preferably including but not limited to acetone, butanone, methyl ethyl ketone, methyl isobutyl ketone, and diethyl ketone. More preferably, the ketone is acetone, and most preferably ice-cold acetone. The final ketone (preferably acetone) concentration (v / v) used for precipitation is preferably 8.5% to 28.5%, 13.5% to 23.5%, 16% to 21%, and 18.5%, with increasing preference towards the later concentrations.

[0046] Means and methods for determining the fraction of precipitated (poly)peptides in a sample are well known and routinely employed in the art, and corresponding methods are also referred to herein above. In an exemplary setting, the precipitated (poly)peptides are separated from the remaining soluble fraction (e.g., by centrifugation, e.g., 21.000xg, 4°C for 1 minute) and then quantified, e.g., by a BCA assay (e.g., by determining their total amount / mass). The remaining soluble fraction is also quantified with respect to its (poly)peptide content, e.g., by a BCA assay. Next, the proportion of precipitated (poly)peptides from the total amount of (poly)peptides in the sample is calculated by dividing the determined amount of precipitated (poly)peptides by the sum of the determined amounts of precipitated (poly)peptides and soluble fractions, and multiplying the result by 100%. In another exemplary setting, the precipitated (poly)peptides are separated from the remaining soluble fraction (e.g., by centrifugation). SDS-PAGE is performed on (i) a specified amount of precipitated (poly)peptide (degraded in SDS sample buffer) and (ii) a specified amount of the remaining soluble fraction. The resulting protein bands can then be quantified by densitometry. Next, the percentage (i.e., ratio) of precipitated (poly)peptide can be calculated from the measured band intensity, the original sample volume, and the volume of the remaining soluble fraction, relative to the total amount of protein originally present in the sample.

[0047] In a third embodiment, the present invention relates to a method for evaluating the response of cancer and / or acute inflammatory diseases to a candidate treatment in a subject, the method comprising: (a) Subject a sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample, wherein the sample is obtained after the candidate treatment has been initiated; (b) Evaluate the level of precipitated (poly)peptides in the sample relative to the level of precipitated (poly)peptides in a reference sample obtained from the subject before the sample in step (a) was obtained from the subject, wherein the reference sample is subjected to the corresponding precipitation-inducing conditions as in step (a); - If the level of precipitated (poly)peptides in the sample is equal to or lower than the level of precipitated (poly)peptides in the reference sample, cancer and / or acute inflammatory diseases are classified as responsive to candidate treatment; and / or - If the level of precipitated (poly)peptides in the sample exceeds the level of precipitated (poly)peptides in the reference sample, cancer and / or acute inflammatory diseases are classified as unresponsive to candidate treatment.

[0048] As used in the present invention, the term "candidate treatment" refers to a treatment being tested for effectiveness against a disease or disorder, namely, in this case, a specific type of cancer and / or acute inflammatory disease to which the subject is suffering.

[0049] As used in the present invention, the term "responsiveness" refers to the occurrence of a favorable response when cells, tissues, organs, or subjects are brought into contact with a drug (e.g., a candidate therapeutic agent). A non-limiting example would be the inhibition or cessation of abnormal cell proliferation when cancer cells are brought into contact with a particular drug, while an unfavorable response would be the promotion of tumor growth when a patient with a tumor is brought into contact with a particular drug.

[0050] In this invention, the term "candidate agent" refers to an agent that is tentatively expected to be suitable for the treatment of cancer and / or inflammatory diseases (preferably acute inflammatory diseases). More specifically, in this invention, the corresponding agents are referred to as "anticancer agents" and "anti-inflammatory agents," respectively.

[0051] Candidate drugs can generally be selected from known drugs, such as those already used in (pre)clinical trials for the treatment and / or prevention of specific pathological conditions (e.g., cancer and / or inflammatory diseases), or drugs approved by one or more regulatory bodies / health authorities (e.g., the European Medicines Agency (EMA), the Food and Drug Administration (FDA), etc.), as well as drugs not yet used as pharmaceuticals, or drugs used as pharmaceuticals but only for the treatment and / or prevention of conditions different from the condition the subject is suffering from (i.e., cancer and / or inflammatory diseases). Exemplary candidate drugs include naturally occurring compounds, such as secondary metabolites or agents produced and isolated by bacteria, fungi, animals or plants, as well as synthetic agents, such as agents from combinatorial libraries or combinatorial display libraries of chemical compounds (e.g., ribosome or phage display antibody / scFv libraries). The term "candidate drug" also refers to derivatives of the above term.

[0052] The term "treatment" specifically refers to the reduction or alleviation of one or more symptoms in a subject, the prevention of the worsening or progression of one or more symptoms, the promotion of recovery or improvement of prognosis, and / or the prevention of disease in subjects without such symptoms, as well as the slowing or mitigation of the progression of a pre-existing disease. For a given subject, improvement, worsening, regression, or progression of symptoms can be determined by objective or subjective measures. The effectiveness of treatment can be measured as an improvement in morbidity or mortality. Palliative (e.g., improvement of quality of life) or preventive (e.g., prevention of disease onset or recurrence) methods are also considered treatments. For example, in the context of cancer treatment, neoadjuvant therapy to shrink the primary tumor and make local therapy (e.g., surgery or radiation therapy) more effective, and adjuvant therapy to reduce the likelihood of recurrence and / or the development of resistance are also considered treatments.

[0053] The term "anti-cancer agent" as used in this invention includes all agents suitable for the treatment of cancer.

[0054] Current strategies in cancer treatment include chemotherapy, radiotherapy, immunotherapy (including adoptive cell therapy (CAR-T cell therapy or TCR-modified T cell therapy, etc.) and immune checkpoint inhibitors), hormone therapy, monoclonal antibodies and antibody-drug conjugates, surgery, and combinations thereof. Furthermore, known anticancer agents include estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyltransferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, and angiogenesis inhibitors.

[0055] An "estrogen receptor modulator" refers to a compound that interferes with or inhibits the binding of estrogen to its receptor, regardless of the mechanism. Examples of estrogen receptor modulators, but not limited to, include tamoxifen, raloxifen, idoxifen, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]phenyl 2,2-dimethylpropanoate, and 4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone.

[0056] An "androgen receptor modulator" refers to a compound that inhibits the binding of androgens to their receptors, regardless of the mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, rialozole, and abiraterone acetate.

[0057] A "retinoid receptor modulator" refers to a compound that interferes with or inhibits the binding of retinoids to their receptors, regardless of the mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cisretinoic acid, 9-cisretinoic acid, α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenylretinamide.

[0058] "Cytotoxic agents" refer to compounds that primarily induce cell death or inhibit or interfere with cellular myosis through direct action on cellular function, such as alkylating agents, tumor necrosis factors, intercalators, microtubule inhibitors, and topoisomerase inhibitors. Examples of cytotoxic drugs, though not limited to them, include tirapazimine, certenef, cachectin, ifosfamide, tasonelmin, ronidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfant sylate, trophosphamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satoraplatin, prophylromycin, cisplatin, ilofluben, dexphosphamide, cis-aminedichloro(2-methylpyridine)platinum, benzylguanine, gluphosphamide, GPX100, and (trans,trans,trans)bi Examples include s-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, dialicydinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, solubicin, idarubicin, daunorubicin, bisanthren, mitoxantrone, pirarubicin, pinafide, barbicin, amrubicin, antineoplaston, 3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, anamycin, galarubicin, erinafide, MEN10755, and 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulfonyldaunorubicin.

[0059] Examples of microtubule inhibitors include paclitaxel, vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaloicoblastine, docetaxol, lyzoxin, dorastatin, mybobrin isethionate, auristatin, semadin, RPR109881, BMS184476, vinflunin, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-N-methyl-L-valyl-L-proline-t-butylamide, TDX258, and BMS188797.

[0060] "Topoisomerase inhibitors" include, for example, topotecan, hicaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3′,4′-O-exobenzylidenchaltreusine, 9-methoxy-NN-dimethyl-5-nitropyrazolo[3,4,5-kl]acridin-2-(6H)propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]indolidino[1,2b]quinoline-10,13(9 H,15H)-Zione, Lulutotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)Camptothecin, BNP1350, BNPI1100, BN80915, BN80942, Etoposide Phosphate, Teniposide, Sobuzoxane, 2′-Dimethylamino-2′-Deoxyetoposide, GL331, N-[2-(dimethylamino)ethyl]-9-Hydroxy-5,6-Dimethyl-6H-Pyrido[4,3-b]Carbazole-1-Carboxamide, Aslacrin, (5a,5aB,8aa,9b)-9 -[2-[N-[2-(di-methylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofloxacin(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylene-dioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]phenantridinium, 6,9-bis[(2-amino-ethyl)amino]benzo[g]isoquinoline-5,10-dione, 5-(3-aminopropyl These are ropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridine-6-one, N-[1-[2-(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthene-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinoline-7-one and dimesna.

[0061] An "angiogenesis inhibitor" refers to a compound that inhibits the formation of new blood vessels, regardless of the mechanism.Examples of angiogenesis inhibitors, though not limited to them, include tyrosine kinase inhibitors, such as inhibitors of tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epithelial, fibroblast, or platelet-derived growth factors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, interferon-α, interleukin-12, pentosan polysulfate, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, and cyclooxygenase inhibitors including selective cyclooxygenase-2 inhibitors such as celecoxib and rofecoxib (PNAS (1992) 89:7384; JNCI (1982) 69:475; Arch. Opthalmol. (1990) 108:573; Anat. Rec. (1994) 238:68; FEBS Letters (1995)). 372:83;Clin, Orthop. (1995) 313:76;J. Mol. Endocrinol. (1996) 16:107;Jpn. J. Pharmacol. (1997) 75:105;Cancer Res. (1997) 57:1625 (1997);Cell (1998) 93:705;Intl. J. Mol. Med. (1998) 2:715;J. Biol. Chem. (1999) 274:9116)), steroidal anti-inflammatory drugs (corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone, etc.), carboxamide triazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagirol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. (1985) 105:141-145), and antibodies against VEGF (e.g., Brower, V. (1999) Nature Biotechnology, 17:963-968; Kim et al. (1993) Nature 362:841-844; WO 00 / 44777; and WO (See 00 / 61186)

[0062] "Antiproliferative agents" include antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231 and INX3001, as well as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimethrexate, fludarabine, capecitabine, gallocitabine, cytarabine ocphosphate, fostiabine sodium hydrate, larcitrexed, and parcitrexed. Emitefur, Thiazofulin, Decitabine, Nolatrexed, Pemetrexed, Nerzarabine, 2′-Deoxy-2′-Methylidencytidine, 2′-Fluoromethylene-2′-Deoxycytidine, N-[5-(2,3-Dihydrobenzofuryl)sulfonyl]-N′-(3,4-Dichlorophenyl)urea, N6-[4-Deoxy-4-[N2-[2(E),4(E)-Tetradecadienoyl]glycylamino]-L-Glycerol-B L-mannoheptopyranosyl]adenine, apridin, ectinacidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b]-1,4-thiadin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil (5-FU), alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formi Examples of antimetabolites include ru-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)tetradeca-2,4,6-triene-9-yl acetate, swinesonin, lometrexol, dexrazoxane, methioninase, 2′-cyano-2′-deoxy-N4-palmitoyl-1-BD-arabinofuranosilcytosine, and 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone. "Antivogenic agents" also include monoclonal antibodies against growth factors other than those listed under "angiogenic inhibitors," such as trastuzumab, and tumor suppressor genes such as p53 that can be delivered by recombinant virus-mediated gene transfer (see, for example, U.S. Patent No. 6,069,134).

[0063] "HMG-CoA reductase inhibitors" refer to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that can be used include, but are not limited to, lovastatin (MEVACOR®; see U.S. Patents 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patents 4,444,784, 4,820,850 and 4,916,239), and pravastatin (PRAVACHO®; see U.S. Patents 4,346,227, 4,537,859 and 4,410,6) Examples include patents 29, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. patents 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), and atorvastatin (LIPITOR®; see U.S. patents 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structural formulas of these and further HMG-CoA reductase inhibitors that can be used in the methods of the present invention are described in M. Yalpani, "Cholesterol Lowering Drugs," p. 87, Chemistry & Industry, pp. 85-89 (1996), and U.S. Patents 4,782,084 and 4,885,314. As used in the present invention, the term HMG-CoA reductase inhibitor includes all pharmaceutically acceptable lactones and open acid forms (i.e., those in which the lactone ring is opened to form a free acid), as well as salts and esters of compounds having HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open acid forms and lactones is within the scope of the present invention.

[0064] A "prenyltransferase inhibitor" refers to a compound that inhibits one or any combination of prenyltransferases, including farnesyltransferase (FPTase), geranylgeranyltransferase type I (GGPTase-I), and geranylgeranyltransferase type II (GGPTase-II, also known as Rab GGPTase).Examples of prenyltransferase inhibitors can be found in the following publications and patents: WO 96 / 30343, WO 97 / 18813, WO 97 / 21701, WO 97 / 23478, WO 97 / 38665, WO 98 / 28980, WO 98 / 29119, WO 95 / 32987, U.S. Patent Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, 5,602,098, European Patent Publication 0618221, European Patent Publication 0675112, European Patent Publication 0604181, European Patent Publication 0696593, WO 94 / 19357, WO 95 / 08542, WO 95 / 11917, WO 95 / 12612, WO 95 / 12572, WO 95 / 10514, U.S. Patent No. 5,661,152, WO 95 / 10515, WO 95 / 10516, WO 95 / 24612, WO 95 / 34535, WO 95 / 25086, WO 96 / 05529, WO 96 / 06138, WO 96 / 06193, WO 96 / 16443, WO 96 / 21701, WO 96 / 21456, WO 96 / 22278, WO 96 / 24611, WO 96 / 24612, WO 96 / 05168, WO 96 / 05169, WO 96 / 00736, US Pat. 96 / 30018, WO 96 / 30362, WO 96 / 30363, WO 96 / 31111, WO 96 / 31477, WO 96 / 31478, WO 96 / 31501, WO 97 / 00252, WO 97 / 03047, WO 97 / 03050, WO Patent Nos. 97 / 04785, WO 97 / 02920, WO 97 / 17070, WO 97 / 23478, WO 97 / 26246, WO 97 / 30053, WO 97 / 44350, WO 98 / 02436, and U.S. Patent No. 5,532,359.

[0065] When used in the present invention, the term "anti-inflammatory agent" includes agents that induce a reaction in a subject that reduces inflammation (either acute or chronic, preferably acute) or downregulates an immune response, for example, by inhibiting enzyme or protein / peptide activity related to inflammation or an immune response (e.g., inhibition of inflammatory markers or reduction of plasma haptoglobin production); improving one or more symptoms of inflammation or an immune response (e.g., pain, redness, heat, or edema); or suppressing an immune response by delaying or stabilizing an inflammatory process or immune response. In some embodiments, "anti-inflammatory agent" is an anti-inflammatory agent known in the art. Examples of anti-inflammatory agents include, but are not limited to, steroid compounds including hydrocortisone; or nonsteroidal anti-inflammatory agents including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin, etc.

[0066] In a fourth embodiment, the present invention relates to a method for evaluating the malignancy of cancer in a subject, the method comprising: (a) Subjecting the sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample in relation to the following: (i) at least one corresponding processed reference sample obtained from one or more reference subjects having cancer of known grade; and / or (ii)(b)(i) A predetermined standard arbitrarily obtained based on one or more reference samples; Here, a level of precipitate in a sample that is high, low, or substantially the same as the level of precipitate in a reference sample indicates that the cancer in the subject has a malignancy that is high, low, or substantially the same as, a reference cancer or a given standard, respectively.

[0067] In the present invention, the term "aggressiveness" refers, in accordance with its general meaning in the art, to the level of aggressiveness of cancer in terms of its ability to progress and / or metastasize, and its likelihood of ultimately causing death. While it is understood that there is no absolute quantitative measure of aggressiveness, it can be determined relative to the aggressiveness of cancer from a reference subject with known medical outcomes or from a given standard, and thus can provide an appropriate qualitative comparative factor for assessing the severity of cancer and establishing prognosis. For example, the level of precipitated (poly)peptides in a sample can be assessed by measuring the change in absorbance caused by the induced precipitation using a spectrophotometer and comparing the change in absorbance to that recorded for cancer patients and / or correspondingly processed samples from cancer patients with certain types of cancer (e.g., brain tumors or pancreatic cancer) that are generally known to have a poor prognosis and high mortality rate.

[0068] The data in Salat et al. (2020), Precision Cancer Medicine, Vol 5, doi: 10.21037 / pcm-21-35 demonstrate that higher, lower, or substantially the same level of precipitate in a sample compared to a reference sample certainly provides an indication that the cancer in question has a higher, lower, or substantially the same degree of malignancy, respectively, compared to a reference cancer or a given standard. A 5-year follow-up study on all-cause mortality data in patients with histologically proven malignancy and healthy volunteers can be referenced, as described in Salat et al. (2020), Precision Cancer Medicine, Vol 5, doi: 10.21037 / pcm-21-35.

[0069] In a preferred embodiment of the method according to the first aspect of the present invention, in step (b), the level of precipitated (poly)peptide in the sample is evaluated based on a relative statistical comparison with the level of precipitated (poly)peptide in a group of correspondingly processed reference samples, where each reference sample is obtained from a reference subject known to be positive for cancer and / or acute inflammatory disease (pathological reference sample) and / or relative to the level of precipitated (poly)peptide in a group of correspondingly processed reference samples, where each reference sample is obtained from a reference subject known to be negative for cancer and acute inflammatory disease (non-pathological reference sample), where the statistical comparison is performed as follows: (a) Calculation of the area under the curve (AUC); where preferably: (a-1) The level of precipitated (poly)peptide in sample (S) is given by the AUC defined by formula (I). P By calculating the value, it is compared to the level of precipitated (poly)peptides in the group of pathological reference samples (P):

number

number

Number

Number

[0070] A preferred embodiment of the method according to the first aspect of the present invention has a unique readout (based on the level of the precipitated (poly)peptide) for diagnosing that the subject is positive for cancer and / or acute inflammatory disease, so it can also be set up as an independent embodiment. Therefore, the present invention also relates to a method for diagnosing cancer and / or acute inflammatory disease in a subject, the method comprising: (a) Subjecting a sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample based on a relative statistical comparison with the level of precipitated (poly)peptides in a group of correspondingly processed reference samples, where each reference sample is obtained from a reference subject known to be positive for cancer and / or acute inflammatory disease (pathological reference sample) and / or relative to the level of precipitated (poly)peptides in a group of correspondingly processed reference samples, where each reference sample is obtained from a reference subject known to be negative for cancer and acute inflammatory disease (non-pathological reference sample), where the statistical comparison is performed as follows: (aa) Calculation of area under the curve (AUC); where preferably: (aa-1) The level of precipitated (poly)peptide in sample (S) is given by the AUC defined by formula (I). P By calculating the value, it is compared to the level of precipitated (poly)peptides in the group of pathological reference samples (P):

number

number

number

number

[0071] In the present invention, the term "pathological reference sample" refers to a correspondingly processed sample obtained from a reference subject known to be positive for cancer and / or acute inflammatory disease, and the term "non-pathological reference sample" refers to a correspondingly processed sample obtained from a reference subject known to be negative for cancer and acute inflammatory disease.

[0072] AUC P Regarding this, it is understood that the specified value preferably eliminates the possibility of detecting a false negative, i.e., the possibility of diagnosing a pathological subject as a non-pathological subject. AUC NIn this regard, it is understood that the specified value more preferably reduces the possibility of detecting false positive results, i.e., the possibility of diagnosing a non-pathological subject as a pathological subject.

[0073] Therefore, in a preferred embodiment, the group of pathological reference samples comprises or consists of at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, and 100 pathological reference samples, the larger the number, the better. Similarly, in a preferred embodiment, the group of non-pathological reference samples includes or consists of at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, and 100 pathological reference samples, with the number increasing more preferably.

[0074] As shown in the experimental results disclosed in this invention (see Examples 1 and 2), applying an acid, particularly 0.4% (v / v) acetic acid, to a sample to bring the sample pH to approximately 4.2 was particularly suitable for precipitation of one or more (poly)peptides and, importantly, at a level that allowed for reliable differentiation between pathological and non-pathological samples.

[0075] Therefore, in a preferred (or even more preferred) embodiment, the precipitation induction conditions are provided by a change in sample pH, preferably by adjusting the sample pH to a value in the range between pH 2 and pH 6, pH 3 and pH 5.5, pH 3.4 and pH 5, pH 3.6 and pH 4.8, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, and pH 4.1 and 4.3, with a greater preference being, and most preferably, a value in the range of pH 4.2 ± 0.05.

[0076] Given the suitability of acetic acid having a pKa of approximately 4.76 as shown in the present invention, other acids having a similar pKa range to acetic acid, preferably other weak acids, and especially other carboxylic acids, are also suitable for use in the methods disclosed in the present invention. This is shown in Example 3 and the corresponding Figures 10.2A-C, where formic acid (a (mono)carboxylic acid, similar to acetic acid, and having a similar pKa of approximately 3.75) is suitably used as a precipitation inducer. The fact that citric acid (pKa 3.13, 4.76, 6.4; Figure 10.1) and perchloric acid (pKa = -10; Figure 10.3) also function in principle is also shown in Example 3.

[0077] In a preferred embodiment of the latter configuration, the pH of the sample is adjusted by adding the following to the sample: (a) At least one acid having a pKa in the range of 2.0 to -10.0, 3.0 to 7.0, 3.5 to 6.5, 3.8 to 5.8, and 4.1 to 5.5, with higher values ​​being preferable, and most preferably in the range of 4.76 ± 0.05; Here, preferably, at least one acid is an organic acid, and here preferably, the organic acid is: (i) a carboxylic acid, more preferably (i-1) a monocarboxylic acid, preferably selected from the group consisting of formic acid, acetic acid, benzoic acid, propionic acid and butyric acid, or (i-2) a dicarboxylic acid or tricarboxylic acid, preferably citric acid; (ii) Barbiturates; and / or (iii) perchloric acid; and / or (b) An aqueous solution containing at least one acid as defined in (a); Here, preferably: - The aqueous solution contains at least one acid in a total concentration within the range of 0.3 vol% and 1.0 vol%, 0.3 vol% and 0.9 vol%, 0.3 vol% and 0.8 vol%, 0.3 vol% and 0.7 vol%, 0.3 vol% and 0.6 vol%, 0.3 vol% and 0.5 vol%, and 0.35 vol% and 0.45 vol%, with higher concentrations being preferable and most preferably within the range of 0.4 vol%; - The aqueous solution has a pH in the range between pH 2 and pH 5, pH 2 and pH 4, pH 2.5 and pH 3.5, pH 2.8 and pH 3.2, and pH 2.9 and pH 3.1, with higher values ​​being preferable, and most preferably having a pH of 2.97 ± 0.05; - The aqueous solution is added to the sample at a concentration of 0.59:1, with the volume-to-volume ratio of the aqueous solution to the sample being between 0.3:1 and 0.9:1, 0.4:1 and 0.8:1, and 0.5:1 and 0.7:1; and / or - The aqueous solution further contains sodium chloride at concentrations in the range of 0.01%(w / v) and 7%(w / v), 0.1%(w / v) and 6%(w / v), 0.2%(w / v) and 5%(w / v), 0.3%(w / v) and 4%(w / v), 0.4%(w / v) and 3%(w / v), 0.5%(w / v) and 2%(w / v), 0.6%(w / v) and 1%(w / v), and 0.7%(w / v) and 0.9%(w / v), most preferably at a concentration of 0.81%(w / v).

[0078] In other or even more preferred embodiments, the pH of the sample is adjusted by adding an acid to the sample, where the acid is selected from the following: - Acetic acid; here, preferably, acetic acid is added to the sample to a final concentration between 0.02 vol% and 0.28%, 0.05 vol% and 0.25 vol%, and between 0.1 vol% and 0.2 vol%, with the greater the better, and most preferably 0.15 vol%; - Citric acid; here, preferably, citric acid is added to the sample to a final concentration of 3.7 vol%, which is more preferable, between 0.1 vol% and 10%, 0.5 vol% and 8 vol%, 1 vol% and 7 vol%, 2 vol% and 6 vol%, and 3 vol% and 5 vol%; - Formic acid, where preferably formic acid is added to the sample to a final concentration between 0.04 vol% and 0.40 vol%, 0.06 vol% and 0.35 vol%, 0.07 vol% and 0.30 vol%, with the greater the better, and most preferably about 0.074 vol%; and / or - Perchloric acid, where preferably, perchloric acid is added to the sample to a final concentration between 0.20 vol% and 0.50 vol%, 0.25 vol% and 0.40 vol%, and 0.30 vol% and 0.35 vol%, with the higher the better, and most preferably about 0.324 vol%.

[0079] In a preferred embodiment, the sample includes or consists of: (a) Body fluids, preferably selected from blood, saliva, mucus, sputum, vomit, sweat, tears, urine, semen, vaginal fluid, feces, and exudates, or any mixture thereof; and / or (b) Body tissue, preferably homogenized body tissue, more preferably a cell-free suspension of homogenized body tissue.

[0080] In a particularly preferred embodiment, the sample contains or consists of blood. As used in the present invention, the term "blood" includes whole blood or any fraction of blood, such as serum and plasma as conventionally defined. The blood (sample) is preferably plasma.

[0081] Therefore, in a more preferred embodiment, the sample contains plasma (plasma), consists essentially of plasma (plasma), or consists of plasma (plasma). As used in the present invention, the term “consistently of” means that the referenced content constitutes, more preferably, at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the total volume or mass.

[0082] Means and methods for separating plasma from whole blood are well known and routinely employed in the art (see, for example, Basu D, Kulkarni R. Overview of blood components and their preparation. Indian J Anaesth. 2014 Sep;58(5):529-37). For example, plasma can be obtained by centrifugation of whole blood to which an anticoagulant has been added, or it can be collected directly by a process called plasmapheresis (see, for example, Madore F. Crit Care Clin. 2002 Apr;18(2):375-92). For example, for the purposes of the present invention, plasma can preferably be obtained by directly collecting whole blood into a commercially available blood collection tube (e.g., S-Monovette® K2-EDTA, 9 ml, Sarstedt; https: / / www.sarstedt.com / ) that has already been manufactured to contain an anticoagulant (e.g., K3-EDTA or K2-EDTA or another anticoagulant, preferably K2-EDTA), and then immediately (i.e., within 20 minutes after blood collection) being subjected to centrifugation (e.g., 3000 × g / 10 minutes) to separate the plasma from the remaining blood components.

[0083] The term "blood plasma," also more simply referred to as "plasma" in this invention, is the fraction of whole blood obtained by centrifugation of blood treated with an anticoagulant as the upper layer. Plasma is the cell-free liquid fraction of blood and contains serum proteins and coagulation factors (including fibrinogen). The term "blood serum," also more simply referred to as "serum" in this invention, refers to the watery portion of the liquid obtained by centrifugation after blood has been treated to coagulate (without the addition of an anticoagulant, i.e., without the addition of an anticoagulant), in which coagulation factors and fibrinolytic factors are absent. In other words, serum is the liquid that remains after blood has coagulated; plasma is the liquid that remains when coagulation is inhibited by the addition of an anticoagulant. Plasma can also be converted to serum by a method called defibrillation (see, for example, Castro AR et al., (2002) Clin Diagn Lab Immunol.;9(6):1376-8).

[0084] When collecting a whole blood sample from a subject, the collected blood is preferably immediately exposed to an anticoagulant to prevent coagulation. In this invention, the term "anticoagulant" refers to a chemical part that prevents blood coagulation. Known anticoagulants include heparin, ethylenediaminetetraacetic acid (EDTA), D-Phe-Pro-Arg chloromethyl ketone dihydrochloride ("PPACK"), and citrate.

[0085] Therefore, in one preferred embodiment, the sample further includes: (i) Anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin; and / or (ii) One or more proteinase inhibitors.

[0086] In a particularly preferred embodiment, the anticoagulant is EDTA or a salt thereof (preferably a sodium salt or a potassium salt), and more preferably tripotassium EDTA (K2-EDTA). In another preferred embodiment, the anticoagulant is trisodium EDTA (Na2-EDTA).

[0087] In other embodiments, the anticoagulant is a citrate or a salt thereof (preferably a sodium salt or a potassium salt), and more preferably trisodium citrate (Na3C6H5O7). In other embodiments, the anticoagulant is tripotassium citrate (K3C6H5O7).

[0088] In other embodiments, the anticoagulant is heparin. "Heparin" is a sulfated glycosaminoglycan used clinically for thrombosis prevention. As used in the present invention, the term "heparin" refers to all forms of heparin, including but not limited to unfractionated heparin, heparinoids, dermatans, chondroitins, low molecular weight heparins (e.g., tinsaparin (including tinsaparin sodium)), very low molecular weight heparin, and ultra-low molecular weight heparin. Non-limiting examples include unfractionated heparins such as heparin sodium (e.g., heparin sodium USP, available from Scientific Protein Labs of Waunakee, WI).

[0089] To prevent unintended proteolysis of (poly)peptides contained in the sample, it may be advantageous to add one or more protease inhibitors to the sample. The term "protease inhibitor," as used in this invention, generally refers to a compound, substance, and / or composition capable of inhibiting the action of one or more proteases. Particularly preferred protease inhibitors that may be employed in connection with the methods of this invention are those capable of inhibiting the activity of proteases known (or typically suspected) to be present in certain types of samples, such as blood.

[0090] Known protease inhibitors that can be suitably used in connection with the disclosed method include, but are not limited to, inhibitors of serine proteases, cysteine ​​proteases, aspartate proteases, metalloproteases, thiol proteases, and exopeptidases. Of these, serine protease inhibitors and cysteine ​​protease inhibitors are of particular interest, and metalloprotease inhibitors are also important. Non-exclusive examples of serine protease inhibitors include antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), AEBSF, TLCK, TPCK, leupeptin, and soybean trypsin inhibitors. Examples of cysteine ​​protease inhibitors include IAA (indoleacetic acid) and E-64. Preferred examples of aspartate protease inhibitors include pepstatin and VdLPFFVdL. Non-limiting examples of metalloproteinase inhibitors include EDTA, 1,10-phenanthroline, and phosphoramodone. Therefore, EDTA may act as an anticoagulant and / or protease inhibitor. Examples of exopeptidase inhibitors include astatin, bestatin, diprotin A, and diprotin B. Further preferred examples of protease inhibitors include α-2-macroglobulin, soy trypsin inhibitors or lima metrypsin inhibitors, pancreatic protease inhibitors, oval ovostatin, and oval cystatin. In a preferred embodiment, a commercially available "protease inhibitor cocktail," e.g., Roche-cOmplete TM Protease Inhibitor Cocktail and SIGMAFAST TM A combination of protease inhibitors, such as Protease Inhibitor Cocktail Tablets and EDTA-Free formulations, can be used.

[0091] In a further preferred embodiment, the sample comprises plasma and EDTA or a salt thereof (preferably potassium EDTA, e.g., di- or tri-potassium EDTA (K2- or K3-EDTA)). The corresponding sample is conventionally referred to as an "EDTA-plasma" sample. In a further preferred embodiment, the sample is a plasma sample further comprising EDTA or a salt thereof (preferably potassium EDTA such as K2- or K3-EDTA) at a concentration between 0.5 and 2.7 mg / ml, more preferably between 1.1 and 2.1 mg / ml, more preferably between 1.2 and 2.0 mg / ml, and most preferably at a concentration of 1.6 mg / ml.

[0092] In the specific assay protocol established in the present invention (shown in Figure 2B), 26 μl of EDTA-plasma sample was first (i) diluted with 70 μl of 0.9% (w / v) NaCl (see Step 1) before being subjected to precipitation induction conditions. The resulting 96 μl reaction mixture was then (ii) further diluted with 40 μl of distilled H2O to a total sample volume of 136 μl. However, in an alternative setting, 26 μl of EDTA-plasma sample was diluted with 110 μl of aqueous solution containing 0.57% NaCl (corresponding to 98 mM NaCl).

[0093] Therefore, according to a preferred embodiment, the sample is diluted with a diluent before being subjected to precipitation induction conditions, where the diluent is preferably: (i) an aqueous solution containing a salt, preferably sodium chloride (NaCl), at concentrations in the range of 5 mM and 200 mM, 20 mM and 175 mM, 45 mM and 150 mM, 65 mM and 130 mM, 75 mM and 120 mM, 80 mM and 115 mM, 85 mM and 110 mM, 91 mM and 105 mM, with the greater the concentration, the more preferably, and most preferably 98 mM; and / or (ii) The diluent is added to the sample in a volume-to-volume (v / v) ratio of diluent to sample, most preferably 4.2:1, between 1:1 and 7:1, 2:1 and 6.5:1, 2.5:1 and 6:1, 3:1 and 5.5:1, 3.4:1 and 5:1, 3.6:1 and 4.8:1, 3.8:1 and 4.6:1, and 4.0:1 and 4.4:1, with the greater the ratio, the better.

[0094] In an alternative, preferred embodiment, the sample is subjected to precipitation-inducing conditions before: (i) Diluted with a first diluent, where the first diluent is: (ia) an aqueous solution containing a salt, preferably sodium chloride (NaCl), at concentrations in the range of 5 mM and 250 mM, 80 mM and 230 mM, 100 mM and 210 mM, 110 mM and 200 mM, 120 mM and 190 mM, 130 mM and 180 mM, 140 mM and 170 mM, 148 mM and 160 mM, with the greater the better, and most preferably 154 mM; and / or (ib) The first diluent is added to the sample in a volume-to-volume (v / v) ratio of the first diluent to the sample, most preferably at a v / v ratio of 2.69:1, between 0.1:1 and 6:1, 0.5:1 and 5.5:1, 0.8:1 and 5:1, 1:1 and 4.8:1, 1.6:1 and 4:1; 1.6:1 and 3.8:1, 1.8:1 and 3.6:1, 2:1 and 3.4:1, and 2.4:1 and 3.1, with the greater the better. and (ii) Diluted with a second diluent, where the second diluent is: (ii-a) containing water or consisting of water; and / or (ii-b) The second diluent is added to the sample obtained in (i) in a volume-to-volume (v / v) ratio of the second diluent to the sample, preferably a second diluent to sample v / v ratio of 0.42:1, between 0.01:1 and 2:1, 0.05:1 and 1:1, 0.075:1 and 0.8:1, 0.1:1 and 0.6:1, 0.2:1 and 0.7:1, and 0.3:1 and 0.5:1, with the larger the ratio, the better.

[0095] In an alternative, preferred embodiment, the sample is subjected to precipitation-inducing conditions before: (i) Diluted with a first diluent, where the first diluent is: (ia) containing water or consisting of water; and / or (ib) The first diluent is added to the sample in a volume-to-volume (v / v) ratio between 0.05:1 and 3:1, 0.5:1 and 1:1, 0.8:1 and 2.2:1; 1.1:1 and 2:1, 1.3:1 and 1.8:1, 1.4:1 and 1.7:1, and 1.5:1 and 1.6:1, with the greater the preferable, and in a first diluent to sample volume-to-volume (v / v) ratio, most preferably 1.54:1; and (ii) Diluted with a second diluent, where the second diluent is: (ii-a) an aqueous solution containing a salt, preferably sodium chloride (NaCl), at concentrations in the range of 5 mM and 250 mM, 80 mM and 230 mM, 100 mM and 210 mM, 110 mM and 200 mM, 120 mM and 190 mM, 130 mM and 180 mM, 140 mM and 170 mM, 148 mM and 160 mM, with the greater the better, and most preferably 154 mM; and / or (ii-b) The second diluent is added to the sample in a second diluent to sample volume-to-volume (v / v) ratio that becomes progressively more preferable, between 0.1:1 and 3:1, 0.3:1 and 2:1, 0.5:1 and 1.8:1, 0.6:1 and 1.5:1, 0.8:1 and 1.3:1, and 0.9:1 and 1.2:1, most preferably at a second diluent to sample v / v ratio of 1.06:1.

[0096] Examples of salts that may be contained in the above aqueous solutions include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), and magnesium chloride (MgCl2).

[0097] In a preferred embodiment, the precipitation induction conditions in step (b) are provided by, or additionally by: (i) Increase in sample temperature; (ii) Addition of at least one salting agent to the sample; where preferably, the salting agent is selected from the following: (ii-a)Ca 2+ , (CH3)4N + , Cs + , Guanidinium (C(NH2)3 + ), Rb + NH4 + , K + kaNa + Mn 2+ and Li + Selected cations from; and OH - SO4 2- HPO4 2- CH3COO - C3H5O(COO)3 3- CO3 2- ClO3 - , BrO3 - , thiocyanate (SCN - ) and Cl - Anions selected from; more preferably salts containing KCl and / or (NH4)2SO4; and / or (ii-b) Alkali metal sulfates; preferably Li2SO4, Na2SO4, K2SO4, Rb2SO4, and / or Cs2SO4; (iii) Addition of one or more other aliphatic alcohols, preferably alkanols, most preferably ethanol; (iv) Concentration of the sample by evaporation; (v) Addition of a ketone, preferably acetone; (vi) Ionizing radiation (IR); and / or (vii) Any combination of (i) to (vi).

[0098] In a preferred embodiment, the rise in sample temperature is provided by exposing the sample to a temperature within the range of 38°C and 99°C, 45°C and 95°C, 50°C and 90°C, 55°C and 85°C, 60°C and 80°C, and 65°C and 75°C, with the greater the preferable, and most preferably 70°C ± 2°C.

[0099] Those skilled in the art will understand that the ideal duration (i.e., time) of thermal exposure to induce the precipitation of one or more (poly)peptides in a sample before determining the level of precipitated (poly)peptides in the sample depends on various factors such as the volume of the sample and / or the material of the reaction tube / apparatus. However, to those skilled in the art, selecting an appropriate duration of thermal exposure is within the realm of ordinary art, i.e., long enough to induce the precipitation of one or more (poly)peptides in the sample, but short enough not to cause the precipitation of all (poly)peptides contained in the sample. However, in a preferred embodiment, the exposure of the sample to the above temperature would be a period between 2 seconds and 10 minutes, 10 seconds and 5 minutes, 20 seconds and 3 minutes, and 30 seconds and 2 minutes, with the greater the better, and most preferably 60 seconds. The above temperatures and times can be combined, for example, 30 seconds to 2 minutes at 65°C to 75°C, 30 seconds to 2 minutes at 70°C ± 2°C, and 60 seconds at 70°C ± 2°C, with the greater the better. In a preferred embodiment, at least one salting agent is selected from the group consisting of (NH4)2SO4, alkali metal chloride (preferably selected from KCl and / or CaCl2), guanidine thiocyanate, and MnCl2, and most preferably (NH4)2SO4.

[0100] In a preferred embodiment, a salting agent, preferably (NH4)2SO4, is added to the sample so that the final concentration is in the range of 1-40%(w / v), 4-30%(w / v), 6-25%(w / v), 8-20%(w / v), 10-16%(w / v), and 12-14%(w / v), with higher concentrations being preferable and most preferably 13%(w / v).

[0101] In preferred embodiments, one or more other aliphatic alcohols are selected from ethanol, isopropanol, and / or methanol, most preferably ethanol.

[0102] The final aliphatic alcohol (preferably ethanol, isopropanol, and / or methanol) concentration used to induce precipitation is between 10.0% (v / v) and 40.0% (v / v), 15.0% (v / v) and 35.0% (v / v), most preferably between 18.5% (v / v) and 27.8% (v / v), with higher concentrations being preferable.

[0103] The final ketone (preferably acetone) concentration used to induce precipitation is preferably between 8.5% (v / v) and 28.5% (v / v), 13.5% (v / v) and 23.5% (v / v), and 16% (v / v) and 21% (v / v), with most preferably 18.5% (v / v).

[0104] In a more preferred embodiment, the salting agent, preferably (NH4)2SO4, is added to the sample in an aqueous solution containing the salting agent, preferably (NH4)2SO4, at a concentration in the range of 10-60% (w / v), 20-50% (w / v), 25-45% (w / v), and 30-40% (w / v), with a higher and more preferably higher concentration in the range of 35% (w / v); wherein the aqueous solution is added to the sample in a volume-to-volume ratio of aqueous solution to sample between 0.3:1 and 0.9:1, 0.4:1 and 0.8:1, and 0.5:1 and 0.7:1, with a higher and more preferably higher concentration in the volume-to-volume ratio of aqueous solution to sample of 0.59:1.

[0105] In preferred embodiments of the methods according to the first, third, and fourth aspects of the present invention, the level of precipitated (poly)peptide is evaluated in step (b) by spectrophotometrics, nephelometry, spectrofluorometry, circular dichroism (CD) spectroscopy, mass spectrometry (MS), and / or NMR spectroscopy.

[0106] In a particularly preferred embodiment, the level of precipitated (poly)peptide is evaluated by spectrophotometric analysis.

[0107] The terms "spectrophotometrics" or "photometrics" refer to well-known and widely used optical analysis techniques for measuring the amount of light absorbed by or transmitted through a sample (i.e., discontinuous wavelengths of ultraviolet (UV, 200-400 nm) or visible light (VIS, 400-800 nm)). Absorbance (A) is interchangeably referred to in this invention as quenching (E), and is the intensity of light before it passes through the sample (I o Absorbance is equal to the logarithm of the ratio of the light intensity (I) after it has passed through the sample. By definition, absorbance is a dimensionless quantity, but it is usually expressed in absorbance units (AU) or extinction units (E), or in milli-absorbance units (mAU) or milli extinction units (mE).

[0108] In this invention, the term "spectrophotometric method," as used in its broadest sense, also includes, and preferably means "turbidimetry," which refers to a well-known method in the art for determining the amount of turbidity in a solution, i.e., turbidity, based on the measurement of the effect of turbidity on the transmission and scattering of light. Turbidity in a liquid is caused by the presence of finely divided suspended particles, such as precipitated (poly)peptides. When light is passed through a turbid sample, its intensity decreases due to scattering, and the amount of scattered light depends on the concentration, size, and particle size distribution of the particles. Therefore, a spectrophotometer can also measure the increase in turbidity based on the decrease in the intensity of transmitted light. This increase in turbidity allows for the direct measurement of the formation of precipitated (poly)peptides.

[0109] The term "nephelometry" in this invention refers to a technique related to "turbidimetry" and is well known in the art. Turbidimetry measures the amount or intensity of light passing through a sample, while nephelometry measures the amount or intensity of light scattered from the incident light ray at a defined angle (usually 90°).

[0110] Generally, for spectrophotometric analysis of liquid samples, the solution must be placed in the optical path of the spectrophotometer in a specific format. A cuvette is a sample container with optical windows and is a standard choice for this application. The distance between the optical windows is precisely defined, so that the optical path length of the sample within the cuvette is known. The most common cuvette is square. This format typically accommodates sample volumes from microliters (ultramicro cuvettes) to milliliters (macro cuvettes). The standard path length of a cuvette is 10 mm. However, cuvettes with shorter optical paths are also available. For example, using cuvettes with shorter optical paths (less than 1 cm) can be convenient to reduce the amount of sample or reagents required for measurement, or to reduce the space in the laboratory, spectrophotometer, or sample changer. As is done with many spectrophotometers, the output generated from measurements using cuvettes with optical path lengths different from 1 cm is generally provided (automatically inversely calculated) as if the measurement had been performed with a cuvette with a 1 cm optical path length. For example, in the following example, a cuvette with a path length of 0.7 cm is used, and the spectrophotometer normalizes its output to correspond to a cuvette with a path length of 1.0 cm. Standard cuvettes made from PMMA, polystyrene, or ordinary glass are generally transparent only in the visible region. When using wavelengths in the ultraviolet region, especially wavelengths below approximately 300 nm, cuvettes made of quartz glass or special plastics that have sufficient transparency in this region should be used. With a spectrophotometer using a cuvette with a path length of 1 cm (10 mm), the linear absorbance range is usually up to approximately 2.5 A (i.e., 2500 mE). When analyzing samples that exhibit absorbances exceeding this value, diluting the sample with a diluent (such as an isotonic salt solution) to reduce the detectable absorbance may be beneficial for improving accuracy.

[0111] In the embodiments disclosed in the present invention, a conventional clinical chemistry analyzer, namely Indiko TMSpectrophotometric analysis was performed using a Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). However, various other instruments suitable for performing each analysis are known and commercially available in the field and can be suitably employed for the purposes disclosed in this invention.

[0112] Exemplary, particularly preferred precipitation induction conditions include the addition of at least one acid having a pKa in the range of 3.5 to 6.0, more preferably 4.1 to 5.5, and even more preferably 4.75 ± 0.05; preferably, the at least one acid is a carboxylic acid, more preferably a monocarboxylic acid, and most preferably 0.4% (v / v) acetic acid in 0.81% NaCl, which is added to the sample so that the pH is preferably between pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, pH 4.1 and pH 4.3, and most preferably between pH 4.2 ± 0.05.

[0113] In a particularly preferred embodiment, the level of precipitated (poly)peptide is evaluated by measuring the change in absorbance with a spectrophotometer, and the determined increase or decrease in absorbance indicates an increase or decrease in the level of precipitated (poly)peptide in the sample, respectively.

[0114] In this invention, the term “change in absorbance” refers to a potential increase or decrease in the detected absorbance due to the provision of precipitation-inducing conditions. Therefore, in a typical setting, and in a particularly preferred embodiment, the absorbance to be measured is set to zero ("blank") immediately before the provision of the precipitation-inducing conditions, or alternatively, at approximately the same time. In such embodiments, the change in absorbance to be measured corresponds to the (net) change in absorbance caused by the provision of the precipitation-inducing conditions, thereby enabling a relative comparison with the change in absorbance measured for a correspondingly processed reference sample and / or a given standard.

[0115] In the experimental evidence disclosed in this invention, the level of precipitated (poly)peptides formed as a result of providing precipitation-inducing conditions was measured, for example, at a wavelength of 340 nm by spectrophotometric method. This wavelength was found to be particularly sensitive for the detection of precipitated (poly)peptides. Similar wavelengths are considered to be essentially equally suitable for the purposes considered in this invention.

[0116] Therefore, in a preferred embodiment, the absorbance is measured at a wavelength of 340 nm, which is more preferably greater, within the range of 200 nm and 800 nm, 260 nm and 420 nm, 280 nm and 400 nm, 300 nm and 380 nm, 320 nm and 360 nm, 330 nm and 350 nm, and 335 nm and 345 nm.

[0117] As reported in the experimental evidence disclosed in the present invention (see Examples 1 and 2, Figures 7A-C and Tables 1 and 2A), due to the differences in precipitation sensitivity reported in the present invention, the inventors further conducted extensive research by systematically evaluating a vast number of plasma samples (355 in total) from subjects with either pathological (114) or non-pathological (241) backgrounds to identify spectrophotometrically observable potential differences that would enable reliable identification of samples in a source-dependent manner (see Table 1). As a result, it was advantageously found that, when applying spectrophotometric methods as shown in Figure 2B, the absorbance changes detected for pathological and non-pathological samples surprisingly fell into two separate, well-separated ranges. As is evident from Table 2A, subsequent calculations for various performance metrics (e.g., specificity, sensitivity, and accuracy) revealed that an accuracy of 95% or higher is achieved when an absorbance increase of at least 78 mE is applied as the minimum threshold for determining a sample to be positive for cancer and / or acute inflammatory disease. This accuracy is essentially maintained up to a threshold of 170 mE, and in some cases even improves further. When thresholds above 170 mE are applied, the calculated specificity remains high, but the sensitivity begins to decrease slightly. The highest accuracy (99.44%) was calculated at thresholds of 105–134 mE. These results demonstrate that accurate identification of pathological and non-pathological samples, and thus reliable diagnosis, can be achieved solely by performing the spectrophotometric method disclosed in this invention and evaluating the sample by comparing the measured absorbance change with the empirically determined thresholds described above.

[0118] Therefore, in a preferred embodiment of the latter, in a method for diagnosing cancer and / or acute inflammatory disease in a subject, if the change in absorbance is an increase in absorbance of 78-170 milli extinction units (mE), 80-160 mE, 83-155 mE, 88-150 mE, 93-145 mE, and 98-140 mE, preferably a larger increase, and most preferably 105-134 mE, then, preferably when measured with a 1 cm pass cuvette or normalized to a measurement with a 1 cm pass cuvette, the subject is diagnosed as positive for cancer and / or acute inflammatory disease.

[0119] The preferred embodiments of the latter embodiment of the method according to the first aspect of the present invention have their own readouts for diagnosing a subject as positive for cancer and / or acute inflammatory disease (based on the level of precipitated (poly)peptides), and therefore they can also be designed as independent embodiments. Accordingly, the present invention also relates to a method for diagnosing cancer and / or acute inflammatory disease in a subject, the method comprising: (a) Subjecting the sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample in relation to the following: (bi) Levels of precipitated (poly)peptides in at least one correspondingly processed reference sample obtained from one or more reference subjects known to be positive for cancer and / or acute inflammatory disease; (b-ii) Levels of precipitated (poly)peptides in at least one corresponding processed reference sample obtained from one or more reference subjects known to be negative for cancer and acute inflammatory disease; and / or A predetermined standard determined based on (b-iii)(bi) and / or (b-ii); Here, the level of precipitated (poly)peptide is evaluated by measuring the change in absorbance with a spectrophotometer, thereby a determined increase in absorbance indicates an increase or decrease in the level of precipitated (poly)peptide in the sample, and the subject is diagnosed as positive for cancer and / or acute inflammatory disease if the increase in absorbance is preferably in the range of 78-170 milli extinction units (mE), 80-160 mE, 83-155 mE, 88-150 mE, 93-145 mE, and 98-140 mE, and most preferably 105-134 mE, when measured with a 1 cm pass cuvette or normalized to the measurement with a 1 cm pass cuvette; Preferably: -The precipitation induction conditions in (a) are provided by the addition of at least one acid having a pKa in the range of 3.5 to 6.0, preferably in the range of 4.1 to 5.5, more preferably 4.75 ± 0.05; the at least one acid is a carboxylic acid, more preferably a monocarboxylic acid, most preferably 0.4% (v / v) acetic acid in 0.81% NaCl, which is added to the sample so that the pH of the sample is between pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, pH 4.1 and pH 4.3, the greater the sample pH, the more preferably, and most preferably pH 4.2 ± 0.05; and / or - The change in absorbance is measured at wavelengths between 200 nm and 800 nm, 260 nm and 420 nm, 280 nm and 400 nm, 300 nm and 380 nm, 320 nm and 360 nm, 330 nm and 350 nm, and 335 nm and 345 nm, most preferably in the range of 340 nm.

[0120] Therefore, those skilled in the art will understand that the absorption rate ranges mentioned above are used as diagnostic determination thresholds and correspond to predetermined standards determined from correspondingly treated reference samples having pathological or non-pathological origins (as described in the method in the first aspect of the invention).

[0121] In a preferred embodiment, the level of precipitated (poly)peptide is evaluated in step (b) from the remaining soluble fraction of the sample, preferably after the precipitated (poly)peptide has been removed.

[0122] When carrying out the methods disclosed in the present invention, the inventors typically perform one or more culture steps, thereby bringing the sample to equilibrium before analysis. More specifically, the inventors typically incubate the sample for, for example, about 300 seconds (see Figure 2B, step 4) before subjecting it to precipitation induction conditions at a temperature of 37°C. Such an equilibrium step can be particularly employed when diluting the sample with the diluent described in the present invention before providing the precipitation induction conditions.

[0123] Furthermore, as is evident from Figure 4, the inventors found that the increase in absorbance resulting from the provision of precipitation-inducing conditions could be detected immediately from the moment the precipitation-inducing conditions were applied to the sample. Moreover, it was found that this increase, although starting immediately, progressed over several seconds, and after further increases in absorbance, reached a plateau where the absorbance remained nearly constant (for several hours). Therefore, the sample after the provision of precipitation-inducing conditions (see Figure 2B, step 6) was typically cultured at 37°C for 300 seconds and then analyzed by spectrophotometry (see Figure 2B, step 7), etc.

[0124] Therefore, in a particularly preferred embodiment, (i) Before subjecting the sample to the precipitation induction conditions in step (a), incubate the sample at a temperature between 2°C and 42°C, preferably 37±2°C, for a period of at least 10 seconds, preferably between 10 and 5000 seconds, 20 and 4000 seconds, 30 and 3000 seconds, 40 and 2000 seconds, 50 and 1000 seconds, 60 and 800 seconds, 80 and 600 seconds, 100 and 500 seconds, 200 and 400 seconds, 250 and 350 seconds, more preferably between 275 and 325 seconds; most preferably in the range of 300 seconds; and / or (ii) After being subjected to the precipitation-inducing conditions in step (a), before step (b), the sample is incubated at a temperature between 2°C and 42°C, preferably 37±2°C, for at least 60 seconds, preferably in the range of 60 and 600 seconds, 100 and 500 seconds, 200 and 400 seconds, 250 and 350 seconds, and 280 and 320 seconds, with the greater the better, and most preferably 300 seconds.

[0125] In other (or even more preferred) embodiments, the absorbance measurement is performed within a time period of 60 to 600 seconds, 100 to 500 seconds, 200 to 400 seconds, 250 to 350 seconds, 280 to 320 seconds, more preferably 300 seconds, after providing the precipitation induction conditions in step (a).

[0126] In a preferred embodiment, the sample and / or diluent further includes: (i) Anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate, and heparin or any salt thereof; (ii) Proteinase inhibitors; (iii) One or more stabilizers, preferably selected from albumin, casein, gelatin, collagen, globulin, and protamine; (iv) Skim milk powder; (v) Surfactants; preferably selected from Tween, preferably Tween 20 or Tween 80, Triton X-100, and sodium dodecylbenzenesulfonate; (vi) polyethylene glycol (PEG); where preferably PEG has a molecular weight in the range of 1,000 and 20,000 Da and / or is selected from PEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, and PEG20000; and / or (vii) Polysaccharides; preferably dextran selected from dextran-1, dextran-10, dextran-20, dextran-30, and dextran-40.

[0127] Of the above options (i) to (vii), option (i) is particularly preferred. In an alternative preferred embodiment, the sample and / or diluent further comprises none of (i) to (vii).

[0128] In preferred embodiments, particularly when the sample is a human sample, the diluent is human-derived or does not contain human-derived components. Therefore, in preferred embodiments, albumin is non-human albumin, such as bovine serum albumin (BSA), and / or heparin is non-human heparin. However, in other preferred embodiments, albumin is human serum albumin. In even more preferred embodiments, milk (poly)peptides and / or skim milk powder are of non-human origin, more preferably milk-derived.

[0129] In relation to the embodiments described above, if the additive is a (poly)peptide, such as serum albumin, or another animal product, such as milk powder, it is understood that the additive does not originate from the same subject and / or species from which the sample was obtained, and does not originate from a subject and / or species that had acute inflammation and / or cancer at the time the additive was obtained. Standardized albumin preparations such as bovine serum albumin, human serum albumin, milk protein, and milk powder are commercially available and can be used for the purposes disclosed in this invention.

[0130] In methods according to the first, second, and / or any further aspects of the present invention, these methods further include evaluating a sample taken from a subject, preferably a blood sample, for the presence or absence of acute inflammation, where, in the case of a subject determined to have cancer and / or acute inflammatory disease, the absence of determined acute inflammation indicates that the subject has cancer and does not have acute inflammatory disease. Thus, in the latter scenario, the subject is diagnosed as positive for cancer and not having acute inflammatory disease.

[0131] Further evaluation for the presence of acute inflammation can be preferably carried out by various conventional methods for assessing the presence of acute inflammation as described below, and such evaluation is particularly considered when the results of the methods according to the first, second and / or further embodiments of the present invention suggest the presence of cancer and / or acute inflammatory disease.

[0132] Therefore, it may be interesting to conduct further analysis to determine whether this finding is caused by the presence of acute inflammation. Only if the latter condition can be ruled out will a clear indicator of the presence of cancer be provided. On the other hand, if the results of the method according to the first, second and / or further embodiments of the invention provide an indicator of the presence of cancer and / or acute inflammatory disease, and such further evaluation confirms the presence of acute inflammation, the possibility that cancer is present simultaneously in addition to acute inflammation cannot be ruled out.

[0133] It will be readily apparent to those skilled in the art that the sample may be the same sample, or a part or fragment thereof, taken from the subject and used in step (a) of the method according to the first, second and / or further aspects of the present invention, if it is convenient and applicable depending on the individual method employed to assess the presence of acute inflammation, or the sample may be a separate further sample obtained from the subject. For example, if the sample employed in the initial analysis carried out in connection with the method according to the first, second and / or further aspects of the present invention is a cell-free sample (e.g., a plasma sample) and inflammation is assessed through the evaluation of the level of certain cells known to indicate the presence of acute inflammation (e.g., an increase in leukocyte levels), then it will be necessary to obtain a further sample from the subject. In practice, it would be convenient to first obtain a whole blood sample from the subject, prepare a plasma sample from only a portion thereof, while using the remaining whole blood sample for the analysis of the presence or absence of acute inflammation.

[0134] In a preferred embodiment of the latter, the presence or absence of acute inflammation is assessed by evaluating the levels of one or more inflammatory markers; here, preferably, one or more inflammatory markers are selected from C-reactive protein (CRP), procalcitonin (PCT), fibrinogen, and leukocytes.

[0135] In a preferred embodiment, the presence or absence of acute inflammation is assessed by evaluating at least one, at least two, at least three, or all four levels of the above-mentioned inflammatory markers, preferably in greater proportions, and preferentially, or by additionally evaluating the levels of further known inflammatory markers.

[0136] In a particularly preferred embodiment, the presence or absence of acute inflammation is assessed by evaluating the level of CRP. The level of CRP is generally determined in a sample, where the sample is preferably a blood sample or a blood-derived sample (e.g., plasma or serum), and most preferably plasma.

[0137] C-reactive protein (CRP) is a liver-produced glycoprotein characterized by precipitation with C-polysaccharides from Streptococcus pneumoniae. This protein is not normally produced. When acute inflammation is present, body tissues are destroyed, leading to the release of interleukins 1 and 6, which stimulates the production of this protein and causes a rapid increase in CRP levels (hence its name as the acute-phase reactive protein). Once the acute inflammation subsides, CRP rapidly disappears. Therefore, CRP is considered a marker reflecting the activation of a systemic inflammatory response. CRP is nonspecific, and its levels are elevated in all acute inflammatory states. Typically, CRP rises within 6 hours of acute inflammation, allowing for early detection of inflammation. Those skilled in the art will know that two types of CRP tests are routinely used in current medical / diagnostic practice: (1) the "conventional or standard CRP test"; and (2) the more sensitive, so-called "high-sensitivity CRP (hs-CRP) test". Given these differing sensitivities, the different levels of CRP detected, depending on which test is applied, are generally considered to indicate the presence or absence of acute inflammation. In this invention, each CRP level is referred to as the "standard CRP level" or the "high-sensitivity CRP (hs-CRP) level," depending on which test is applied for detection.

[0138] Therefore, in a preferred embodiment: - A determined standard CRP level up to 5 mg / dl, a determined hsCRP level up to 0.5 mg / dl, a determined fibrinogen level up to 400 mg / dl, and / or a determined white blood cell count up to 10,000 per microliter indicates the absence of acute inflammation; and / or A determined CRP level greater than 5 mg / dL, a determined hsCRP level greater than 0.5 mg / dL, a determined fibrinogen level greater than 400 mg / dL, and / or a determined white blood cell count greater than 10,000 per microliter indicate the presence of acute inflammation.

[0139] In alternative or more preferred embodiments, the presence or absence of acute inflammation is assessed by evaluating procalcitonin (PCT) levels, or additionally by other means.

[0140] Procalcitonin (PCT) is an established biomarker for the diagnosis of acute inflammation, particularly for the early diagnosis of sepsis. PCT levels are known to reflect the severity of bacterial infection and are used to monitor the progression of infection to sepsis, severe sepsis, and septic shock (see, e.g., Meisner M. Ann Lab Med. 2014 Jul;34(4):263-73). In healthy individuals, plasma PCT concentrations are typically less than 0.1 ng / mL. A useful reference range is a concentration of ≤0.2 ng / mL to rule out sepsis and acute inflammation. A useful reference range is a concentration of >10 ng / mL to diagnose sepsis and acute inflammation with a high probability. PCT levels are generally determined in a sample, which is preferably a blood sample or a blood-derived sample (e.g., plasma or serum), most preferably plasma.

[0141] Therefore, in a preferred embodiment: - A determined PCT level (preferably PCT plasma level) of ≤0.2 ng / mL (preferably less than 0.1 ng / mL) indicates the absence of acute inflammation (especially sepsis); and / or A determined PCT level of >0.10 ng / mL (preferably a PCT plasma level) indicates the presence of acute inflammation (especially sepsis).

[0142] In a preferred embodiment of the method according to the first, second, third, fourth and / or any further embodiment of the present invention, the method is carried out as follows: (i) On a (bio) chip; (ii) In a minimized form, for example, using a sample volume in the range of 1 picoliters to 100 microliters. (iii) in an automated or semi-automated manner; (iv) Using microfluidic devices / systems; and / or (v) In a high-throughput and / or multiplex format, multiple samples are placed in separate wells of one or more microtiter plates (also known as microwell plates or multiwell plates, e.g., 6-well, 12-well, 24-well, 48-well, or 96-well plates) and analyzed sequentially (continuously) or in parallel. A miniaturized form of the method of the present invention is shown in Example 6. In a further embodiment, the present invention provides a system, apparatus, or device (preferably a diagnostic apparatus or diagnostic device) configured to carry out a method according to any first, second, third, fourth, fifth, or other aspect of the present invention disclosed herein. [Brief explanation of the drawing]

[0143] [Figure 1] Environmental stimuli have different effects on pathological plasma proteins. Pathological (n=5 or 8) and non-pathological (n=4 or 7) plasma was exposed to extreme stimuli known to impair protein structure. Precipitated proteins were collected by centrifugation, and the amount of protein present in the pellet ("Pellet") and supernatant ("SN") was measured by bicinchoninate assay (BCA). Plasma was exposed to A) ~0.2% acetic acid ("ACID") at 37°C for 5 minutes; B) 70°C ("HEAT") for 1 minute; C) ~13% ammonium sulfate ("SALT") at room temperature for 5 minutes; D) ~18.5% acetone ("Ketone"); or E) ~18.5% ethanol ("Alcohol") at 4°C for 5 minutes. A two-sided independent student's t test was performed. Data for (AE) are expressed as mean ± SD from at least four biological copies. *P<0.05,**P<0.01,***P<0.001,ns=non-significant. [Figure 2]A spectrophotometer-based assay for quantifying protein precipitation. A) Illustration of the Indiko™ Plus Clinical Chemistry analyzer. B) Workflow of the default assay setup. A seven-step protocol was established to monitor protein precipitation by measuring absorbance at 340 nm, the wavelength of light scattering. Note that the addition of acetic acid in step 6 initiates precipitation. In step 7, the precipitation can be measured kinetically once, for example, over 1 hour (see Figure 4), or once after the time it takes for the precipitation to form (e.g., 300 seconds as illustrated in the attached example). C) Lyophilized plasma was reconstituted to contain the indicated amount of protein, and precipitation was carried out as outlined in (B). Absorbance was measured 5 minutes after exposure to acetic acid. Each data point represents the mean of two technical replicates. The coefficient of determination (r²) shows a linear relationship between the amount of plasma protein and the absorbance subtracted from the background. [Figure 3]Major determinants of acetic acid-induced protein precipitation. A) Plasma proteins from pathological and non-pathological (n=3) subjects were precipitated and quantified according to the method shown in Figure 2. As shown, the concentration of added acetic acid increased and the pH of the precipitant decreased. This demonstrated that precipitation conditions can be optimized to differentiate based on the pathological state of the plasma. A two-sided independent sample t-test with Benjamini-Hochberg false positive rate (FDR) correction was performed to compare pathological and non-pathological samples. B) The experiment was performed as in A), but hydrochloric acid (HCl) was used instead of acetic acid as the precipitant. The x-axis shows the pH of HCl before mixing with plasma, showing that simply lowering the pH does not cause sufficient precipitation. A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed to compare pathological (n=3) and non-pathological (n=3) samples at each specified pH value. C) The experiment was carried out in the same manner as in A), except that acetic acid was dissolved in a specified amount of sodium chloride (NaCl). As the amount of salt increased, the pH decreased. Two-sided independent Student's t-tests were performed to compare pathological (n=4) and non-pathological (n=3) samples for each specified NaCl concentration. Data in (A-C) are shown as mean ± standard deviation (SD) obtained from at least three biological copies. *P<0.05, **P<0.01, ***P<0.001. [Figure 4] Acetic acid induces precipitation via a self-propelled chain reaction in plasma. The experiment was performed as shown in Figure 2. Precipitation was monitored for 1 hour. Addition of acetic acid caused rapid precipitation in pathological samples but had little effect on non-pathological samples. Repeated measures two-way ANOVA and Sidak's posthoc test were used for multiple comparison correction. Data were expressed as the mean ± SD of 5 biological replications. *P<0.05, **P<0.01, ***P<0.001. [Figure 5]Acetic acid induces reversible precipitation of plasma proteins. A) Workflow of the assay setup. In contrast to the default assay (see Figure 2B), acetic acid was present 1 minute before further manipulation of the reaction by adding 16 mM NaOH or vehicle (dH2O). B-E) Two pathological samples (B, C) or two non-pathological samples (D, E) were exposed to the workflow described in 5A. Acetic acid induced rapid precipitation in the pathological samples over 11 minutes, while little reaction was observed in the non-pathological samples. Addition of 16 mM NaOH to equilibrate pH to 7.4 completely reversed the reaction, but NaOH did not affect the acetone (18.5%)-induced precipitation. 5B) Addition of NaOH immediately reversed the acetic acid-induced protein precipitation. This indicates that acetic acid-induced protein precipitation is pH-dependent. All biological replications are shown (B-E). Data points connected by lines represent repeated measurements from the same biological replication. [Figure 6]Pathological plasma proteins exhibit impaired structural stability. Experiments were conducted as described in Figure 2A. Absorbance was measured 5 minutes after acetic acid addition to assess the level of precipitated protein. A-B) Pathological plasma (n=3) or non-pathological plasma (n=3) was pre-incubated at 37°C or 50°C for 5 minutes before acetic acid addition. Non-pathological samples became more susceptible to precipitation by acetic acid after incubation at 50°C. This indicates that pathological samples are composed of structurally different proteins that are more prone to precipitation. A two-tailed paired t-test was performed to compare samples incubated at 37°C and 50°C. C-D) Pathological samples (n=3) or non-pathological samples (n=3) were centrifuged multiple times (10x) through a 3kDa cutoff filter, either untreated or with the non-protein-binding material (=plasma matrix) replaced. The plasma matrix was either replaced with 0.9% NaCl ("exchange") or the separated matrix was reintroduced ("simulated exchange"). One-way ANOVA with repeated measures and Tukey's posthoc test for multiple comparisons were performed. All biological copies are shown (A-E). Data points connected by lines represent repeated measures from the same biological copy. *P<0.05, **P<0.01, ***P<0.001. [Figure 7]Evaluation of clinical performance and optimization of thresholds. A) Based on a training set of samples known to be cancer-positive (pathological) or cancer-negative (non-pathological) and free from inflammatory disease, the optimal clinical indicator was evaluated using receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) index. It was shown that subtraction (i.e., extinction subtracted by the blank value) performed best. The performance of random chance (AUC=0.5) is shown for illustrative purposes (in black). B) Based on the distribution of a non-pathological reference group (n=241) that could be almost completely separated from the pathological reference group (n=114), a reference range of subtraction values ​​from 0 to 84.7 was determined. The optimal subtraction threshold of 134 was determined using the Youden index J shown in A). D-E) The p-value was determined based on the subtraction value by comparing one subject to the non-pathological reference group using the Wilcoxon signed-rank test. To assess whether a subject is diagnosed as pathological, the p-value threshold and the size of the respected reference group subset were optimized for the number of misclassifications (C) and balanced accuracy (D). F-I) Similarly, the AUC threshold for comparing a single subject to either the pathological reference group (F-G) or the non-pathological reference group (H-I) was optimized (similar to D-E), and denoted as AUCP and AUCN, respectively. J-K) Using AUCP (red) and AUCN (blue), respectively, the minimum required sample sizes for the pathological and non-pathological reference groups were estimated. [Figure 8]Acetate-induced protein precipitation is a useful test for the diagnosis of various cancers. A total of 555 "blind" samples (from which subjects were obtained were unknown) were tested using the workflow described in Figure 2 with a 5-minute endpoint readout. The clinical diagnoses of the subjects were as follows: healthy (n=214), brain tumor (n=14), head and neck cancer (n=14), esophageal cancer (n=18), gastric cancer (n=19), colorectal cancer (n=14), rectal cancer (n=14), pancreatic cancer (n=18), colon cancer (n=15), lung cancer (n=26), breast cancer (n=14), uterine cancer (n=14), prostate cancer (n=14), bladder cancer (n=14), kidney cancer (n=14), and cutaneous malignancies. Tumors (n=18), sarcomas (n=5), ovarian cancer (n=14), neuroendocrine tumors (n=14), testicular cancer (n=14), laryngeal cancer (n=14), bone cancer (n=14), lymphoma (n=5), cervical cancer (n=14), gastrointestinal stromal tumors (n=5), squamous cell carcinoma (n=1), bile duct / gallbladder cancer (n=5), peritoneal cancer (n=5), fibrosis (n=2), pulmonary fibrosis (n=3), sarcoidosis (n=4), bronchitis (n=2). The black dashed line indicates the extinction threshold (120 mm expansion units) at which the subject is judged to be pathological. This threshold is within a range that can be equally used to distinguish between pathological and non-pathological samples, as shown in Figure 7C. [Figure 9]A) Protein concentrations in K2-EDTA plasma from pathological (n=8) or non-pathological (n=8) sources were measured by BCA. Two-sided independent student's t-test. B) Osmotic pressure of K2-EDTA plasma from pathological (n=10) or non-pathological (n=10) sources was measured by vapor pressure osmosis. Two-sided independent student's t-test. C) Osmotic pressure of K2-EDTA plasma from pathological (n=10) or non-pathological (n=10) sources was measured by vapor pressure osmosis. Two-sided independent student's t-test. D) The experiment was performed as shown in Figure 2B. Absorbance at 340 nm was measured for pathological (n=10) or non-pathological (n=10) samples before acetic acid addition (=blank absorbance). This experiment shows that there are no existing protein aggregates that can explain the differences observed between pathological and non-pathological samples. Two-sided independent student's t-test. E) The pH of K2-EDTA plasma from either pathological (n=7) or non-pathological (n=7) sources was measured after the addition of acetate. Two-sided independent student's t-test. Data (A-E) are expressed as the mean ± SD of 8-10 biological copies. *P<0.05,**P<0.01,***P<0.001,ns=non-significant. [Figure 10]Environmental stimuli have different effects on plasma proteins of pathological origin. Plasma from healthy controls and cancer patients was handled as shown in Figure 2B. Step 6 was modified to initiate protein precipitation with different acids of varying concentrations. Measurements were taken at 340 nm after 5 minutes (300 seconds) and 10 minutes, respectively. Carcimune concentration (y-axis) is shown in milli-quenching units using an Indiko™Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are expressed as mean ± SD from at least three biological copies using one-sided or two-sided t-tests. *P<0.05, **P<0.01, ***P<0.001, ns=non-significant. Figure 10.1: Citrate in 0.9% NaCl aq. used in Step 6. A) 10% citrate. B) 5% citrate. C) Measurement of precipitation kinetics over 12 time points when 10% and 5% citric acid are used as precipitating agents. Time points are divided at 54-second intervals. Figure 10.2: Formic acid in 0.9% NaCl aq. A) 0.8% formic acid. B) 0.4% formic acid. C) 0.2% formic acid. D) Measurement of precipitation dynamics over 12 time points using 0.8%, 0.4%, and 0.2% formic acid as precipitating agents. Time points are divided at 54-second intervals. Figure 10.3: Perchloric acid in 0.9% NaCl aq. A) 0.875% perchloric acid. B) Measurement of precipitation rate over 12 hours using 0.875% perchloric acid as a precipitating agent. Time points are divided at 54-second intervals. [Figure 11]Environmental stimuli have different effects on plasma proteins of pathological origin. Plasma from healthy controls and cancer patients was handled as shown in Figure 2B. Step 6 was modified to initiate protein precipitation with alcohols of different concentrations. Measurements were taken at 340 nm after 5 and 10 minutes, respectively. Calcium quenching concentration (y-axis) is shown in milli-quenching units using an Indiko™Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are expressed as mean ± SD from at least three biological copies by one-sided or two-sided t-tests. *P<0.05, **P<0.01, ***P<0.001, ns=non-significant. Figure 11.1: Ethanol used in Step 6. A) 75% ethanol. B) 50% ethanol. C) Precipitation dynamics measured over 12 time points using 75% and 50% ethanol as precipitants. Time points are spaced at 54-second intervals. Figure 11.2: Isopropanol used in Step 6. A) 75% isopropanol. B) Measurement of precipitation rate over 12 time points using 75% isopropanol as the precipitating agent. Time points are spaced at 54-second intervals. Figure 11.3: Methanol used in step 6. A) 75% methanol. B) 50% methanol. C) Measurement of precipitation kinetics over 12 time points using 75% and 50% methanol as precipitating agents. Time points are spaced at 54-second intervals. [Figure 12]Environmental stimuli have different effects on plasma proteins of pathological origin. Plasma from healthy controls and cancer patients was handled as shown in Figure 2B. Step 6 was modified to initiate protein precipitation using salts of different concentrations. Measurements were taken at 340 nm at 5 and 10 minutes, respectively. Calcium quenching concentration (y-axis) is shown in milli-quenching units using an Indiko™Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are expressed as mean ± SD from at least three biological copies using one-sided or two-sided t-tests. *P<0.05, **P<0.01, ***P<0.001, ns=non-significant. Figure 12.1: CaCl2 used in Step 6. A) 5% CaCl2. B) 2.5% CaCl2. C) Measurement of precipitation dynamics at 12 time points using 5% and 2.5% CaCl2 as precipitating agents. Time points are spaced at 54-second intervals. Figure 12.2: Guanidine thiocyanate used in Step 6. A) 10% guanidine thiocyanate. B) Measurement of precipitation rate at 12 time points using 10% guanidine thiocyanate as the precipitating agent. Time points are spaced at 54-second intervals. Figure 12.3: MnCl2 used in Step 6. A) 5% MnCl2. B) 2.5% MnCl2. C) Measurement of precipitation rate over 12 time points using both 5% and 2.5% MnCl2 as precipitating agents. Time points are spaced at 54-second intervals. [Figure 13] Densitometry analysis of Coomassie blue stained plasma precipitates from healthy individuals (N=3) and cancer patients (N=3) after SDS-PAGE, using ImageJ. [Figure 14]Acetic acid induces reversible precipitation of plasma proteins. The assay setup workflow shown in Figure 5A was extended by adding step 10. In contrast to the assay (see Figure 5A), acetic acid was present for 5 minutes (step 7) before further manipulating the reaction by adding a dilution series of NaOH or vehicle (dH2O). Addition of 17 mM and 12 mM NaOH, respectively, reversed the reaction (similar to the results shown in Figure 5B). Addition of 7.5 mM NaOH stopped the reaction but did not reverse it. Samples stopped with 17 mM and 12 mM NaOH were further manipulated in step 10 with the addition of 0.4% acetic acid or vehicle (dH2O). It is noteworthy that the amount of acetic acid added was chosen so that the final concentration of acetic acid in the reaction mixture was approximately 0.15%, representing the predetermined assay conditions. Precipitation was initiated again (SN1AA and SN2AA), demonstrating the reversibility of this reaction. [Figure 15] Miniaturization of calcium simulators using nanodrop measurement: Dilution series table [Figure 16] Miniaturization of the calcium simulator test using nanodrop measurement: (a) Nanodrop readouts measured at 340 nm show a significant difference between samples from healthy patients and samples from cancer patients; (b) The test remains robust even when the total reaction volume is reduced by several times. [Examples]

[0144] The following examples illustrate the present invention:

[0145] Example 1: Materials and methods Plasma sampling: Human K2-EDTA plasma was purchased from ProteoGenex, Inc. (460 Hindry Ave., Unit A Inglewood, CA 90301, USA).

[0146] Bicinchoninate assay (BCA): To measure the protein concentration in blood serum, Thermo Fisher's Pierce TMThe bicinchoninic acid assay (BCA) was performed using the BCA protein assay kit (#23227) according to the manufacturer's instructions. Briefly, plasma was diluted 1:100 with STET (50mM Tris, pH 7.5, 150mM NaCl, 2mM EDTA, 1% NP40) lysis buffer. Bovine serum albumin was serially diluted to create a standard curve for 0–2 mg / mL. Standards and samples were incubated in kit reagents A and B at 37°C for 30 minutes. Absorbance was measured at 562 nm using a Tecan Infinite M200 Nanoquant.

[0147] Plasma protein precipitation: To quantify protein precipitation using the BCA assay, plasma was exposed to various stimuli known to induce protein precipitation. All samples were diluted in the same manner before inducing precipitation. 110 µl of 0.57% NaCl was added to 26 µl of K2-EDTA plasma and incubated in a 1.5 mL Eppendorf tube at 37°C for 30 minutes. For heat-induced protein precipitation, the sample was incubated at 70°C for 1 minute. For ketone and alcohol-induced precipitation, 80 µl of 50% ice-cold acetone or ethanol was added and incubated at 4°C for 5 minutes. For salt-induced precipitation, 80 µl of 35% ammonium sulfate was added at room temperature for 5 minutes. For acid-induced precipitation, 80 µl of 0.4% acetic acid dissolved in 0.81% NaCl was added at 37°C for 5 minutes. After incubation with the precipitating agent, the sample was immediately centrifuged at 21.000xg at 4°C for 1 minute. The supernatant was transferred to a new 1.5 mL Eppendorf tube. The supernatant was immediately diluted 1:200 with STET buffer and stored at 4°C to limit precipitation. The protein pellet was resuspended in STET buffer containing 1% SDS and sonicated until completely dissolved. 5 mM NaOH was added to STET buffer to reverse acid precipitation. Protein concentrations were determined using the BCA assay as described.

[0148] Measurement of pH and osmotic pressure of blood samples. Orion TMThe pH of K2-EDTA plasma samples was measured at room temperature (21°C) using a Seven pH-meter (Mettler Toledo) equipped with a micro-pH electrode (9810BN, Thermo Fisher). The osmolality of the plasma was measured using a VAPRO 5600 pressure osmometer according to the supplier's manual.

[0149] UV / VIS spectrophotometer assay for quantifying protein precipitates: Indiko TM Protein precipitation was quantified using a Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). A seven-step protocol was programmed using Indiko's default software (see Figure 2B). Briefly, plasma samples were diluted with approximately 0.57% (w / v) NaCl to a final volume of 136 ul and an NaCl concentration of approximately 0.63%. The samples were then incubated at 37°C for 5 minutes in a cuvette with a path length of 0.7 cm. The absorbance of the diluted sample at 340 nm was measured and used as the blank value. Subsequently, 80 ul of 0.4% (v / v) acetic acid containing 0.81% (w / v) NaCl was added to the diluted sample. The absorbance at 340 nm was measured kinetically once, for example, over 1 hour (see Figure 4), or once after the time required for precipitation (e.g., 300 seconds as illustrated here). The blank value was subtracted as background. The changes in the concentrations of acetic acid and salt are shown in the legend of each figure.

[0150] In experiments using reagents intended to stop the precipitation reaction with acid, NaOH was added to the reaction 60 seconds after adding 0.4% acetic acid or 50% acetone.

[0151] statistics: The statistical tests used in the analysis are shown in the legend of each figure. Statistics were calculated using GraphPad 9 software. For threshold optimization (see Figure 7), data processing, data visualization, and statistical analysis were performed using Python 3.8 with pandas 1.1.4, matplotlib 3.3.4, seaborn 0.11, scipy 1.6.2, and statsmodels 0.12.2.

[0152] Optimization of evaluation metrics and thresholds: To evaluate the quality of this assay, five performance metrics were used, defined as follows: Sensitivity (SEN), Specificity (SPE), Accuracy (ACC), Balanced Accuracy (bACC), and F1 score (F1): SEN = TP / (TP + FN) SPE = TN / (TN + FP) ACC = (TP + TN) / (TP + TN + FP + FN) bACC=(SEN+SPC) / 2=(TP / (TP+FN)+TN / (TN+FP)) / 2 F1 = 2TP / (2TP + FP + FN) Here, TP, FP, TN, and FN represent the number of true positives, false positives, true negatives, and false negatives, respectively. Sensitivity and specificity are also called the true positive rate (TPR) and true negative rate (TNR), respectively.

[0153] Apply the area under the curve (AUC) to the subtracted value S of the test sample (i.e., the value obtained by subtracting the measured UV quenching from the blank value), and set S to the set of subtracted values ​​(AUC) of the positive / pathological reference group P. P (as indicated) was compared with AUC. P It was calculated as follows:

number

number

[0154] Alternatively, AUC can be applied to compare S against a set of subtracted values ​​from a non-pathological reference group N, and AUC N It is written as follows and calculated as follows:

number

[0155] Example 2: Results and Discussion Because protein conformation is crucial for the biological function of proteins, different conformational states of proteins are characteristic of many pathological conditions. Therefore, protein stability (the ability to maintain conformation under given conditions) is extremely important. There are various methods for experimentally investigating and quantifying protein stability, but the most common method utilizes the fact that changes in conformation are accompanied by changes in protein solubility. However, while conformation is mainly determined by primary, secondary, tertiary, and quaternary structures, protein conformation is also affected by environmental factors such as pH, temperature, and the presence of ligands and other molecules. Since pathological conditions have been found to be reflected in changes in plasma composition (Geyer, PE et al., Cell systems (2016) 2:185-195), this study investigates whether changes in plasma composition can affect the solubility of plasma proteins.

[0156] To verify this, K2-EDTA plasma from four individuals with no reported pathological conditions and diseased samples from five individuals were exposed to conditions known to affect protein solubility (Figure 1). The precipitate was collected using a bicinchoninate assay (1 min, 14.000 x g), and the amount of insoluble protein was quantified. As shown in Figure 1A, the presence of acetic acid (~0.15%) resulted in approximately 2% of the sample becoming insoluble in the diseased samples, while only about 1.5% precipitated in the non-pathological samples. As a result, less protein remained in the supernatant in the diseased samples than in the non-pathological samples, but the detected difference was not statistically significant, which is thought to be due to the specific assay conditions applied in the BCA assay.

[0157] Similar results were observed when other protein precipitation strategies were used (Figures 1B-E). When using heat to denature proteins, exposure to 70°C for 1 minute resulted in precipitation of approximately 7% from pathological samples, but less in healthy samples (~4%, Figure 1B). Similar differences were observed when precipitation was induced using ethanol, ammonium sulfate, and acetone (Figures 1C, E). Interestingly, a fairly large portion of plasma proteins (up to 30%) were affected in the presence of ethanol or ammonium sulfate (Figures 1C, E), while less than 10% of total proteins were affected by acetone (Figure 1D). It is noteworthy that only a portion of the proteome was affected under each precipitation condition. However, it was surprising that acetic acid, in particular, affected only about 1-2% of total proteins at a concentration of about 0.15%, and that a small number of samples were already statistically significant enough to distinguish between pathological and non-pathological samples. Because plasma protein concentrations vary from person to person, the precipitated protein was normalized relative to the total protein amount before precipitation was induced (Figures 1 and 9).

[0158] From these results, we concluded that the presence of a pathological condition alters protein stability, and that the difference in protein precipitation could potentially be used to distinguish between samples taken from individuals with a pathological condition and those without. Furthermore, we considered that the ability to distinguish samples with specific pathological conditions under precipitation conditions optimized to affect only a small portion of the plasma proteome could be an interesting diagnostic approach. Therefore, we decided to investigate protein precipitation under weakly acidic conditions using acetic acid as a denaturing agent.

[0159] Next, we began to further characterize and optimize the protein precipitation using acetic acid. A common method for observing protein precipitation is to measure the absorbance at 340 nm. For this purpose, Indiko TM An assay specifically tracking protein precipitation was set up using the Plus Clinical Chemistry Analyzer (Thermo Fisher) (Figures 2A, B). Serial dilutions of lyophilized plasma were performed to determine the optimal plasma protein concentration required for detection of protein precipitation at 340 nm. As shown in Figure 2C, adding 0.4% (v / v) acetic acid to pre-diluted plasma samples for 5 minutes established a linear relationship between absorbance and input protein. The assay showed almost perfect linearity in the input protein range of 100–4500 μg.

[0160] After confirming that plasma from a pathological background exposed to extreme stimuli is more prone to precipitation than plasma from a non-pathological background, and that this assay can elicit a robust and linear effect on plasma proteins, we sought to investigate the major determinants of acetic acid-induced precipitation. Acetic acid-induced protein precipitation is known to be affected by its concentration, pH, ionic strength, and temperature. First, the concentration of acetic acid added to the undiluted protein sample was gradually increased over 5 minutes at 37°C. As expected, protein precipitation increased with increasing acetic acid levels (Figure 3A). However, it was surprising that almost no precipitation was observed at concentrations below 0.2%. At high concentrations (1.6%), protein precipitation was significant, but it was no longer possible to distinguish between the plasma origin (pathological or non-pathological). The most significant difference was observed at 0.4%, corresponding to a final concentration of 0.15% (Figure 3A). After determining the optimal concentration of acetic acid, we investigated whether this was induced by the acidic environment or by a specific effect induced by acetic acid itself. A strong acid, hydrochloric acid (HCl), was used to trigger precipitation. Surprisingly, even when HCl was added at pH 1.5, no strong precipitation was induced. This is good evidence that pH is not the only factor determining plasma-derived protein precipitation. It is known that increasing salt concentration lowers the pH of an aqueous solution. Therefore, it was expected that precipitation would increase as the amount of salt increased. Unexpectedly, precipitation of pathological and non-pathological plasma proteins decreased with moderate amounts of sodium chloride, but increased strongly with no added salt or in the presence of very large amounts of salt (Figure 3C). It is tempting to speculate that a so-called "salting-out" effect was observed, where sodium chloride enhances protein stability at low concentrations, as is known with Hofmeister series salts, which are neither chaotropic nor cosmotropic (Gregory, KP et al., Physical chemistry chemical physics: PCCP (2022).24, 12682-12718), as is known with sodium chloride, etc. Following this line of reasoning, at high concentrations (9% in this case), salts "salt out" proteins, and this, combined with a decrease in pH, induces protein precipitation.Interestingly, physiological NaCl concentrations (~0.9%) had far less effect on the precipitation behavior of healthy and pathological samples than low or high concentrations. We concluded that specific precipitation conditions are necessary to distinguish plasma in a source-dependent manner. Optimal conditions can be achieved by manipulating the concentrations of acid, salt, and pH, which are the main determinants of acetate-induced protein precipitation. It is noteworthy that protein solubility may also be influenced by the body's electrolyte-water balance, which is interconnected with salt concentration and blood pH (Auton, M. et al. (2011). Biophysical chemistry 159, 90-99). The normal reference range for human plasma osmolality is 275-300 mOsm / kg. To investigate whether the structural instability of plasma-derived proteins stems from pH changes or from the body's endogenous water balance, we experimentally measured the pH and osmolality of K2-EDTA plasma, but found no significant differences (Figure 9B, C).

[0161] Next, we wanted to kinetically map the precipitation reaction because protein precipitation starts at specific hotspots but quickly spreads to the surrounding area, precipitating nearby, unaffected proteins as well. From this, we hypothesized that the specific precipitation behavior of plasma could be explained by the presence of tiny protein aggregates, so-called "seeds," that could act as hubs for protein precipitation. To answer this question, we conducted a 1-hour time-course experiment and found that both pathological and non-pathological samples exhibited an acetic acid reaction that rapidly increased for the first 10 minutes, then flattened out and remained almost completely stable (Figure 4). Surprisingly, the rate and extent of the reaction differed significantly between pathological and non-pathological samples. Importantly, we did not observe any increase in baseline absorbance at 340 nm before the addition of acetic acid (Figure 9D). This suggests that the observed differences are not due to the presence of aggregate species that are present in pathological samples but absent or very infrequent in non-pathological samples. To verify this idea, we wanted to investigate whether acetic acid transiently insolubilizes proteins or causes irreversible denaturation and subsequent aggregation. Indeed, acetic acid is known to transiently precipitate proteins when used at low concentrations. Therefore, if there are aggregation species that need to be induced by the denaturant, the reaction is expected to proceed self-initiated as the level of aggregated protein increases. Conversely, if this phenomenon is not due to a self-initiated chain reaction by pre-existing species, the reaction should proceed reversibly upon neutralization of the acetic acid. To verify this, we added NaOH to stop the precipitation reaction (Figure 5A). We utilized the fact that when the pH becomes neutral, acetic acid loses its acidic properties because most of its protons dissociate. As expected, the precipitation was completely reversed by pH neutralization, but the precipitation with acetone was not reversed (Figures 5B-E). This indicates that acetic acid induces a chain reaction dependent on its acidic form. We concluded that endogenous structural instability, rather than pre-existing aggregation species, is the reason for this effect.However, since plasma is a complex mixture of thousands of components, we considered whether the structural instability was due to endogenous or exogenous protein phenomena. To verify this, we gently heated the plasma (50°C for 5 minutes) before exposing it to acetic acid. Surprisingly, the pathological sample did not change its precipitation behavior, while the non-pathological sample precipitated similarly. This suggests that the conformation of the pathological protein is altered, making it more prone to precipitation. To further substantiate the idea that the observed effect is dependent on protein conformation, we replaced the plasma matrix with 0.9% NaCl by multi-step centrifugation through a 3kDa cutoff column. The precipitation behavior remained unchanged under all conditions (Figure 6C,D). Most importantly, the difference between pathological and non-pathological precipitation was conserved, indicating that the observed phenomenon is due to endogenous protein effects affecting the structural integrity of plasma proteins.

[0162] After developing assays and their underlying mechanisms for distinguishing between pathological and non-pathological samples, we chose to significantly increase the sample size to establish thresholds for distinguishing between pathological and non-pathological precipitation behavior.

[0163] To optimize the clinical performance of this assay, we evaluated the measured UV quenching, the value obtained by subtracting this quenching from the blank value (hereinafter referred to as the "subtracted value"), and the ratio of quenching to the blank value. Assuming a positive / pathological reference group P (n=114) and a negative / non-pathological reference group N (n=241), we calculated ROC (Receiver Operating Characteristic) curves (Sciences & Kumar, 2011) for these three measurements (Figure 7A). The ROC curves indicate that the subtraction (AUC=0.999) takes precedence over the ratio (AUC=0.997) and quenching (AUC=0.948). Next, we defined the clinical reference range (Haeggstroem, 2014) of this measurement method using the subtracted value of the non-pathological reference group N (mean = 42.3 ± 24.5). Therefore, we removed two outliers from N that were 3 standard deviations (SD) higher than the mean subtracted value. As a result, 228 out of 241 non-pathological samples fell within this range (95% required for clinical testing), and the reference range was defined as the mean ± 2SD (0 to 84.7; n=239, mean = 41.3 ± 21.7) excluding outliers. The reference range is shown in Figure 7B.

[0164] Our objective was to evaluate whether a subject was diagnosed with cancer and / or acute inflammatory disease (hereinafter referred to as "diagnosed as pathological") based on the subtraction values ​​S, P, and N for the subject, pathological reference group, and non-pathological reference group, respectively. Therefore, we optimized the subtraction threshold using the Euden exponent J (Fluss, Faraggi, & Reiser, 2005; Schisterman, Perkins, Liu, & Bondell, 2005). Given the sensitivity (SEN, true positive rate) and specificity (SPE, true negative rate) of the test, the Euden exponent is defined as J = SEN + SEP - 1 (see Example 1, Materials and Methods). We calculated J over a range of subtraction thresholds and obtained an optimal threshold of 134 with a maximum J = 0.99, as shown in the ROC curve (Figure 7B). These values ​​are within the range of the optimal specificity threshold (105) and the optimal sensitivity threshold (134), and are defined as the highest value (no outliers) observed at N and the lowest value observed at P, respectively (Figure 7C). As a result, newly tested subjects are diagnosed as positive if S > x, and x is in the subtraction range of 105 to 134.

[0165] This threshold depends on the measurement values ​​of the reference group and changes when the assay setting is modified, so we tested three threshold-less approaches (Yuan, Su, & Zhu, 2015).

[0166] First, we attempted a Bayesian statistical approach called the likelihood ratio (LR) (Fierz & Bossuyt, 2021), which represents the probability of a test result being positive or negative. However, the LR is merely a general estimate of the probability that a subject will be diagnosed as pathological or non-pathological, and does not take into account the actual readout of the test result. Therefore, it is not suitable for direct application to evaluate the assay readout of a subject.

[0167] Secondly, as a simple alternative, we used the Wilcoxon signed-rank test to determine whether S (i.e., the subtraction value measured for the subject) was significantly greater than N (i.e., the subtraction value for the non-pathological reference group or healthy control group). Using a p-value threshold >= 0.05, we calculated the number of subjects misclassified from the two reference groups (n=355) and analyzed the applicability of this strategy, finding 110 misclassifications (Figure 7D). Since the p-value depends on the underlying reference group, we used a subset of N (given as the percentage of non-pathological subjects with the highest subtraction value) with different commonly used p-value thresholds (0.05, 0.01, 0.001, 10). -4 , 10 -5 , 10 -6 , 10 -7 We tested this in combination with [another method]. Our optimization showed that using only 5% of the total reference group and setting the p-value threshold to 0.01 or 0.001 resulted in only two false positives or a balanced accuracy of 99.6% (Figure 7E). While this demonstrates near-perfect discriminative power, the drawbacks of this approach are that the p-value threshold is replaced by the subtraction threshold, and the size of the reference group must be further optimized, which also depends on the assay setup. For this reason, we determined that this approach is not feasible as a general performance measure for our assay.

[0168] Thirdly, to overcome the potential problems of classical statistical approaches such as Wilcoxon's signed-rank test, we adopted an unconventional alternative by using the area under the curve (AUC) (Mason & Graham, 2002; Yuan et al., 2015) to assess whether S is different from the reference group. To avoid confusion with the AUC used above in relation to the optimization of the subtraction threshold, the AUC of this single-value comparison is defined as the AUC used depending on whether P or N is used as the reference group. P or AUC N This is what we will do (see Example 1). Below, we will discuss AUC. P I will explain this, but AUC N AUC works similarly. P The value ranges from 0 to 1, representing the extreme cases where all subtracted values ​​of P are higher or lower than S, respectively. AUC of 0.5 P This reflects the fact that 50% of the subtracted values ​​of P are higher than S, and 50% are lower than S. AUC P The results are consistent with non-parametric tests such as Wilcoxon's signed-rank test (Mason & Graham, 2002). Furthermore, they are easier to interpret and more robust because they vary between 0 and 1 regardless of assay settings. Therefore, newly tested subjects are more robust in terms of AUC. P A pathological diagnosis is made when the AUC exceeds a certain threshold. P To optimize the threshold, the entire set P(n=114) is used as the pathological reference group, and the AUC is calculated for each value of P and N. P Calculate different AUCs P Various performance indicators, such as accuracy, were calculated using thresholds (see Example 1, Materials and Methods). Similarly, AUC was calculated using the entire set N (n=241). N The threshold was optimized. AUC P Optimizing (Figure 7F, G) yields similar results to our statistical approach (Figure 7D, E), and theoretically optimal AUC. P I supported a threshold of 0.

[0169] As a result, the number of new test subjects was AUC P>0, that is, when the subtracted value is within the distribution of the subtracted values of the diseased reference group, it is suggested that it is diagnosed as diseased. Similarly, the optimization of AUC N (Figs. 7H - I) suggests an optimal threshold of 1. Therefore, the newly tested subject is diagnosed as diseased with AUC N = 1. This means that its subtracted value is greater than all the subtracted values of the non - diseased reference group. However, our data also show that even with less stringent thresholds of AUC P (>=0.77) and AUC N (>=0.64), it is sufficient to achieve an accuracy of 75%. The values of AUC P and AUC N of both reference groups are shown in Table 1. For a detailed overview of the threshold optimization results regarding origin, AUC P , AUC N , refer to Tables 2A, B, C.

[0170] Determination of the minimum required size of the diseased reference group and non - diseased reference group Finally, using AUC P or AUC N , the minimum size required for the reference group to achieve good performance was evaluated. Therefore, for the diseased and non - diseased reference groups, a training - test set split was performed by randomly selecting 50 subtracted values each. These 100 values were used as the training set, and the remaining 255 values were used for testing (64 from P and 191 from N). The n values of the diseased and non - diseased training sets were randomly sampled and increased from n = 1 to n = 50. For each n, the training subset of the subtracted values was used as the reference group, and AUC N , AUC P were calculated for all samples of the test set. Our proposed thresholds (AUC P >0, AUC NBy using (1), different predictive indicators were evaluated as before. To ensure statistically robust results, random sampling was repeated 10 times for each n, and the results were averaged. Also, the training and test set splits were repeated 10 times, and the average values including the standard deviation for each n were calculated and used to determine the required minimum sample size. As a result, AUC N and AUC P It was shown that a minimum sample size of 2 is required for each of the non-diseased reference group and the diseased reference group to achieve 75% accuracy using (Figure 7J). To achieve 95% accuracy, a minimum sample size of 4 is sufficient for the diseased reference group, and a minimum sample size of 22 is required for the non-diseased reference group (Figure 7K). For details, refer to Tables 3A - D.

[0171] Based on our results, we recommend the following applications: The subject is diagnosed as having a high risk of suffering from cancer and / or acute inflammatory diseases in the following cases: · The measured value S of the test sample for each subject satisfies AUC P (S, P)>x, where AUC P is defined in the materials and methods, P is all the measured values of the diseased reference group having at least two reference samples, and x is the AUC P threshold within the range from 0 to 0.77. · The measured value S of the test sample for each subject satisfies AUC N (S, N)>=x, where AUC N is defined in the materials and methods, N is all the measured values of the non-diseased reference group having at least two reference samples, and x is the AUC N threshold of 0.64 or more.

[0172] It is noted that generally, the reference range and the optimized threshold depend on the assay setup and the reference groups of diseased and non-diseased specimens. Therefore, it needs to be adjusted before clinical application. In contrast, AUC P without a threshold and AUC NBecause it is independent of assay readout, it can be interpreted across different assay platforms (Fierz & Bossuyt, 2021).

[0173] Having established a threshold for distinguishing between pathological and non-pathological samples, we aimed to utilize this tool to streamline the processing of pathological samples. For this purpose, plasma samples were obtained from individuals with defined medical conditions, and their precipitation behavior was measured (Figure 8). Precipitation with acetic acid proved to be a strong indicator of distinguishing between samples from malignant tumor patients and non-pathological samples without reported medical conditions. Notably, samples from non-cancerous (e.g., bronchitis) or benign (e.g., sarcoidosis) diseases showed similar results to non-pathological samples from subjects generally considered healthy.

[0174] Based on the above, we established a protocol that utilizes the ability to selectively precipitate plasma proteins under weakly acidic conditions. This phenomenon can be used to investigate the structural stability of plasma-derived proteins and enables identification based on the origin of the plasma sample. Most importantly, our protocol successfully detected 27 types of cancer. The results disclosed in this invention demonstrate that differential protein precipitation can be used as a highly sensitive tool for the detection of not only acute inflammatory diseases but also pan-cancer.

[0175] Example 3: Further testing of precipitation-inducing conditions As shown in Figures 2A, B, and C, Indiko TM Using the Plus Clinical Chemistry Analyzer (Thermo Fisher), we were able to set up an assay that specifically tracks protein precipitation. Next, various acids, alcohols, and salts were used as precipitating agents in step 6 (Figure 2B). As shown in Figure 10.1, citric acid is suitable for inducing distinguishable protein precipitation when comparing healthy individuals with cancer patients. This also explains the dilution range for formic acid and perchloric acid (see Figures 10.2 and 10.3). To determine if alcohols could be used as precipitating agents, a dilution series of ethanol, isopropanol, and methanol was tested in Step 6. As shown in Figures 11.1-11.3, each alcohol tested was also suitable for inducing protein precipitation in a manner that was distinguishable when comparing healthy samples with cancer samples. • In step 6, this procedure was repeated with various salts to induce protein precipitation. CaCl2, guanidine thiocyanate, and MnCl2 were used as “salting-out” agents (see Figures 12.1-12.3). In conclusion, it is preferable to use a different precipitation method instead of the standard protocol of applying acetic acid in physiological saline. In the context of this invention, the term “physiological saline” is also referred to as “0.9% NaCl aq.” or “0.9% physiological saline,” and refers to an aqueous solution containing 0.9% (w / v) NaCl. However, it is preferable to optimize the threshold for each compound.

[0176] Figures 10-12 show both 5-minute and 10-minute endpoint measurements. Dynamic measurements consisting of 12 measurements spaced 54 seconds apart are provided in conjunction with the endpoint measurements described above.

[0177] BCA (Figure 1) and Indiko TM Since the Plus analyzer (Figure 2C) demonstrated its suitability for quantifying precipitates, we wanted to show that this is possible with another standard protein quantification procedure. As shown in Figure 13, precipitates (starting with the standard method) and corresponding plasma from three patients each (healthy vs. cancer) were diluted with gel loading dye before performing SDS-PAGE. The gel was then stained with Coomassie Brilliant Blue. Total protein content (meaning each lane) was quantified by densitometry analysis using ImageJ software. As shown in Figure 13, there is a significant difference between the healthy and cancer samples.

[0178] Example 4: Reversibility of precipitation Furthermore, we were able to demonstrate that acetic acid in the presence of 0.9% (w / v) NaCl induces reversible precipitation of plasma proteins (Figure 14). The assay setup workflow is shown in Figure 5A, but was extended by an additional step 10. In contrast to the assay (see Figure 5A), acetic acid was present for 5 minutes before further manipulating the reaction by adding a dilution series of NaOH or vehicle (dH2O) (step 7). Adding 17 mM and 12 mM NaOH, respectively, reversed the reaction (similar results are shown in Figure 5B). Adding 7.5 mM NaOH stopped the reaction but did not reverse it. Samples whose reactions were stopped with 17 mM and 12 mM NaOH were further manipulated in step 10 by adding 4% acetic acid or vehicle (dH2O). The amount of acetic acid added was selected so that the final concentration of acetic acid in the reaction mixture was approximately 0.15% v / v. Precipitation began again (SN1AA and SN2AA). This demonstrates the reversibility of this reaction.

[0179] Example 5: Chemical Sensitivity Table 4 below shows that the carcimun levels obtained by the method described in this invention correlate with the clinical classification of cancer during chemotherapy. Tumors in remission and stable stages showed significantly lower carcimun levels compared to tumors in advanced and rapidly progressing stages.

[0180] [Table 1] JPEG2026509792000013.jpg92170

[0181] Example 6: Miniaturization of the CalcimunTest To demonstrate that the calcium simulator test can be miniaturized, the standard protocol for the calcium simulator test (Figure 2b) was performed using samples from healthy individuals (N=3) and cancer patients (N=3). Readouts were performed using a NanoDrop spectrometer (NanoQuant infinite M200pro, Tecan Trading AG, Switzerland) with 1 μl of reaction sample. As shown in Figure 16a, the NanoDrop readouts measured at 340 nm showed a significant difference between samples from healthy individuals and those from cancer patients. A table of the dilution series is shown in Figure 15. Next, to demonstrate that the volume of the reaction mixture can be reduced, a fourth dilution (Figure 15) was performed (the concentration and step were the same, but the total reaction volume was smaller). For this purpose, two cancer samples were compared to healthy controls. As shown in Figure 16b, the test remains robust even when the total reaction volume is reduced by several times.

[0182] These results highlight the possibility of scaling down the test protocol shown in Figure 2b. Test assay readout can be performed using a NanoDrop spectrometer, with the only limiting factor being the pipetteable volume of each compound at each step, which can be overcome by an automated system.

[0183] Additional References Fierz, W., & Bossuyt, X. (2021). Likelihood Ratio Approach and Clinical Interpretation of Laboratory Tests. Frontiers in Immunology, 12(April), 1-5. https: / / doi.org / 10.3389 / fimmu.2021.655262 Fluss, R., Faraggi, D., & Reiser, B. (2005). Estimation of the Youden Index and its associated cutoff point. Biometrical Journal, 47(4), 458-472. https: / / doi.org / 10.1002 / bimj.200410135 Haeggstroem, M. (2014). Establishment and clinical use of reference ranges. WikiJournal of Medicine, 1(1), 1-7. https: / / doi.org / 10.15347 / wjm / 2014.003 Mason, S. J., & Graham, N. E. (2002). Areas beneath the relative operating characteristics (ROC) and relative operating levels (ROL) curves. Quarterly Journal of the Royal Meteorological Society, 128, 2145-2166. Schisterman, E. F., Perkins, N. J., Liu, A., & Bondell, H. (2005). Optimal cut-point and its corresponding Youden index to discriminate individuals using pooled blood samples. Epidemiology, 16(1), 73-81. https: / / doi.org / 10.1097 / 01.ede.0000147512.81966.ba Sciences, M., & Kumar, R. (2011). Receiver Operating Characteristic (ROC) Curve for Medical Researchers. Encyclopedia of Machine Learning and Data Mining, 1116-1116. https: / / doi.org / 10.1007 / 978-1-4899-7687-1_735 Yuan, Y., Su, W., & Zhu, M. (2015). Threshold-Free Measures for Assessing the Performance of Medical Screening Tests. Frontiers in Public Health, 3(April). https: / / doi.org / 10.3389 / fpubh.2015.00057

[0184]

Table 2

[0185]

Table 3

[0186] [Table 4] JPEG2026509792000030.jpg254170JPEG2026509792000031.jpg122170

[0187] [Table 5] JPEG2026509792000033.jpg254170JPEG2026509792000034.jpg122170

[0188] [Table 6] JPEG2026509792000036.jpg63170

[0189] [Table 7] JPEG2026509792000038.jpg64170

[0190] [Table 8] JPEG2026509792000040.jpg64170

[0191] [Table 9] JPEG2026509792000042.jpg64170

[0192] It is understood that the definitions and embodiments described above in relation to the first aspect of the present invention also apply, as far as possible, to the second, third, fourth, fifth, and sixth aspects of the present invention.

[0193] In the present invention, for the purpose of exemplarily explaining preferred embodiments of the present invention, the present invention will be described only exemplarily with reference to the accompanying drawings.

[0194] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In case of conflict, the patent specification including the definitions shall prevail.

[0195] Regarding the embodiments characterized in this specification, particularly in the claims, each embodiment described in a dependent claim is intended to be combined with each embodiment of each claim (independent claim or dependent claim) to which the said dependent claim depends. For example, in the case of independent claim 1 describing three options A, B, and C, dependent claim 2 describing three options D, E, and F, and claim 3 depending on claims 1 and 2 and describing three options G, H, and I, this specification is to be understood as clearly disclosing the embodiments corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless otherwise specified.

[0196] Similarly, even if an independent claim and / or dependent claim does not describe an alternative, if the dependent claim refers to multiple prior claims, it is understood that any combination of subject matter covered by it is deemed to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to both claim 2 and 1, the combination of subject matter of claim 3 and 1 is explicitly and uniquely disclosed, as is the combination of subject matter of claim 3, 2, and 1. If there is further dependent claim 4 referring to any one of claims 1 through 3, the combinations of subject matter of claim 4 and claim 1, subject matter of claim 4, claim 2, and claim 1, subject matter of claim 4, claim 3, and claim 1, and subject matter of claim 4, claim 3, claim 2, and claim 1 are explicitly and uniquely disclosed.

[0197] The above considerations apply to all attached claims.

[0198] Each patent, patent application, publication, and document referenced herein is incorporated by reference. The above references to patents, patent applications, publications, and documents do not constitute an endorsement of any of them as appropriate prior art, nor do they endorse the content or date of any of these publications or documents. The above references do not constitute a search for relevant disclosures. All statements regarding the date or content of documents are based on available information and do not constitute an endorsement of their accuracy or correctness.

[0199] Modifications to the foregoing can be made without departing from the basic aspects of the present technology. Although the present technology has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to the embodiments specifically disclosed herein, but such modifications and improvements will remain within the scope and spirit of the present technology.

[0200] The techniques described exemplary in this invention can preferably be implemented even when there are no elements not specifically disclosed herein. Therefore, for example, in each example herein, any of the words “including,” “essentially consisting of,” and “consisting of” can be replaced with any of the other two terms. The terms and expressions used are for illustrative purposes only, not limitation, and the use of such terms and expressions does not preclude equivalents of the exhibited and described features or parts thereof, and various modifications are possible within the scope of the claimed technique. In this specification, the terms “method” and “process” are used interchangeably.

[0201] The words "a" or "an" can refer to one or more of the elements they modify (for example, "a cell" can mean "one or more cells"). The word "about" as used in this invention refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and when the word "about" is used at the beginning of a string of values, each value is modified (i.e., "about 1, 2, 3" refers to about 1, about 2, and about 3). For example, a weight of "about 100 grams" can include weights between 90 grams and 110 grams. Furthermore, if a list of values ​​is provided herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), that list includes all intermediate and fractional values ​​(e.g., 54%, 85.4%). Thus, although this art is specifically disclosed by representative embodiments and optional features, those skilled in the art should understand that modifications and variations of the concepts disclosed herein can be relied upon, and such modifications and variations are considered within the scope of this art.

[0202] Specific embodiments of this technology are described in the following claims.

Claims

1. A method for diagnosing cancer and / or acute inflammatory disease in a subject, including the following: (a) Subjecting the sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample in relation to the following: (b-i) Levels of precipitated (poly)peptides in at least one correspondingly processed reference sample obtained from one or more reference subjects known to be positive for cancer and / or acute inflammatory disease; (b-ii) Levels of precipitated (poly)peptides in at least one correspondingly processed reference sample obtained from one or more reference subjects known to be negative for cancer and acute inflammatory disease; and / or A predetermined standard determined based on (b-iii)(b-i) and / or (b-ii); Here, the subjects are those diagnosed with a positive result for cancer and / or acute inflammatory disease in the following cases: The level of precipitated (poly)peptide in the sample is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, compared to the level of precipitated (poly)peptide in reference sample (b-i) or to a predetermined standard, and is preferably higher, most preferably at least 100%; and / or The level of precipitated (poly)peptides in the sample is higher than the level of precipitated (poly)peptides in the reference sample (b-ii), or, more preferably, at least 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, and most preferably at least 30%, compared to the respective specified standards.

2. A method for diagnosing cancer and / or acute inflammatory disease in a subject, including the following: (a) Subjecting a sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample, the conditions for inducing precipitation are provided as follows: (i) Adjust the pH of the sample to a value within the range of pH 2 and pH 6, pH 3 and pH 5.5, pH 3.4 and pH 5, pH 3.6 and pH 4.8, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, and pH 4.1 and pH 4.3, with the greater the value, the more preferable, and most preferably within the range of pH 4.2 ± 0.05; Preferably, this is done by adding an acid or an aqueous solution containing an acid, where the acid has a pKa in the range of 3.0 to 7.0, 3.5 to 6.5, 3.8 to 5.8, and 4.1 to 5.5, with higher values ​​being preferable, and most preferably having a pKa of 4.75 ± 0.05; where preferably, the acid is an organic acid, and the organic acid is preferably a carboxylic acid, more preferably a monocarboxylic acid, and most preferably acetic acid; (ii) Increase in sample temperature; (iii) Addition of at least one salting agent; (iv) Addition of one or more aliphatic alcohols, preferably alkanols, most preferably ethanol; and / or (vi) Addition of a ketone, preferably acetone; and (b) Determine the fraction of precipitated (poly)peptides from the total amount of (poly)peptides in the sample by the following: - When precipitation is induced by a decrease in the pH of the sample, the determined fraction of the precipitated (poly)peptide is preferably greater than 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the total amount of (poly)peptides in the sample, and more preferably greater than 2.1%; - When precipitation is induced by an increase in sample temperature, the determined fraction of precipitated (poly)peptides is preferably greater than 5%, 5.5%, 6%, or 6.5% of the total amount of (poly)peptides in the sample, and more preferably greater than 7%; - When precipitation is induced by the addition of at least one salting-out agent, the determined fraction of the precipitated (poly)peptide is preferably greater than 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the total amount of (poly)peptides in the sample, and more preferably greater than 2.1%; - If precipitation is induced by the addition of ethanol and / or other aliphatic alcohols, the determined fraction of the precipitated (poly)peptide is more preferably 4%, 6%, 8%, 10%, 11%, and more preferably 12% of the total amount of (poly)peptides in the sample; and / or - When precipitation is induced by the addition of ketones, the determined fraction of precipitated (poly)peptides is preferably greater than 1%, 2%, 3%, or 4%, and more preferably greater than 5%, of the total amount of (poly)peptides in the sample; The subjects are methods diagnosed as positive for cancer and / or acute inflammatory diseases.

3. A method for evaluating the response of cancer and / or acute inflammatory diseases to a candidate treatment in a subject, including the following: (a) Subject a sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample, wherein the sample is obtained after the candidate treatment has been initiated; (b) Evaluate the level of precipitated (poly)peptides in the sample relative to the level of precipitated (poly)peptides in a reference sample obtained from the subject before the sample in step (a) was obtained from the subject, wherein the reference sample is subjected to the corresponding precipitation-inducing conditions as in step (a); - If the level of precipitated (poly)peptides in the sample is equal to or lower than the level of precipitated (poly)peptides in the reference sample, cancer and / or acute inflammatory disease is classified as responsive to candidate treatment; and / or - A method by which cancer and / or acute inflammatory disease is classified as unresponsive to candidate treatment if the level of precipitated (poly)peptides in the sample exceeds the level of precipitated (poly)peptides in the reference sample.

4. A method for evaluating the malignancy of cancer in a subject, including the following: (a) Subjecting the sample obtained from the subject to conditions that induce precipitation of one or more (poly)peptides contained in the sample; and (b) Evaluate the level of precipitated (poly)peptides in the sample in relation to the following: (i) at least one corresponding processed reference sample obtained from one or more reference subjects having cancer of known grade; and / or (ii)(b)A predetermined standard arbitrarily obtained based on one or more reference samples in accordance with (ii)(i); Herein, a method in which a level of precipitate in a sample that is high, low, or substantially the same as the level of precipitate in a reference sample indicates that the cancer in the subject has a degree of malignancy that is high, low, or substantially the same as a reference cancer or a given standard, respectively.

5. In step (b), the level of precipitated (poly)peptide in the sample is evaluated based on a relative statistical comparison with the level of precipitated (poly)peptide in a group of correspondingly treated reference samples, where each reference sample is obtained from a reference subject known to be positive for cancer and / or acute inflammatory disease (pathological reference sample) and / or relative to the level of precipitated (poly)peptide in a group of correspondingly treated reference samples, where each reference sample is obtained from a reference subject known to be negative for cancer and acute inflammatory disease (non-pathological reference sample), where the statistical comparison is performed as follows: (a) Calculation of area under the curve (AUC); where preferably: (a-1) The level of precipitated (poly)peptide in sample (S) is defined by the AUC of formula (I). Ｐ By calculating the value, it is compared to the level of precipitated (poly)peptides in the group of pathological reference samples (P): [Math 1] In the formula, n Ｐ x is the number of reference samples in the group of pathological reference samples P, and x ｉ is the level of precipitated (poly)peptide for the i-th pathological reference sample in P, and k is x as defined by formula (II). ｉ This is an index function for comparing S with respect to: [Math 2] Regarding S, AUC Ｐ If the value is calculated within the range of >0.75 to 1.0, >0.7 to 1.0, >0.6 to 1.0, >0.5 to 1.0, >0.4 to 1.0, >0.3 to 1.0, >0.2 to 1.0, and most preferably within the range of >0 to 1.0, the subject is diagnosed as positive for cancer and / or acute inflammatory disease; and / or (a-2) The level of precipitated (poly)peptide in sample (S) is defined by the AUC of formula (III). Ｎ By calculating the value, it is compared to the level of precipitated (poly)peptides in a group of non-pathological reference samples (N): [Math 3] where n Ｎ is the number of samples in the group of non-diseased reference samples N, and x ｉ is the level of the precipitated (poly)peptide for the i-th non-diseased reference sample in N, and k is an index function for comparing S to x ｉ as defined by equation (II): [Math 4] Regarding S, AUC Ｎ The method according to claim 1, wherein the subject is diagnosed as positive for cancer and / or acute inflammatory disease if the value is calculated in the range of 0.65 and 1.0, 0.70 and 1.0, 0.75 and 1.0, 0.80 and 1.0, 0.85 and 1.0, 0.95 and 1.0, 0.96 and 1.0, 0.97 and 1.0, 0.98 and 1.0, and most preferably in the range of 1.

0.

6. The method according to any one of claims 1 and 3 to 5, wherein the conditions for inducing precipitation are provided by changing the sample pH, preferably by adjusting the sample pH to a value in the range of pH 2 and pH 6, pH 3 and pH 5.5, pH 3.4 and pH 5, pH 3.6 and pH 4.8, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, and pH 4.1 and 4.3, the greater the haphazardly, and most preferably within the range of pH 4.2 ± 0.

05.

7. The method according to claim 6, wherein the adjustment of the sample pH is provided by adding the following to the sample: (a) At least one acid having a pKa in the range of 2.0 to -10.0, 3.0 to 7.0, 3.5 to 6.5, 3.8 to 5.8, and 4.1 to 5.5, with higher values ​​being preferable, and most preferably 4.76 ± 0.05; Here, preferably, at least one acid is an organic acid, and here preferably, the organic acid is: (i) a carboxylic acid, more preferably (i-1) a monocarboxylic acid, preferably selected from the group consisting of formic acid, acetic acid, benzoic acid, propionic acid and butyric acid, or (i-2) a dicarboxylic acid or tricarboxylic acid, preferably citric acid; (ii) Barbiturates; and / or (iii) perchloric acid; And / or (b) an aqueous solution containing at least one acid as defined in (a); Here, preferably: - The aqueous solution contains at least one acid in a total concentration within the range of 0.3 vol% and 1.0 vol%, 0.3 vol% and 0.9 vol%, 0.3 vol% and 0.8 vol%, 0.3 vol% and 0.7 vol%, 0.3 vol% and 0.6 vol%, 0.3 vol% and 0.5 vol%, and 0.35 vol% and 0.45 vol%, with a higher concentration being preferable, and most preferably 0.4 vol%; - The aqueous solution has a pH in the range between pH 2 and pH 5, pH 2 and pH 4, pH 2.5 and pH 3.5, pH 2.8 and pH 3.2, and pH 2.9 and pH 3.1, with higher values ​​being preferable, and most preferably a pH of 2.97 ± 0.05; - The aqueous solution is added to the sample at a volume-to-volume ratio of the aqueous solution to the sample, most preferably at a concentration of 0.59:1, between 0.3:1 and 0.9:1, 0.4:1 and 0.8:1, and 0.5:1 and 0.7:1; and / or - A method wherein the aqueous solution further contains sodium chloride at concentrations in the range of 0.01% (w / v) and 7% (w / v), 0.1% (w / v) and 6% (w / v), 0.2% (w / v) and 5% (w / v), 0.3% (w / v) and 4% (w / v), 0.4% (w / v) and 3% (w / v), 0.5% (w / v) and 2% (w / v), 0.6% (w / v) and 1% (w / v), and 0.7% (w / v) and 0.9% (w / v), most preferably at a concentration of 0.81% (w / v).

8. The method according to any one of claims 1 to 7, wherein the sample includes or consists of: (a) Body fluids, preferably blood (preferably plasma), saliva, mucus, sputum, vomit, sweat, tears, urine, semen, vaginal fluid, feces, and exudates, or any mixture thereof; and / or (b) Body tissue, preferably homogenized body tissue, more preferably a cell-free suspension of homogenized body tissue.

9. The sample is diluted with a diluent before being subjected to conditions that induce precipitation, where the diluent is preferably: (i) an aqueous solution containing a salt, preferably sodium chloride, at a concentration in the range of 5 mM and 200 mM, 20 mM and 175 mM, 45 mM and 150 mM, 65 mM and 130 mM, 75 mM and 120 mM, 80 mM and 115 mM, 85 mM and 110 mM, 91 mM and 105 mM, with the greater the concentration, the more preferably, and most preferably 98 mM; and / or (ii) The method according to any one of claims 1 to 8, wherein the diluent is added to the sample in a volume-to-volume (v / v) ratio of the diluent to the sample, most preferably at a v / v ratio of 4.2:1 between the following: 1:1 and 7:1, 2:1 and 6.5:1, 2.5:1 and 6:1, 3:1 and 5.5:1; 3.4:1 and 5:1, 3.6:1 and 4.8:1, 3.8:1 and 4.6:1, 4.0:1 and 4.4:1, with the greater the preferred ratio.

10. The method according to any one of claims 1 to 9, wherein conditions for inducing precipitation are provided by, or additionally provided by: (i) Increase in sample temperature; (ii) Addition of at least one salting agent to the sample; where preferably, the salting agent is selected from the following methods: (ii-a)Ca ２＋ , (CH ３ ) ４ N ＋ , Cs ＋ , Rb ＋ NH ４ ＋ _K ＋ Na ＋ Mn ２＋ , and Li ＋ A cation selected from; and OH － SO ４ ２－ HPO ４ ２－ ,CH ３ COO － , C ３ H ５ O(COO) ３ ３－ CO ３ ２－ , ClO ３ － , BrO ３ － , thiocyanate (SCN － ), and Cl － An anion selected from; more preferably KCl and / or (NH ４ ) ２ SO ４ Salts containing; and / or (ii-b) Alkali metal sulfate; preferably Li ２ SO ４ Na ２ SO ４ _K ２ SO ４ , Rb ２ SO ４ , and / or Cs ２ SO ４ ; (iii) Addition of one or more other aliphatic alcohols, preferably alkanols, most preferably ethanol; (iv) Concentration of the sample by evaporation; (v) Addition of a ketone, preferably acetone; and / or Any combination of (vi)(i) to (v).

11. The method according to any one of claims 1 and 3 to 10, wherein in step (b), the level of precipitated (poly)peptide is evaluated by spectrophotometric, nephelometry, spectrofluorometry, circular dichroism (CD) spectroscopy, mass spectrometry (MS) and / or NMR spectroscopy.

12. In step (b), the level of precipitated (poly)peptide is assessed by measuring the change in absorbance with a spectrophotometer, and the determined increase or decrease in absorbance indicates an increase or decrease in the level of precipitated (poly)peptide in the sample, respectively; The method according to claim 11, wherein the absorbance is measured at wavelengths in the range of 200 nm and 800 nm, 260 nm and 420 nm, 280 nm and 400 nm, 300 nm and 380 nm, 320 nm and 360 nm, 330 nm and 350 nm, and 335 nm and 345 nm, preferably in the range of 340 nm.

13. A method for diagnosing cancer and / or acute inflammatory disease in a subject, wherein the change in absorbance is preferably a larger increase, most preferably in the range of 78-170 milli extinction unit (mE), 80-160 mE, 83-155 mE, 88-150 mE, 93-145 mE, and 98-140 mE, and the subject is diagnosed as positive for cancer and / or acute inflammatory disease, preferably when measured with a 1 cm pass cuvette or normalized to a measurement taken with a 1 cm pass cuvette.

14. The method according to any one of claims 1 to 13, wherein in step (b), the level of precipitated (poly)peptide is evaluated in reverse from the remaining soluble fraction of the sample, preferably after the precipitated (poly)peptide has been removed.

15. A method according to any one of claims 1 to 14: (i) Before subjecting the sample to conditions that induce precipitation in step (a), incubate the sample at a temperature between 2°C and 42°C, preferably 37±2°C, for a period of at least 10 seconds, preferably between 10 and 5000 seconds, 20 and 4000 seconds, 30 and 3000 seconds, 40 and 2000 seconds, 50 and 1000 seconds, 60 and 800 seconds, 80 and 600 seconds, 100 and 500 seconds, 200 and 400 seconds, 250 and 350 seconds, more preferably between 275 and 325 seconds; most preferably in the range of 300 seconds; and / or (ii) A method comprising: (i) After subjecting the sample to conditions that induce precipitation in step (a), before step (b), incubating the sample at a temperature between 2°C and 42°C, preferably 37±2°C, for a period of at least 60 seconds, preferably between 60 and 600 seconds, 100 and 500 seconds, 200 and 400 seconds, 250 and 350 seconds, and 280 and 320 seconds, with the greater the haphazardly, and most preferably between 300 seconds.

16. The method according to any one of claims 1 to 15, wherein the sample and / or dilution medium further comprises: (i) Anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citric acid, and heparin or any salt thereof; (ii) Proteinase inhibitors; (iii) One or more stabilizers, preferably selected from albumin, casein, gelatin, collagen, globulin, and protamine; (iv) Skim milk powder; (v) Surfactants; preferably selected from Tween, preferably Tween 20 or Tween 80, Triton X-100, and sodium dodecylbenzenesulfonate; (vi) polyethylene glycol (PEG); where preferably the PEG has a molecular weight in the range of 1,000 and 20,000 Da and / or is selected from PEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, and PEG20000; and / or (vii) Polysaccharides; preferably dextran selected from dextran-1, dextran-10, dextran-20, dextran-30, and dextran-40.

17. Furthermore, the method according to any one of claims 1, 2, and 5 to 16, further comprising evaluating a sample taken from the subject, preferably a blood sample, for the presence or absence of acute inflammation, wherein in an example of a subject in which a finding of having cancer and / or acute inflammatory disease has been determined, the absence of determined acute inflammation indicates that the subject has cancer and / or does not have acute inflammatory disease.

18. The presence or absence of acute inflammation is assessed by evaluating the levels of one or more inflammatory markers; Preferably, the method according to claim 17, wherein one or more inflammatory markers are selected from C-reactive protein (CRP), procalcitonin (PCT), fibrinogen, and leukocytes.

Images