Means and methods for diagnosing cancer and / or an acute inflammatory disease

Abstract

The present invention relates to methods for diagnosing cancer and / or an acute inflammatory disease in a subject. The invention further relates to a method for evaluating the responsiveness of a cancer and / or an acute inflammatory disease to a candidate treatment in a subject. In addition, the invention relates to a method for assessing the malignancy level of a cancer in a subject.

Description

[0001] Means and methods for diagnosing cancer and / or an acute inflammatory disease

[0002] The present invention relates to methods for diagnosing cancer and / or an acute inflammatory disease in a subject. The invention further relates to a method for evaluating the responsiveness of a cancer and / or an acute inflammatory disease to a candidate treatment in a subject. In addition, the invention relates to a method for assessing the malignancy level of a cancer in a subject.

[0003] In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Cancer is one of the leading causes of death worldwide, with an estimated 12.7 million cases around the world affecting both sexes equally. This number is even expected to increase to 21 million by 2030 (Vinay DS. Semin Cancer Biol. (2015);35 Suppl:S185-S198). Timely detection of malignancy by early diagnosis and cancer screening provides patients with the opportunity to effectively benefit from cancer therapy because treatment response and, accordingly, survival gains are by far more pronounced at early stages of cancer (Siegel RL et al., Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30). Decreased mortality rates of some cancers, such as colorectal and breast cancer, can at least be partially attributed to the establishment of corresponding cancer screening tests (Byers T et al., The American Cancer Society; 2016;66(5):359-69).

[0005] However, the majority of cancer patients is currently being diagnosed after the onset of symptoms at advanced stages when tumors have already progressed to a point where treatment options are limited. Diagnostic tools for screening only exist for a small subset of cancers and include microscopic analysis combined with DNA testing (cervical cancer (Tsikouras P et al. Journal of BIION: official journal of the Balkan Union of Oncology. 2016;21 (2):320-5)), skin exams (Wolff T et al., Annals of internal medicine. 2009; 150(3): 194-8), imaging techniques (breast cancer mammography (van den Biggelaar FJ et al., Breast (Edinburgh, Scotland). 2008;17(1):85-90.5), computed tomography (CT) coIonography and colonoscopy (Pickhardt PJ et al., Radiology. 2011 ;259(2):393-405), chest low-dose computed tomography (Hoffman RM et al., The Medical clinics of North America. 2017; 101 (4):769-85.)), blood-based tests (e.g., prostate-specific antigen (Brawer MK, Seminars in surgical oncology. 2000; 18(1):3-9), carcinoembryonic antigen (Young GP etal., Cancer medicine. 2016;5(10):2763-72)), and developing methods such as 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 are limited by rather low sensitivity and specificity that range from approximately 70% to 80% and 60% to 70%, respectively (Schiffman JD etal., American Society of Clinical Oncology Educational Book. 2015(35):57-65). In addition, sensitivity depends on tumor size, the patient’s age, disease history, and tissue 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).

[0006] Most currently available cancer screening strategies focus on the detection of specific tumor entities, contradicting the ‘paradox’ of cancer epidemiology, which says that the risk for any cancer over the course of the lifespan is generally high, while the risk for one specific cancer at a certain time is rather low (Schiffman JD et al. American Society of Clinical Oncology Educational Book. 2015(35):57-65). A major drawback of the current screening tests is hence that only those specific cancers may be detected for which reliable markers are known and for which a specific test is available. In order to extend the diagnosis to a larger group of cancers, a multiplicity of biomarkers needs to be analyzed in parallel thereby multiplying the laboratory work, costs for the healthcare system and risk of false results.

[0007] Many research efforts have been devoted towards the development of more universally applicable cancer screening tests (so-called “multi-cancer” or “pan-cancer” screening tests) that are capable of early stage diagnosing of most frequent tumor entities with high sensitivity, specificity, and accuracy and with clinically adequate applicability and accessibility (Shapley M et al. Br J Gen Pract. 2010;60(578):e366-77). For example, recent progress has been made in the development of approaches based on the detection of circulating cell-free DNA, including DNA methylation aberrations. However, reported overall performances remain unsatisfying, particularly in terms of sensitivities, not least due to intrinsically poor quantities of tumor-derived DNA in the circulation (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).

[0008] A related major burden on global health are inflammatory diseases. Inflammation is the body’s response to protect itself against invading pathogens or tissue injury. Acute inflammation is an immediate immune response that triggers cytokines and chemokines to promote the migration of immune cells to the infected or injured area to eliminate pathogens and regenerate tissues. When the initial, acute inflammatory response fails to remove the pathogen or source of tissue / cellular injury, chronic inflammation may evolve and can lead to further complications, including metabolic diseases, such as coronary heart disease, type 2 diabetes, and rheumatoid arthritis. Besides its previously ascribed function in tissue regeneration and host defense, inflammation has more recently also 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). It is believed that an early detection of inflammation, in particular at early acute stages, can also be helpful for assessing a subject’s predisposition for a future cancer development. It would hence be ideal to having a laboratory screening test at hand that can detect a broad bandwidth of cancers but also acute inflammatory disease with high specificity, sensitivity, and reliability.

[0009] There is, hence, an unmet need in the art for means and methods for diagnosing the presence of cancer and / or an acute inflammatory disease in a subject which overcome the limitations of currently available approaches. The present invention addresses this and other needs and provides related advantages as well.

[0010] Accordingly, the invention relates in a first aspect to a method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:

[0011] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and

[0012] (b) assessing the level of the precipitated (poly)peptides in the sample relative to:

[0013] (b-i) the level 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 an acute inflammatory disease;

[0014] (b-ii) the level of precipitated (poly)peptides in at least one correspondingly treated reference sample obtained from one or more reference subjects known to be negative for cancer and an acute inflammatory disease; and / or

[0015] (b-iii) a predetermined standard which has been determined based on (b-i) and / or (b-ii); whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if: the level of the precipitated (poly)peptides in the sample is at least, with increasing preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, most preferably at least 100% of the level of the precipitated (poly)peptides in the reference sample (b-i), or as compared to a respective predetermined standard; and / or the level of the precipitated (poly)peptides in the sample exceeds the level of the precipitated (poly)peptides in the reference sample (b-ii), or as compared to a respective predetermined standard, by at least, with increasing preference, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, most preferably by at least 30%.

[0016] The at least one correspondingly treated reference sample is, with increasing preference, at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75 and 100 correspondingly treated reference samples. Similarly, and independently of the numbers of reference samples, the one or more reference subjects are obtained from, with increasing preference, at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75 and 100 reference subjects. In this connection, it is to be understood that each reference sample can be obtained from one reference subject or from more than one reference subject. In the latter case the reference sample may be called a “pooled” reference sample obtained from more than one reference subject. It is also conceivable, and particularly contemplated herein, that the reference sample has been obtained from different body regions / tissues and / or different body fluids of one reference subject or more than one reference subject.

[0017] The term “correspondingly treated” means that the condition inducing precipitation of one or more (poly)peptides is the same as used in step (a).

[0018] The level of precipitated (poly)peptides is preferably defined as the weight of level of precipitated (poly)peptides and / or the percentage of precipitated (poly)peptides as compared to the total amount of (poly)peptides in the sample.

[0019] The term “cancer”, as used herein, refers to any malignant abnormal growth of cells. Examples include, without limitation, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of tumor-forming cancers.

[0020] In preferred embodiments, the cancer is selected from the group of solid cancers. In other embodiments, the cancer is selected from the group of liquid cancers. The term “solid cancer”, as used herein, refers to cancers in which a plurality of malignant cells is associated with one another, i.e., contiguous and localized within a confined site. As used herein, the term "solid cancer" includes, but is not limited to, "carcinomas", "adenocarcinomas", and "sarcomas". "Sarcomas" are cancers of the connective tissue, cartilage, bone and / or muscle. "Carcinomas" are cancers of epithelial (lining) cells. “Adenocarcinoma” refers to carcinoma derived from cells of glandular origin. “Solid cancers” are to be contrasted with “liquid cancers”, alternatively referred to as "fluid cancers" or "hematogenous cancers”, in which the malignant cells occur primarily as unassociated or individual cells. Liquid cancers are cancers that develop in the blood, bone marrow, or lymph nodes and include leukemia, lymphoma, and myeloma, such as the specific forms of these conditions referred to above.

[0021] As is evident from the herein disclosed experimental results (cf. Examples 1 and 2, and specifically Figure 8), the assay of the invention was proven to be effective for distinguishing samples originating from patients suffering from a broad spectrum of different cancers, including bile duct cancer, gallbladder cancer, bladder cancer, bone cancer, brain tumor, breast cancer, cervical carcinoma, colorectal cancer, esophageal cancer, gastric cancer, gastro intestinal stromal tumor (GIST), head and neck cancer, kidney cancer, laryngeal carcinoma, lung cancer, lymphoma, skin cancer, neuroendocrine tumor, ovarian cancer, pancreatic cancer, peritoneal carcinosis, prostate cancer, rectal cancer, sarcoma, squamous cell carcinoma, testicular cancer, and uterine cancer. In view of this disclosed evidence, in particularly preferred embodiments, the cancer may be selected from any of these specific kinds of cancers.

[0022] However, because of the absence of any indication that any particular kind of cancer would not be detectable by the method of the invention, it is expected that the assay is capable for the detection of any kind of cancer, and that the assay may thus be suitably employed as a “pan- cancer”-screening test (as mentioned in the introduction).

[0023] The terms "inflammation" and "inflammatory", as used herein, refer to a biological response involving an upregulation of the immune system, which may include an increase in the expression and / or activity of (poly)peptides related to inflammation or an immune response (e.g., pro- inflammatory markers such as chemokines and cytokines, production of plasma haptoglobin) and symptoms of inflammation (e.g., pain, heat, redness and / or edema). Generally, inflammation may be acute or chronic.

[0024] The term “inflammatory disease”, as used herein, has its general meaning in the art and refers to any disease and condition associated with acute or chronic inflammation, or both. The term may include, but is not limited to, (1) inflammatory or allergic diseases, such as systemic anaphylaxis or hypersensitivity responses, drug allergies, insect sting allergies; inflammatory bowel diseases, such as Crohn’s disease, ulcerative colitis, ileitis and enteritis; vaginitis; psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis; spondyloarthropathies; scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung diseases, and the like, (2) autoimmune diseases, such as arthritis (rheumatoid and psoriatic), osteoarthritis, multiple sclerosis, systemic lupus erythematosus, diabetes mellitus, glomerulonephritis, and the like, (3) graft rejection (including allograft rejection and graft-versus-host disease (GvHD)), and (4) other diseases in which undesired inflammatory responses may occur (e.g., atherosclerosis, myositis, inflammatory CNS disorders, such as stroke and closed-head injuries, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis and Bechet's syndrome). In particularly preferred embodiments, the “inflammatory disease” is sepsis.

[0025] The term “sepsis”, as used herein, has its general meaning in the art and represents a serious medical condition that is characterized by a whole-body inflammatory state. In addition to symptoms related to the provoking infection, sepsis is characterized by presence of acute inflammation present throughout the entire body. In particular, sepsis is defined as a deregulated immune response to infection, translating into life-threatening organs dysfunction, and may thus also be referred to a deleterious systemic inflammatory response to infection. The term “sepsis”, as used herein, also encompasses specific forms and complications thereof, such as “severe sepsis” and “septic shock”.

[0026] In accordance with the method according to the first and / or second aspect of the invention, the “inflammatory disease” which may be diagnosed is an “acute inflammatory disease”, i.e., characterized by a presently i.e., at the time when the sample is obtained from the subject) ongoing inflammatory process which manifests inter alia by the presence of elevated levels of one or more inflammatory markers, e.g., in the blood of a subject. A variety of markers of inflammation, such as C-reactive protein (GRP) and / or procalcitonin (PCT), are well known in the art and their measurement is nowadays routinely performed. Specifically preferred inflammatory markers and their corresponding levels that are commonly considered indicative of an acute inflammation are described in preferred embodiments herein below.

[0027] It will be appreciated that the herein disclosed methods and applications are useful in the fields of human medicine and veterinary medicine. Thus, the term “subject” or “patient” as interchangeably used herein refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (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), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In preferred embodiments, the subject is a mammal. In even more preferred embodiments, the subject is a human.

[0028] The term “sample” or “body sample”, as used herein, generally refers to any biological sample obtainable from a subject’s body, such as a body fluid (e.g., blood, such as whole blood, plasma or serum) or a body tissue (e.g., a tissue sample obtained by biopsy). The term “sample” may be a purely biological sample or a processed form thereof. For example, a “sample” may comprise or consist of a certain fraction of a body fluid (e.g., plasma or serum obtained from whole blood) or a body tissue, and / or may comprise additional constituents such as an anticoagulant agent and / or a stabilizing agent, for example, a protease inhibitor. Particularly preferred samples and additional constituents that may preferably be comprised therein or which may be added thereto are defined herein below.

[0029] In view of the herein disclosed methods being based on the assessment of precipitated (poly)peptides resulting from the provision of precipitation-inducing condition(s), it is understood that the sample which is to be subjected for the herein described methods is preferably substantially free of any precipitate, in particular, substantially free of any precipitated (poly)peptides. Thus, the sample before being employed in the herein disclosed methods may preferably by subjected to a treatment (e.g., a centrifugation or filtration) to remove any potentially present precipitate.

[0030] The term "(poly)peptide", as used herein, refers to a linear polymer of amino acid residues linked by peptide bonds in a specific sequence and embraces both, the group of “polypeptides” and the group of “peptides”. The group of “polypeptides”, as interchangeably used herein with the term "protein", consists of molecules with more than 30 amino acids, which is in distinction to the group of “peptides” which consists of molecules with up to 30 amino acids. The group of “peptides” also refers to fragments of proteins of a length of 30 amino acids or less. (Poly)peptides may further form dimers, trimers and higher oligomers, i.e., consisting of more than one (poly)peptide molecule. (Poly)peptide molecules forming such dimers, trimers etc. may be identical or nonidentical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. Homo- or heterodimers etc. also fall under the definition of the term “(poly)peptide”. The term “(poly)peptide” also refers to chemically or post- translationally modified peptides and polypeptides.

[0031] The term “precipitation” refers in line with its common general meaning to a phase change from a colloid dispersion to a solid mass when it is subjected to a perturbation. More specifically, the term “precipitation” refers to the formation of an insoluble solid mass by a reaction which occurs in solution. For example, precipitation can occur upon addition of a suitable precipitation agent to a solution. When precipitation occurs, the solid mass formed as a result is called the “precipitate”. The precipitate can be collected or separated from the remaining solution by various methods, such as filtration, decanting, centrifuging and the like. As used herein in connection with the disclosed methods, the term “precipitation” refers even more specifically to a process by which one or more (poly)peptides, which is / are initially comprised in the sample in a dissolved ( / .e., soluble) form, in consequence of being subjected to an environmental stimulus ( / .e., a precipitation-inducing condition, as described below), undergo(es) a phase change to become insoluble.

[0032] The term “condition inducing precipitation” or “precipitation-inducing condition”, as used herein, is intended to refer to any physical and / or chemical condition that may cause one or more (poly)peptides comprised in the sample to precipitate. Typically, in the context of the present invention, precipitation occurs as a result of the provision of a precipitation-inducing condition to the sample, as described below, such as, the addition of a precipitating agent (e.g., an acid).

[0033] It has surprisingly and strikingly been found by the present inventor that (poly)peptides comprised in a body sample (e.g., blood plasma) from a subject afflicted by cancer and / or an acute inflammatory disease are differentially susceptible to certain precipitation-inducing conditions as compared to (poly)peptides comprised in a sample from a healthy subject ( / .e., not afflicted by a cancer and / or an acute inflammatory disease). In particular, it has been found that - upon an exposure of a body sample from a cancerous and / or acute inflammatory subject to such a precipitation-inducing condition - (poly)peptides comprised in said body sample will undergo precipitation to a significantly more pronounced extent compared to (poly)peptides comprised in a correspondingly treated body sample from a healthy subject. Although the precise underlying molecular details of this phenomenon are yet to be elucidated, as disclosed herein, further explorations conducted by the present inventor revealed that this “differential precipitation behavior” is consistently and reproducibly observed with body samples originating from subjects suffering from a broad variety of cancers and / or acute inflammatory conditions. It is demonstrated herein that this mechanism can be effectively exploited as a diagnostic means for discriminating with high accuracy whether a body sample stems from a healthy subject or from a subject afflicted by a cancer and / or an acute inflammatory disease.

[0034] Moreover, further investigations conducted by the inventor revealed that this differential precipitation is most pronounced, and accordingly, a distinction between samples originating from a cancer / acute inflammatory subject vs. samples originating from a healthy subject is best possible, if the precipitation-inducing condition which is employed is rather mild, i.e., a condition that is just sufficient to initiate a precipitation process, in distinction to harsh precipitation-inducing conditions which would result in an immediate precipitation and / or aggregation (or even in a hydrolysis) of most or even substantially all (poly)peptides in both, the sample from a cancerous / acute inflammatory subject and the sample of a healthy subjects, and thereby impede a differentiation based on the herein disclosed effects.

[0035] Thus, in preferred embodiments, the precipitation-inducing condition is a condition upon which application at least, with increasing preference, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95% of the total amount or mass of the (poly)peptides comprised in the sample remain soluble, but wherein preferably at least 1% of the total amount or mass of the (poly)peptides comprised in the sample precipitate.

[0036] As used herein, the term “assessing the level of precipitated (poly)peptides” ( / .e., relative to a reference sample and / or a predetermined standard) is intended to refer to any direct or indirect assessment technique which allows for a detection of a quantitative signal or other measurement parameter that correlates with the formation of precipitated (poly)peptide(s) and which thus allows for a comparison between the levels of precipitated (poly)peptides in the sample of the subject to be tested and a correspondingly treated reference sample from a subject of known health condition ( / .e., a subject known to have a cancer and / or acute inflammatory disease or a subject known to not have any of the referred conditions) and / or a respective predetermined standard.

[0037] Exemplary methods suitable for such an assessment include, among others, separation of the precipitated (poly)peptide(s) from the sample (e.g., by centrifugation or filtration), determining the weight or volume of the precipitate, and comparing it with the weight or volume of the precipitated (poly)peptides obtained from a correspondingly treated reference sample. Alternatively, the separated precipitated (poly)peptides may be subjected to a gel-electrophoresis (e.g., polyacrylamide gel-electrophoresis (PAGE)), such as an SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), possibly followed by Coomassie-Blue-staining (or other staining approaches, e.g., silver staining) or Western Blotting, wherein the obtained band intensities can be quantified (e.g., by densitometry) and compared with those from a correspondingly treated reference sample. A corresponding assessment by SDS-PAGE (and staining, such as Coomassie-Blue staining) followed by densitometric quantification of band intensities has been conducted in Example 4 and corresponding Figure 13 and is particularly preferred. Other well-known (poly)peptide quantification methods include the bicinchoninic acid (BCA) assay or other copper-based assays, the Bradford assay, the Lowry assay, and chromatographic approaches, such as reverse phase-high performance liquid chromatography (RP-HPLC). Further methods by which the level of (poly)peptide(s) may be quantitatively assessed include mass-spectrometry, liquid chromatography-mass spectrometry (LC-MS), Matrix Assisted Laser Desorption / lonization (MALDI; in particular MLDI-MS and MALDI-TOF- MS), light scattering (in particular Dynamic Light Scattering (DLS)) and nuclear magnetic resonance (NMR) spectroscopy (e.g., solution-state NMR spectroscopy).

[0038] The skilled person will understand that the level of the precipitated (poly)peptide(s) may alternatively also be assessed indirectly or reversely from the remaining soluble fractions of the sample and reference sample, e.g., by (i) determining the total amount / mass of (poly)peptides initially comprised in the sample or reference sample, and (ii) determining the amount / mass of soluble (poly)peptides comprised in the remaining soluble fraction of the sample and reference sample after the provision of the precipitation-inducing condition, preferably after removal of the precipitate (e.g., by centrifugation and / or filtration). Any of the above referred quantification methods may analogously also be employed for that purpose.

[0039] One further exemplary approach to that end is to measure the UV absorbance at 280 nm wavelength (A280nm) of the initial sample ( / .e., before providing the precipitation-inducing condition) and of the remaining soluble (poly)peptide fraction ( / .e., after providing the precipitation-inducing condition, and preferably after removal of the precipitated polypeptide(s)) and to thereby determine the fraction of precipitated (poly)peptide from the total amount of (poly)peptides comprised in the sample; and to compare said fraction with the respectively determined fraction determined of precipitated (poly)peptide in a correspondingly treated reference sample. The skilled person will understand that, even though in the absence of knowledge of the extinction coefficients of the complex mixture of (poly)peptides comprised in the sample, a respective assessment will still allow a relative assessment for comparing the level of the precipitated (poly)peptides in the sample relative to the level of the precipitated (poly)peptides in the reference sample.

[0040] Further preferred approaches for assessing the level of the one or more precipitated (poly)peptides in the sample, in particular, further absorbance-based approaches, are defined in various embodiments herein below.

[0041] It will be readily understood by the skilled artisan that the term “diagnosing a subject of being positive for cancer and / or an acute inflammatory disease”, as used herein, is not intended to mean to provide an absolute ( / .e., 100%) certainty of the presence of cancer and / or an acute inflammatory disease in the subject but rather as to provide a strong indication / suspicion for the subject to have cancer and / or an acute inflammatory disease, and that, in the instance of such a positive diagnosis by the methods of the present invention, a further assessment ( / .e., follow-up examination) may be conducted for the sake of a further verification either confirming or refuting that initial diagnosis; and in the former case, possibly also for gathering further information on the type of condition, e.g., in the case of cancer, the type of the cancer and / or its localization in the subject’s body. Such follow-up examination may be conducted, for example, by physical examination, blood and / or urine tests for an assessment of known biomarkers, ultrasound (sonography), mammography and / or other imaging technologies, such as by X-rays, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Thus, the recited method purpose “for diagnosing cancer and / or an acute inflammatory disease” as referred to herein in connection with the method of the first aspect of the invention (and also referred to below in connection with the method of the second aspect of the invention) may, alternatively and interchangeably, be formulated as “for assessing whether a subject has an increased risk of having cancer and / or an acute inflammatory disease”, or “for screening a subject for the presence of cancer and / or an acute inflammatory disease” or “for identifying a subject having or suspected of having cancer and / or an acute inflammatory disease”.

[0042] It has further been advantageously found by the present inventor that, on account of the observed differential precipitation susceptibility, a discrimination of whether a sample originates from a subject afflicted by cancer and / or an acute inflammatory disease or from a healthy subject ( / .e., a subject not afflicted by any of the referred conditions) can alternatively also reliably be established from an assessment of the sample to be tested alone ( / .e., without the need of a comparison to any reference sample and / or predetermined standard), namely by determining the fraction of the precipitated (poly)peptides from the total amount of (poly)peptides comprised in the sample. As demonstrated herein (see, e.g., Figure 1A-E and Figures 10-13), the fraction of (poly)peptides in the sample which, upon exposure to a precipitation-inducing condition, undergo precipitation 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, whereby, however, the extent of the observable difference is dependent on the individual precipitation-inducing condition applied. For example, as shown in Figure 1A, when precipitation was induced by addition of 0.4% (v / v) acetic acid to a final concentration in the sample of about 0.15% (v / v), the fraction of precipitated (poly)peptides in the pathological sample was about 2%, whereas the fraction of precipitated (poly)peptides in the non-pathological sample corresponded to only 1.5% of the total amount of (poly)peptides comprised in the sample. In a similar vein, as shown in Figure 1 B, an exposure to heat (70 °C / 1 min) led to a precipitation of 7% of the (poly)peptides in the pathological sample vs. 4% in the non-pathological sample. It is, hence, plausible that this differential precipitation, when assessed based on the determination of the fraction of the precipitated (poly)peptides relative to the total amount / mass of proteins comprised in the sample, also provides a highly discriminative determinant for the envisaged diagnostic purposes. It is furthermore believed that differential precipitation can also be induced by ionizing radiation (I R), noting that it is well-known hat IR can induce protein precipitation.

[0043] Accordingly, in a second related aspect, the invention provides a method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:

[0044] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample, wherein the precipitation-inducing condition is provided by:

[0045] (i) adjusting (preferably lowering) the pH of the sample to a value in the range of between, with increasing preference, 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, pH 4.1 and 4.3, most preferably pH 4.2±0.05; preferably by addition of an acid, or an aqueous solution comprising the acid, wherein the acid has a pKain the range of between, with increasing preference, 3.0 and 7.0, 3.5 and 6.5, 3.8 and 5.8, 4.1 and 5.5, and most preferably has a pKaof 4.75±0.05; wherein preferably the acid is an organic acid, wherein the organic acid is preferably a carboxylic acid, more preferably a monocarboxylic acid, most preferably acetic acid;

[0046] (ii) an increase of the sample temperature;

[0047] (iii) addition of at least one salting-out agent;

[0048] (iv) addition of one or more aliphatic alcohol(s), preferably alkanol(s), most preferably ethanol; and / or

[0049] (vi) addition of ketone, preferably acetone; and

[0050] (b) determining the fraction of the precipitated (poly)peptides from the total amount of (poly)peptides in the sample; whereby:

[0051] - in the instance of the precipitation being induced by lowering of the sample pH, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, more preferably more than 2.1 % of the total amount of (poly)peptides in the sample;

[0052] - in the instance of the precipitation being induced by an increase of the sample temperature, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 5%, 5.5%, 6%, 6.5%, more preferably more than 7% of the total amount of (poly)peptides in the sample;

[0053] - in the instance of the precipitation being induced by addition of at least one salting-out agent, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, more preferably more than 2.1 % of the total amount of (poly)peptides in the sample;

[0054] - in the instance of the precipitation being induced by addition of ethanol and / or other aliphatic alcohol, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 4%, 6%, 8%, 10%, 11%, more preferably more than 12% of the total amount of (poly)peptides in the sample; and / or - in the instance of the precipitation being induced by addition of a ketone, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1 %, 2%, 3%, 4%, more preferably more than 5% of the total amount of (poly)peptides in the sample; the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease.

[0055] The definitions and preferred embodiments described herein in connection with the first aspect of the invention apply mutatis mutandis to the second aspect of the invention. For instance, below further details and preferred embodiments of the increase of the sample temperature (e.g., temperature and / or time) as well as the addition of at least one salting-out agent (e.g., examples and amounts of salting-out agent) are described, which are also meant to apply to the second aspect of the invention.

[0056] The term “fraction of precipitated (poly)peptides” in a sample, as used herein, refers to the proportion the precipitated (poly)peptides make from entire amount of (poly)peptides comprised in the sample, expressed in percentage or decimal.

[0057] As shown in the herein disclosed experimental evidence (see, Example 1 and 2, and Fig. 1A-E), irrespective of which specific condition was employed for effecting the precipitation, the fraction of precipitated (poly)peptides was consistently higher in samples originating from a pathological subject compared to the fraction of precipitated (poly)peptides measured for correspondingly treated non-pathological reference samples. However, the individually determined fraction of precipitated (poly)peptides was found to be dependent on the specific precipitation-inducing condition applied.

[0058] For example, when the precipitation was effected by addition of an acid (in particular 0.4% (v / v) acetic acid to a final concentration of about 0.15% (v / v)), the fraction of precipitated (poly)peptides was determined to amount to about 2% of the total amount of (poly)peptides comprised in the pathological sample, whereas the fraction of precipitated (poly)peptides in the non-pathological samples was determined to amount to only about 1.5%; see Fig. 1 A.

[0059] When the precipitation was instead effected by an increase of the sample temperature to 70°C for 1min, the fraction of precipitated (poly)peptides was determined to amount to about 7% of the total amount of (poly)peptides comprised in the pathological sample, whereas the fraction of precipitated (poly)peptides in the non-pathological samples was determined to amount to only about 5%; see Fig. 1 B.

[0060] When the precipitation was instead effected by addition of a salting-out agent (in particular, 35% (NH4)2SO4 to a final concentration of 20.6%), the fraction of precipitated (poly)peptides was determined to amount to about 13.5% of the total amount of (poly)peptides comprised in the pathological sample, whereas the fraction of precipitated (poly)peptides in the non-pathological samples was determined to amount to only about 10%; see Fig. 1C.

[0061] When the precipitation was instead effected by addition of a ketone or an alcohol (in particular, 50% ice-cold acetone or 50% EtOH to a final concentration of 18.5% (v / v)), the fraction of precipitated (poly)peptides was determined to amount to about 6.2% and about 28%, respectively, of the total amount of (poly)peptides comprised in the pathological sample, whereas the fraction of precipitated (poly)peptides in the non-pathological samples was determined to amount to only about 5% or about 23%, respectively; see Fig. 1C and D, respectively.

[0062] The term “ketone”, as used herein, preferably means ketones having 3 to 6 carbon atoms, including, but not limited to, acetone, butanone, methyl ethyl ketone, methyl isobutyl ketone, and diethyl ketone. More preferably, the ketone is acetone, most preferably ice-cold acetone. The final ketone (preferably acetone) concentration (v / v) as used for precipitation is with increasing preference 8.5% to of 28.5%, 13.5% to of 23.5%, 16% to of 21% and 18.5%.

[0063] 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 will be separated from the remaining soluble fraction (e.g., by centrifugation, e.g., at 21.000xg for 1 min at 4°C) and then be quantified (e.g., by determination of their total amount / mass), e.g., by using a BCA assay. The remaining soluble fraction will also be quantified with respect to its (poly)peptide content, e.g., by using a BCA assay. The fraction of the precipitated (poly)peptides from the total amount of (poly)peptides comprised in the sample is then calculated by dividing the determined quantity of precipitated (poly)peptides by the sum of the determined quantities of the precipitated (poly)peptides and the soluble fraction, and multiplying the result by 100%. In an alternative exemplary setting, the precipitated (poly)peptides are separated from the remaining soluble fraction (e.g., by centrifugation). An SDS-PAGE is then conducted from (i) a defined volume of the precipitated (poly)peptides (resolved in SDS sample buffer), and from (ii) a defined volume of the remaining soluble fraction. The obtained protein bands may then be densitometrically quantified. The fraction ( / .e., the proportion) of precipitated (poly)peptides from the total amount of proteins which were initially comprised in the sample can then be calculated from the measured band intensities and the knowledge of the original sample volume and the volume of the remaining soluble fraction.

[0064] In a third aspect, the invention relates to a method for evaluating the responsiveness of a cancer and / or an acute inflammatory disease to a candidate treatment in a subject, the method comprising:

[0065] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample, wherein said sample has been obtained at a time point after which the candidate treatment has been initiated;

[0066] (b) assessing the level of the 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 of step (a) was obtained from the subject, wherein the reference sample has been subjected to a corresponding precipitation-inducing condition as in step (a); and whereby:

[0067] - if the level of the precipitated (poly)peptides in the sample is equal to or lower than the level of the precipitated (poly)peptides in reference sample, the cancer and / or the acute inflammatory disease is classified as being responsive to the candidate treatment; and / or

[0068] - if the level of the precipitated (poly)peptides in the sample exceeds the level of the precipitated (poly)peptides in the reference sample, the cancer and / or the acute inflammatory disease is classified as being non-responsive to the candidate treatment.

[0069] As used herein, the term “candidate treatment” refers to a treatment that is being tested for its effectiveness against a disease or disorder, i.e., in the present case the specific kind or cancer and / or acute inflammatory disease by which the subject is afflicted.

[0070] As used herein, the term “responsiveness” refers to the development of a favorable response when a cell, tissue, organ, or subject is contacted with an agent (e.g., a candidate therapeutic agent). By way of non-limiting example, a favorable response can be inhibition or stop of abnormal cell growth when cancer cells are contacted with a particular agent and an unfavorable response can be an accelerated growth of a tumor when a patient with a tumor is contacted with a particular agent.

[0071] The term “candidate agent”, as used herein, refers to any agent that is prima facie expected suitable for treating a cancer and / or an inflammatory disease (preferably an acute inflammatory disease). Corresponding agents are more specifically referred to herein as “anti-cancer agents” and “anti-inflammatory agents”, respectively.

[0072] Candidate agents may generally be selected from any known pharmaceuticals, e.g., agents that are already in use in (pre-)clinical studies or even approved by one or more regulatory agencies / medical authorities (e.g., European Medicines Agency (EMA), Food and Drug Administration (FDA) etc.) for the treatment and / or prevention of a certain pathological condition (e.g., a cancer and / or inflammatory disease), but also from such agents which have not yet been employed as a medicament, or which have been employed as a medicament, yet only for the purpose of treating and / or preventing a pathological condition distinct to the condition ( / .e., the cancer and / or the inflammatory disease) from which the subject is afflicted. Exemplary candidate agents include naturally occurring compounds, such a secondary metabolites or agents produced by and isolated from bacteria, fungi, animals or plants, as well as synthetic agents, such as agents from a combinatorial library of chemical compounds or from a combinatorial display library (e.g., a ribosome- or phage-display antibody / scFv library). The term candidate agent also refers to derivatives of the above-mentioned terms.

[0073] The term “treatment” refers to, inter alia, reducing or alleviating one or more symptoms in a subject, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis, and / or preventing disease in a subject who is free therefrom as well as slowing or reducing progression of an existing disease. For a given subject, improvement in a symptom, its worsening, regression, or progression may be determined by objective or subjective measure. Efficacy of treatment may be measured as an improvement in morbidity or mortality. Palliative (e.g., improving quality of life) or preventative (e.g., preventing development of disease or the incidence of relapse) methods are also considered treatment. For example, in connection with the treatment of a cancer, a neo-adjuvant therapy to shrink a primary tumor and to make local therapy (e.g., surgery or radiation therapy) more effective and adjuvant therapy to reduce recurrence and / or the chance of resistance developing are also considered treatment.

[0074] The term “anti-cancer agent”, as used herein, includes any agent that may be suitable for the treatment of a cancer.

[0075] Current strategies in cancer treatment include chemotherapy, radiotherapy, immunotherapy (including adoptive cellular therapies (such as CAR-T cell therapy or TCR-engineered T cell therapy) and immune checkpoint inhibitors), hormone therapy, monoclonal antibodies and antibody-drug conjugates, and surgery, as well as combinations thereof. Further known anticancer agents include the following: oestrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and further angiogenesis inhibitors.

[0076] “Oestrogen receptor modulators” refers to compounds which interfere with or inhibit the binding of oestrogen to the receptor, regardless of mechanism. Examples of oestrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381 , LY 117081 , 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.

[0077] “Androgen receptor modulators” refers to compounds which interfere with, or inhibit the binding of, androgens to the receptor, regardless of the mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole and abiraterone acetate.

[0078] “Retinoid receptor modulators” refers to compounds which interfere with or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a- difluoromethylornithine, ILX23-7553, trans-N-(4'-hydroxyphenyl)retinamide and N-4- carboxyphenylretinamide.

[0079] “Cytotoxic agents” refers to compounds which result in cell death primarily through direct action on the cellular function or inhibit or interfere with cell myosis, including alkylating agents, tumour necrosis factors, intercalators, microtubulin inhibitors and topoisomerase inhibitors. Examples of cytotoxic agents include, but are not limited to, tirapazimine, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosylate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methylpyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)bis-mu-(hexane-1 , 6-diamine)-mu- [diamine-platinum(ll)]bis[diamine(chloro)platinum(ll)]tetrachloride, diarisidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755 and 4-demethoxy-3-deamino-3-aziridinyl-4- methylsulfonyldaunorubicin.

[0080] Examples of microtubulin inhibitors include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy- 8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881 , BMS184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro- 4-methoxyphenyl)benzenesulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N- methyl-L-valyl-L-prolyl-L-proline-t-butylamide, TDX258 and BMS188797.

[0081] “Topoisomerase inhibitors” are, for example, topotecan, hycaptamine, irinotecan, rubitecan, 6- ethoxypropionyl-3',4'-O-exobenzylidenechartreusin, 9-methoxy-N,N-dimethyl-5- nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9- hydroxy-4-methyl-1 H,12H-benzo [de]pyrano[3',4':b,7]indolizino[1 ,2b]quinoline-10,13(9H,15H)- dione, lurtotecan, 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, asulacrine, (5a,5aB,8aa,9b)-9-[2-[N-[2-(di-methylamino)ethyl]-N- methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxy phenyl]-5,5a,6,8,8a,9-hexohydro furo(3',4':6,7)naphtho(2,3-d)-1 ,3-dioxol-6-one, 2,3-(methylene-dioxy)-5-methyl-7-hydroxy-8- methoxy benzo[c]phenanthridinium, 6, 9-bis[(2-amino-ethyl)amino]benzo[g]isoquinoline-5, 10- dione, 5-(3-amino propylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H- pyrazolo[4,5,1-de]acridin-6-one, N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H- thioxanthen-4-ylmethyl]formamide, N-(2-(dimethyl amino)ethyl)acridine-4-carbox amide, 6-[[2- (dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c] quinolin-7-one and dimesna.

[0082] "Angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal antiinflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like 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; Inti. J. Mol. Med. (1998) 2:715; J. Biol. Chem. (1999) 274:9116)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1 , angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. (1985) 105:141- 145), and antibodies to VEGF (see, e.g., Brower, V. (1999) Nature Biotechnology, 17:963-968; Kim et al. (1993) Nature 362:841-844; WO 00 / 44777; and WO 00 / 61186).

[0083] “Antiproliferative agents” include antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231 and INX3001 and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'- fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydrobenzofuryl)sulfonyl]-N'-(3,4- dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradeca dienoyl]glycylamino]-L-glycero-B-L- mannohepto-pyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8- tetrahydro-3H-pyrimidino[5,4-b]-1 ,4-thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-fluorouracil (5-Fll), alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6- methoxy-14-oxa-1 ,11-diazatetracyclo(7.4.1 ,0.0)tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1-B-D- arabinofuranosyl cytosine and 3-aminopyridine-2-carboxaldehyde thiosemicarbazone. “Antiproliferative agents” also include monoclonal antibodies to growth factors other than those listed under “angiogenesis inhibitors”, such as trastuzumab, and tumour suppressor genes, such as p53, which can be delivered via recombinant virus-mediated gene transfer (see, e.g., U.S. Pat. No. 6,069,134).

[0084] "HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231 ,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589 ), fluvastatin (LESCOL®; see U.S. Pat. Nos. 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. Pat. Nos. 5,273,995, 4,681 ,893, 5,489,691 and 5,342,952). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (1996) and U.S. Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms ( / .e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG- CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

[0085] "Prenyl-protein transferase inhibitor" refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-l), and geranylgeranyl-protein transferase type-ll (GGPTase-l I, also called Rab GGPTase). Examples of prenyl-protein transferase 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. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S. Pat. No. 5,602,098, European Patent Publ. 0 618 221 , European Patent Publ. 0675 112, European Patent Publ. 0604 181 , European Patent Publ. 0 696 593, WO 94 / 19357, WO 95 / 08542, WO 95 / 11917, WO 95 / 12612, WO 95 / 12572, WO 95 / 10514, U.S. Pat. No. 5,661 ,152 , WO 95 / 10515, WO 95 / 10516, WO 95 / 24612, WO 95 / 34535, \N0 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, U.S. Pat. No. 5,571 ,792, WO 96 / 17861 , WO 96 / 33159, WO 96 / 34850, WO 96 / 34851 , WO 96 / 30017, WO 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 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. Pat. No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see GU WZ et al., European J. of Cancer (1999), 35(9): 1394-1401.

[0086] The term “anti-inflammatory agent”, as used herein, includes agents that elicit a response in a subject that reduces inflammation (either acute or chronic, preferably acute) or downregulates the immune response, for example, by reducing or inhibiting enzyme or protein / peptide activity related to inflammation or an immune response (e.g., inhibition of pro-inflammatory markers or reduction in the production of plasma haptoglobin); by ameliorating one or more symptoms of inflammation or an immune response (e.g., pain, redness, heat or edema); or by slowing or delaying of the inflammatory process or the immune response. In some embodiments, the “antiinflammatory agent” is an anti-inflammatory agent known in the art. Anti-inflammatory agents include, but are not limited to, steroidal compounds, including hydrocortisone and the like; or nonsteroidal anti-inflammatory agents, including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin, and the like.

[0087] In a fourth aspect, the invention relates to a method for assessing the malignancy level of a cancer in a subject, the method comprising:

[0088] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and

[0089] (b) assessing the level of the precipitated (poly)peptides in the sample relative to:

[0090] (i) at least one correspondingly treated reference sample obtained from one or more reference subjects having a cancer with a known malignancy level; and / or

[0091] (ii) a predetermined standard that has optionally been obtained based on one or more reference sample(s) according to (b)(i); wherein a higher, lower, or substantially same level of the precipitate in the sample relative to the level of precipitate in the reference sample provides an indication that the cancer in the subject has a higher, lower, or substantially same malignancy, respectively, relative to the cancer of the reference subject or the predetermined standard.

[0092] As used herein, the term “malignancy level” refers in line with its common meaning in the art to the level of aggressiveness of a cancer with respect to its ability to progress and / or to spread and its potential to eventually cause mortality. Whereas it is understood that there is no absolute quantitative measure for the level of malignancy in terms of any unit, a malignancy level may be determined relative to the malignancy level of a cancer from a reference subject with known medical outcome or a respective predetermined standard, and can thus provide a suitable qualitative comparator for assessing the severity of a cancer and establishing a prognosis. For example, the level of the precipitated (poly)peptides in the sample may be assessed by spectrophotometrically measuring a change in the absorbance caused by the induced precipitation and comparing said change of absorbance to a change of absorbance which had been recorded for a correspondingly treated sample from a cancer patient with known medical outcome and / or from a cancer patient afflicted by a specific kind of cancer that is commonly known to be characterized by a poor prognosis and high mortality (e.g., brain cancer or pancreatic cancer).

[0093] The data in Salat et al. (2020), Precision Cancer Medicine, Vol 5, doi: 10.21037 / pcm-21-35 indicate that a higher, lower, or substantially same level of the precipitate in the sample relative to the level of precipitate in the reference sample indeed provides an indication that the cancer in the subject has a higher, lower, or substantially same malignancy, respectively, relative to the cancer of the reference subject or the predetermined standard. Reference is made to the 5-year follow-up study on all-cause mortality data in patients with a histologically proven malignant disease and healthy volunteers as described in Salat et al. (2020), Precision Cancer Medicine, Vol 5, doi: 10.21037 / pcm-21-35.

[0094] In preferred embodiments of the method according to the first aspect of the invention, in step (b) the level of the precipitated (poly)peptides in the sample is assessed based on a statistical comparison relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be positive for cancer and / or an acute inflammatory disease (pathological reference samples) and / or relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be negative for cancer and an acute inflammatory disease (non-pathological reference samples), wherein the statistical comparison is conducted by: (a) an area under the curve (AUC) calculation; wherein preferably:

[0095] (a-1) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of pathological reference samples (P) by calculating an ALICp value as defined by formula (I): wherein np is the number of reference samples in the group of pathological reference samples P, xtis the level of the precipitated (poly)peptides for the ithpathological reference sample in P, and k is the indicator function for comparing S against x, as defined by formula (II): and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an ALICp value is calculated in the range of between, with increasing preference, >0.75 and 1.0, >0.7 and 1.0, >0.6 and 1.0, >0.5 and 1.0, >0.4 and 1.0, >0.3 and 1.0, >0.2 and 1.0, >0.001 and 1.0, and most preferably between >0 and 1.0; and / or

[0096] (a-2) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of non-pathological reference samples ( / V) by calculating an AUCw value as defined by formula (III): nw

[0097] A UCN= — y fc(5, xz) nN

[0098] (HI) is the number of samples in the group non-pathological reference samples A / , Xt is the level of the precipitated (poly)peptides for the Ithnon-pathological reference sample in N, and k is the indicator function for comparing S against x, as defined by formula (II): and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an ALICN value is calculated in the range of between, with increasing preference, 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, 0.99 and 1.0, most preferably of 1.0.

[0099] Since the above preferred embodiment of the method according to the first aspect of the invention has its own read-out (based on the level of precipitated (poly)peptides) for diagnosing a subject as being positive for cancer and / or an acute inflammatory disease it may also be formulated as independent embodiment. Accordingly, the invention also relates to a method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:

[0100] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and

[0101] (b) assessing the level of the precipitated (poly)peptides in the sample based on a statistical comparison relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be positive for cancer and / or an acute inflammatory disease (pathological reference samples) and / or relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be negative for cancer and an acute inflammatory disease (non-pathological reference samples), wherein the statistical comparison is conducted by:

[0102] (aa) an area under the curve (AUC) calculation; wherein preferably:

[0103] (aa-1) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of pathological reference samples (P) by calculating an ALICp value as defined by formula (I): wherein np is the number of reference samples in the group of pathological reference samples P, x;is the level of the precipitated (poly)peptides for the ithpathological reference sample in P, and k is the indicator function for comparing S against x, as defined by formula (II): and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an AUCp value is calculated in the range of between, with increasing preference, >0.75 and 1.0; >0.7 and 1.0, >0.6 and 1.0, >0.5 and 1.0, >0.4 and 1.0, >0.3 and 1.0, >0.2 and 1.0, >0.001 and 1.0, and most preferably between >0 and 1.0; and / or

[0104] (aa-2) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of non-pathological reference samples ( / V) by calculating an AUCw value as defined by formula (III): where n«is the number of samples in the group non-pathological reference samples N, Xt is the level of the precipitated (poly)peptides for the Ithnon-pathological reference sample in A / , and k is the indicator function for comparing S against x, as defined by formula (II): and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an AUCw value is calculated in the range of between, with increasing preference, 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, 0.99 and 1.0, most preferably of 1.0.

[0105] As used herein, the term “pathological reference sample” refers to a correspondingly treated sample obtained from a reference subject known to be positive for cancer and / or an acute inflammatory disease, and the term “non-pathological reference sample” refers to a correspondingly treated sample obtained from a reference subject known to be negative for cancer and an acute inflammatory disease.

[0106] With respect to the ALICp, it is to be understood that the specified values with increasing preference more and more exclude the possibility of detecting a false negative result, i.e., the diagnosis of a pathological subject as a non-pathological subject. With respect to ALICN, it is to be understood that the specified values with increasing preference more and more exclude the possibly of detecting a false positive result, i.e., the diagnosis of a non-pathological subject as a pathological subject.

[0107] Thus, in preferred embodiments, the group of pathological reference samples comprises or consists of at least, with increasing preference, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75 and 100 pathological reference samples.

[0108] Likewise in preferred embodiments, the group of non-pathological reference samples comprises or consists of at least, with increasing preference, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75 and 100 pathological reference samples.

[0109] As shown in the herein disclosed experimental evidence (cf. Examples 1 and 2), applying an acid, in particular 0.4% (v / v) acetic acid, to the sample to result in a sample pH of about pH 4.2 was particularly suitable for effecting the precipitation of one or more (poly)peptides and, importantly, at levels on which basis a reliable discrimination between pathological and non-pathological samples can be established.

[0110] Thus, in preferred (or even more preferred) embodiments, the precipitation-inducing condition is provided by an alteration of the sample pH, preferably by adjusting the sample pH to a value in the range of between, with increasing preference, 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, pH 4.1 and 4.3, most preferably pH 4.2±0.05.

[0111] In view of the herein demonstrated suitability of acetic acid having a pKa of about 4.76, also other acids, preferably other weak acids, in particular other carboxylic acids, having a pKa in a similar range as acetic acid, are suitable for being employed in the herein disclosed methods. This is demonstrated in Example 3 and corresponding Figures 10.2A-C, wherein formic acid - which like acetic acid, is a (mono-)carboxylic acid and has a similar pKa of about 3.75 - has been suitably employed as a precipitation-inducing agent. It is also demonstrated in Example 3 that citric acid (pKa 3.13, 4.76, 6.4; Fig 10.1) and perchloric acid (pKa = -10; Fig. 10.3) in principle work.

[0112] In preferred embodiments of the latter embodiments, the adjustment of the sample pH is provided by adding, to the sample:

[0113] (a) at least one acid having a pKa in the range of between, with increasing preference, 2.0 and -10.0, 3.0 and 7.0, 3.5 and 6.5, 3.8 and 5.8, 4.1 and 5.5, and most preferably a pKa of 4.76±0.05; wherein preferably the at least one acid is an organic acid, wherein preferably the organic acid is:

[0114] (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 di- or tri-carboxylic acid, preferably citric acid;

[0115] (ii) barbituric acid; and / or

[0116] (iii) perchloric acid; and / or

[0117] (b) an aqueous solution comprising at least one acid as defined in (a); wherein preferably:

[0118] - the aqueous solution comprises the at least one acid at a total concentration in the range of between, with increasing preference, 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%, 0.35 vol% and 0.45 vol%, most preferably at a concentration of 0.4 vol%;

[0119] - the aqueous solution has a pH in the range of between, with increasing preference, pH 2 and pH 5, pH 2 and pH 4, pH 2.5 and pH 3.5, pH 2.8 and pH 3.2, pH 2.9 and 3.1 , and most preferably to a value of pH 2.97±0.05;

[0120] - the aqueous solution is added to the sample at a volume-to-volume ratio of aqueous solution to sample of between, with increasing preference, 0.3:1 and 0.9:1 , 0.4:1 and 0.8:1 , 0.5:1 and 0.7:1 , most preferably at a concentration of 0.59:1 ; and / or

[0121] - the aqueous solution additionally comprises sodium chloride at a concentration in the range of between, with increasing preference, 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), 0.7% (w / v) and 0.9% (w / v), most preferably at a concentration of 0.81 % (w / v).

[0122] In other or even more preferred embodiments, the adjustment of the sample pH is provided by adding to the sample an acid, wherein the acid is selected from: acetic acid; wherein preferably the acetic acid is added to the sample to result in a final concentration of between, with increasing preference, 0.02 vol% and 0.28%, 0.05 vol% and 0.25 vol%, 0.1 vol% and 0.2 vol%, and most preferably 0.15 vol%; citric acid; wherein preferably the citric acid is added to the sample to result in a final concentration of between, with with increasing preference, 0.1 vol% and 10%, 0.5 vol% and 8 vol%, 1 vol% and 7 vol%, 2 vol% and 6 vol%, 3 vol% and 5 vol%, and most preferably 3.7 vol%; formic acid, wherein preferably the formic acid is added to the sample to result in a final concentration of between, with increasing preference, 0.04 vol% and 0.40 vol%, 0.06 vol% and 0.35 vol%, 0.07 vol% and 0.30 vol%, and most preferably about 0.074 vol%; and / or perchloric acid, wherein preferably the perchloric acid is added to the sample to result in a final concentration of between, with increasing preference, with increasing preference, 0.20 vol% and 0.50 vol%, 0.25 vol% and 0.40 vol%, 0.30 vol% and 0.35 vol%, and most preferably about 0.324 vol%.

[0123] In preferred embodiments, the sample comprises or consists of:

[0124] (a) a body fluid, preferably selected from blood, saliva, mucus, sputum, vomitus, sweat, tear, urine, semen, vaginal fluid, feces, and exudate, or any mixture thereof; and / or

[0125] (b) a body tissue, preferably a homogenized body tissue, more preferably a cell-free suspension of a homogenized body tissue.

[0126] In particularly preferred embodiments, the sample comprises or consists of blood. As used herein, the term “blood” encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined. The blood (sample) is preferably blood plasma.

[0127] Hence, in an even more preferred embodiment, the sample comprises, consists essentially of, or consists of plasma (blood plasma). As used herein, the term “consists essentially of” means that the referred content makes up at least, with increasing preference, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the entire volume or mass. Means and methods for separating plasma from whole blood are well known and routinely employed in the art (see, e.g., 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 a whole blood to which an anticoagulant has been added, or alternatively be collected directly through a process called plasmapheresis (see, e.g., Madore F. Grit Care Clin. 2002 Apr;18(2):375-92). For example, for the purposes of the present invention, plasma may suitably be obtained by collection of whole blood directly into commercially available blood collection tubes (e.g., S-Monovette® K2-EDTA, 9 ml, Sarstedt; https: / / www.sarstedt.com / ) which are manufactured to already comprise an anticoagulant (e.g., K3-EDTA or K2-EDTA or other anticoagulant, preferably K2-EDTA), and which may then be immediately ( / .e., within < 20 min after blood drawing) subjected to centrifugation (e.g., 3000 x g / 10 min) to separate plasma from remaining blood components.

[0128] The term “blood plasma”, also referred to herein more briefly as “plasma”, is the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants as upper liquid layer. Plasma is the cell-free liquid fraction of blood and contains serum proteins and clotting factors (including fibrinogen). The term “blood serum”, also referred to herein more briefly as “serum”, refers to the watery portion of fluid obtained through centrifugation after coagulation (no addition of anticoagulants, i.e., no clotting inhibitor is added) of blood, so that the coagulation and fibrinolytic factors are not present. In other words, serum is the liquid that remains after the blood has clotted; and plasma is the liquid that remains when clotting is prevented with the addition of an anticoagulant. Plasma can also be converted to serum by the method of defibrination (see, e.g., Castro AR et al., (2002) Clin Diagn Lab Immunol. ; 9 (6): 1376-8).

[0129] Upon obtaining a whole blood sample from a subject, the drawn blood is preferably exposed immediately to an anticoagulant to preclude coagulation. The term “anticoagulant”, as used herein, refers to a chemical moiety that hinders the clotting of blood. Known anticoagulants include heparin, ethylenediaminetetraacetic acid (EDTA), D-Phe-Pro-Arg chloromethyl ketone dihydrochloride (“PPACK”), and citrate.

[0130] Thus, in certain preferred embodiments, the sample additionally comprises:

[0131] (i) an anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin; and / or

[0132] (ii) one or more proteinase inhibitors.

[0133] In particularly preferred embodiments, the anticoagulant is EDTA or a salt (preferably a sodium or potassium salt) thereof, more preferably tri-potassium EDTA (K2-EDTA). In other preferred embodiments, the anticoagulant is tri-sodium EDTA (Na2-EDTA).

[0134] In other embodiments, the anticoagulant is citrate or a salt (preferably a sodium or potassium salt) thereof, more preferably tri-sodium citrate (NasCeHsO?). In other embodiments, the anticoagulant is tri-potassium citrate (K3C6H5O7).

[0135] In other embodiments, the anticoagulant is heparin. “Heparin” is a sulfated glycosaminoglycan and is used clinically for the prevention of blood clots. The term “heparin”, as used herein, refers to all forms of heparin, including, but not limited to, unfractionated heparin, heparinoids, dermatans, chondroitins, low molecular weight heparin (e.g., tinzaparin (including tinzaparin sodium)), very low molecular weight heparin, and ultra-low molecular weight heparin. Non-limiting examples include unfractionated heparin, such as heparin sodium (e.g., heparin sodium USP, available from Scientific Protein Labs of Waunakee, Wl).

[0136] In order to prevent an unintended proteolytic degradation of the (poly)peptides comprised the sample, it may be advantageous to add one or more protease inhibitors to the sample. The term ’’protease inhibitor", as used herein, refers generally to a compound, substance and / or composition which is capable of inhibiting the action of one or more proteases. Particularly preferred protease inhibitors which may be employed in connection with the methods of the present invention are those capable of inhibiting the activity of proteases known (or typically suspected) to be comprised in the particular kind of sample, e.g., in blood.

[0137] Known protease inhibitors which may be suitably employed in connection with the disclosed methods include, but are not limited to, inhibitors of serine proteases, cysteine proteases, aspartic proteases, metalloproteases, thiol proteases, exopeptidases and the like. Of these, serine and cysteine protease inhibitors are of particular interest, with metalloprotease inhibitors also being significant. Non-limiting examples of serine protease inhibitors include antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), AEBSF, TLCK, TPCK, leupeptin and soybean trypsin inhibitor. Inhibitors of cysteine proteases include, for example, IAA (indoleacetic acid) and E-64. Suitable examples of aspartic protease inhibitors include pepstatin and VdLPFFVdL. Non-limiting examples of inhibitors of metalloproteases include EDTA, as well as 1 ,10-phenanthroline and phosphoramodon. Hence, EDTA may act as anticoagulant and / or proteinase inhibitor. Inhibitors of exopeptidases include, for example, amastatin, bestatin, diprotin A and diprotin B. Additional suitable examples of protease inhibitors include alpha-2- macroglobulin, soybean or lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin and egg white cystatin. In preferred embodiments, combinations of protease inhibitors may be employed, such as commercially available “protease inhibitor cocktails”, for example, Roche - complete™ Protease Inhibitor Cocktail and SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free.

[0138] In even further preferred embodiments, the sample comprises plasma and EDTA or a salt thereof (preferably potassium EDTA, such as di- or tri-potassium EDTA (K2- or K3-EDTA)). A corresponding sample is conventionally referred to as “EDTA-plasma” sample. In even more preferred embodiments, the sample is a plasma sample which additionally comprises EDTA or a salt thereof (preferably potassium EDTA, such as K2- or K3-EDTA) at a concentration of between 0.5 and 2.7 mg / ml, even more preferably at a concentration of between 1.1 and 2.1 mg / ml, even more preferably at a concentration of between 1.2 and 2.0 mg / ml, most preferably at a concentration of 1.6 mg / ml.

[0139] In the specific assay-protocol established herein (as illustrated in Fig. 2B), a 26 pl EDTA-plasma sample, prior to being subjected to the precipitation-inducing condition, was first diluted with (i) 70 pl of 0.9 % (w / v) NaCI (cf. step 1), and the obtained reaction mixture of 96 pl was then (ii) further diluted with 40 pl distilled H2O to result in a total sample volume of 136 pl. However, in alternative settings, a 26 pl EDTA-plasma sample was diluted with 110 pl of an aqueous solution comprising 0.57% NaCI (corresponding to 98 mM NaCI).

[0140] Thus, in accordance with preferred embodiments, the sample, prior to being subjected to the precipitation-inducing condition, is diluted with a dilution medium, wherein the dilution medium preferably:

[0141] (i) is an aqueous solution comprising a salt, preferably sodium chloride (NaCI), at a concentration in the range of between, with increasing preference, 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, and most preferably 98 mM; and / or

[0142] (ii) is added to the sample at a volume-to-volume(v / v)-ratio of dilution medium to sample of between, with increasing preference, 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 , and most preferably at a v / v-ratio of dilution medium to sample of 4.2: 1.

[0143] In alternative preferred embodiments, the sample, prior to being subjected to the precipitationinducing condition, is:

[0144] (i) diluted with a first dilution medium, wherein the first dilution medium:

[0145] (i-a) is an aqueous solution comprising a salt, preferably sodium chloride (NaCI), at a concentration in the range of between, with increasing preference, 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, and most preferably 154 mM; and / or (i-b) is added to the sample at a volume-to-volume(v / v)-ratio of first dilution medium to sample of between, with increasing preference, 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 4.2:1 , 1.4:1 and 4:1 ; 1.6:1 and 3.8:1 , 1.8:1 and 3.6:1 , 2:1 and 3.4:1 , 2.4:1 and 3:1 , and most preferably at a v / v-ratio of first dilution medium to sample of 2.69:1 ; and

[0146] (ii) diluted with a second dilution medium, wherein the second dilution medium:

[0147] (ii-a) comprises or consists of water; and / or

[0148] (ii-b) is added to the sample obtained in (i) at a volume-to-volume(v / v)-ratio of second dilution medium to sample of between, with increasing preference, 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 , 0.3:1 and 0.5:1 , and most preferably at a v / v-ratio of second dilution medium to sample of 0.42:1 .

[0149] In other alternative preferred embodiments, the sample, prior to being subjected to the precipitation-inducing condition, is:

[0150] (i) diluted with a first dilution medium, wherein the first dilution medium:

[0151] (i-a) comprises or consists of water; and / or

[0152] (i-b) is added to the sample obtained in (i) at a volume-to-volume(v / v)-ratio of first dilution medium to sample of between, with increasing preference, 0.05:1 and 3:1 , 0.5:1 and 2.5:1 , 0.8:1 and 2.2:1 ; 1 :1 and 2:1 , 1.3:1 and 1.8:1 , 1.4:1 and 1.7:1 , 1.5:1 and 1.6:1 , and most preferably at a v / v-ratio of first dilution medium to sample of 1.54:1 ; and

[0153] (ii) diluted with a second dilution medium, wherein the second dilution medium:

[0154] (ii-a) is an aqueous solution comprising a salt, preferably sodium chloride (NaCI), at a concentration in the range of between, with increasing preference, 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, and most preferably 154 mM; and / or

[0155] (ii-b) is added to the sample at a volume-to-volume(v / v)-ratio of second dilution medium to sample of between, with increasing preference, 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 , 0.9:1 and 1.2:1 , and most preferably at a v / v-ratio of second dilution medium to sample of 1.06:1.

[0156] Exemplary salts which may be comprised in the above-referred aqueous solutions include, without intention to be limiting, sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCh), and magnesium chloride (MgCh).

[0157] In preferred embodiments, the precipitation-inducing condition in step (b) is provided, or additionally provided, by:

[0158] (i) an increase of the sample temperature;

[0159] (ii) addition of at least one salting-out agent to the sample; wherein preferably the salting-out agent is selected from:

[0160] (ii-a) a salt comprising a cation selected from Ca2+, (CH3)4N+, Cs+, guanidinium (C(NH2)3+), Rb+, NH4+, K+, Na+, Mn2+, and Li+; and an anion selected from OH", SO42", HPO42", CH3COO“, C3H5O(COO)33", CO32", CIO3", BrOs", thiocyanate (SON"), and Cl"; more preferably KCI and / or (NH4)2SO4; and / or

[0161] (ii-b) an alkali metal sulfate; preferably U2SO4, Na2SO4, K2SO4, Rb2SO4, and / or CS2SO4;

[0162] (iii) addition of one or more other aliphatic alcohol(s), preferably alkanol(s), and most preferably ethanol;

[0163] (iv) concentration of the sample by evaporation;

[0164] (v) addition of a ketone, preferably acetone;

[0165] (vi) ionizing radiation (IR); and / or

[0166] (vii) any combination of any of (i) to (vi).

[0167] In preferred embodiments, the increase of the sample temperature is provided by exposing the sample to a temperature in the range of between, with increasing preference, 38°C and 99°C, 45°C to 95°, 50°C to 90°, 55°C to 85°C, 60°C to 80°C, 65°C to 75°, and most preferably to a temperature of 70°C±2°C.

[0168] The skilled person will understand that the ideal duration ( / .e., time period) of the heat exposure for inducing precipitation of one or more (poly)peptides in the sample before conducting the assessment of the level of the precipitated (poly)peptides in the sample will depend on various factors, such as the sample volume and / or the material of the reaction tube / device. It will, however, be within the routine skills of the skilled artisan to select a suitable duration for the heat exposure, i.e., sufficiently long to induce precipitation of one or more (poly)peptides in the sample, yet short enough to not result in a precipitation of all (poly)peptides comprised in the sample. Yet, in preferred embodiments, the exposure of the sample to the above-referred temperature will be for a duration of between, with increasing preference, 2 s to 10 min, 10 s to 5 min, 20s to 3min, 30s to 2min, most preferably 60s. The above temperatures and times can be combined, for example, with increased preference 65°C to 75° for 30s to 2min, 70°C±2°C for 30s to 2min, and 70°C±2°C for 60s.

[0169] In preferred embodiments, the at least one salting-out agent is selected from the group of (NH4)2SO4, alkali-metal chloride salts (preferably selected from KCI and / or CaCh), guanidine thiocyanate, and MnCh, and is most preferably (NH4)2SO4.

[0170] In preferred embodiments, the salting-out agent, preferably (NH4)2SO4, is added to the sample to result in a final concentration in a range of between, with increasing preference, 1-40% (w / v), 4- 30% (w / v), 6-25% (w / v), 8-20% (w / v), 10-16% (w / v), 12-14% (w / v), most preferably in a final concentration of 13% (w / v).

[0171] In preferred embodiments, the one or more other aliphatic alcohol(s) is / are selected from ethanol, isopropanol, and / or methanol, and is most preferably ethanol.

[0172] The final aliphatic alcohols (preferably ethanol, isopropanol, and / or methanol) concentration as used for inducing the precipitation is between, with increasing preference, between 10.0% (v / v) and 40.0% (v / v), 15.0% (v / v) and 35.0% (v / v), and most preferably between 18.5% (v / v) and 27.8% (v / v).

[0173] The final ketone (preferably acetone) concentration as used for inducing the precipitation is between, with increasing preference, 8.5% (v / v) and 28.5% (v / v), 13.5% (v / v) and 23.5% (v / v), 16% (v / v) and 21 % (v / v), and most preferably 18.5% (v / v).

[0174] In an even more preferred embodiment, the salting-out agent, preferably (NH^SOt, is added to the sample as comprised in an aqueous solution comprising the salting-out agent, preferably (NH4)2SO4, at a concentration in a range of between, with increasing preference, 10-60% (w / v), 20-50% (w / v), 25-45% (w / v), 30-40% (w / v), most preferably at a concentration of 35% (w / v); and wherein the aqueous solution is added to the sample at a volume-to-volume ratio of aqueous solution to sample of between, with increasing preference, 0.3:1 and 0.9:1 , 0.4:1 and 0.8:1 , 0.5:1 and 0.7:1 , most preferably at a volume-to-volume ratio of aqueous solution to sample of 0.59: 1 .

[0175] In preferred embodiments of the methods according to the first, third and fourth aspect of the invention, the level of the precipitated (poly)peptides is assessed in step (b) by spectrophotometry, nephelometry, spectrofluorometry, circular dichroism (CD) spectroscopy, mass spectrometry (MS) and / or NMR spectroscopy.

[0176] In particularly preferred embodiments, the level of the precipitated (poly)peptides is assessed by spectrophotometry.

[0177] The term “spectrophotometry” or “photometry” refers to a well-known and widely used optical analytical technique that measures the amount of light ( / .e., discrete wavelengths of ultraviolet (UV, 200-400 nm) or visible (vis, 400-800 nm) light) that is absorbed by, or transmitted through, a sample. The absorbance (A), herein also interchangeably referred to as extinction (E), is equal to the logarithm of the ratio of the intensity of light before passing through the sample ( / 0) to the intensity of light after passing through the sample ( / ). Although absorbance is per definition a dimensionless quantity, it is typically expressed as absorbance unit (AU) or extinction unit (E), or milli-absorbance unit (mAU) or milli extinction unit (mE).

[0178] The term “spectrophotometry”, as used herein in its broadest sense, also encompasses and preferably means “turbidimetry”, which refers to a method well-known in the art for determining the amount of cloudiness, or turbidity, in a solution based upon measurement of the effect of this turbidity upon 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. If a beam of light is passed through a turbid sample, its intensity is reduced by scattering, and the quantity of light scattered is dependent upon the concentration, size and size distribution of the particles. The spectrophotometer can thus also measure an increased turbidity based on the reduction of the intensity of the transmitted light. This increased turbidity thus can provide a direct measure of the formation of precipitated (poly)peptides.

[0179] The term “nephelometry”, as used herein, refers to a technique related to “turbidimetry” and is also well known in the art. Whereas in turbidimetry, the amount / intensity of light transmitted through the sample is measured, in nephelometry, the amount / intensity of the light scattered is measured at a defined angle (typically 90°) from the incident beam.

[0180] Generally, for the purpose of spectrophotometric analyses of liquid samples, the solution must be placed into the light path of a photometer in a defined format. Cuvettes, i.e., sample containers featuring optical windows, are the standard choice for this application. The distance between the optical windows is accurately defined; in this way, the path length of the sample inside the cuvette is known. The most common type of cuvette is square. This format typically accommodates sample volumes from the microliter range (ultra-micro cuvettes) to the milliliter range (macro cuvettes). The standard pathlength of a cuvette is 10 mm. However, cuvettes that provide a shorter light path through the sample are also available. For example, it may be convenient in terms of reducing the volumes / amounts of sample and reagents needed for a measurement, and / or for the sake of reducing space in the laboratory or within a spectrophotometer or sample changer, to using a cuvette which has a shorter pathlength (< 1 cm). As is done by many spectrophotometric instruments, the output generated from the measurement using a cuvette which has a pathlength distinct to 1 cm is typically provided (automatically back-calculated), if desired, as if the measurement would have been conducted in a 1 cm pathlength-cuvette. For example, in the examples herein below a cuvette which has a pathlength of 0.7 cm is used and the spectrophotometric instrument normalizes the output to correspond to a cuvette which has a pathlength of 1.0 cm. Standard cuvettes made from PMMA, polystyrene or normal glass are typically only transparent in the visible range. If wavelengths in the UV-range, in particular, at wavelengths below approximately 300 nm, are employed, cuvettes made from quartz glass, or a special type of plastic, which provide sufficient transparency in this range, should be used. Spectrophotometers, when using a cuvette with a pathlength of 1 cm (10 mm), typically have a linear absorbance range up to about 2.5 A ( / .e., 2500 mE). If a sample is to be analyzed which gives rise to an absorbance exceeding this value, it may be beneficial for enhancement of the accuracy to dilute the sample, e.g., with a dilution medium (such as an isotonic salt solution) so that the detectable absorbance will be reduced.

[0181] In the herein disclosed examples, the spectrophotometric analysis was conducted using a conventional clinical chemistry analyzer, i.e., the Indiko™ Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). However, a variety of further devices suitable for conducting respective analyses are known in the art and commercially available and may also suitably be employed for the herein disclosed purposes.

[0182] An exemplary, particularly preferred precipitation-inducing condition is the addition of at least one acid having a pKa in the range of between 3.5 and 6.0, more preferably between 4.1 and 5.5, and even more preferably a pKa of 4.75±0.05; wherein 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% NaCI, which is added to the sample to result in a sample pH of between, with increasing preference, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, pH 4.1 and 4.3, and most preferably pH 4.2±0.05.

[0183] In particularly preferred embodiments, the level of the precipitated (poly)peptides is assessed by spectrophotometrically measuring a change of absorbance, whereby a determined increase or decrease of the absorbance is indicative of an increase or decrease, respectively, of the level of precipitated (poly)peptides in the sample.

[0184] As used herein, the term “change of absorbance” refers to a potential increase or decrease of the detected absorbance caused by the provision of a precipitation-inducing condition. In a typical setting, and thus in particularly preferred embodiments, the measured absorbance will be set to zero (“blank”) at a timepoint shortly before, or alternatively at the (approximate) same timepoint when, the precipitation-inducing condition is provided. In such an embodiment, the measured change of absorbance will thus correspond to the (net-)change of absorbance caused by the provision of the precipitation-inducing condition, and thereby allowing a directed comparison relative to the change of absorbance measured for a correspondingly treated reference sample and / or a predetermined standard.

[0185] In the herein disclosed experimental evidence, the level of precipitated (poly)peptides formed as a result of the provision of the precipitation-inducing condition was, inter alia, measured spectrophotometrically at a wavelength of 340 nm. This wavelength was found particularly sensitive for the detection of precipitated (poly)peptides. It is believed that also similar wavelengths will be essentially equally suitable for the herein contemplated purposes.

[0186] Thus, in preferred embodiments, the absorbance is measured at a wavelength in the range of between, with increasing preference, 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, 335 nm and 345 nm and most preferably at 340 nm.

[0187] As reported in the herein disclosed experimental evidence (cf. Example 1 and 2, Figure 7A-C and Table 1 and 2A), on account of the herein reported differential precipitation susceptibility, the present inventor further conducted extensive research by systematically assessing a vast amount plasma samples (355 in total) originating from subjects of either pathological (114) or non- pathological (241) background in order to identify potential spectrophotometrically observable differences which would allow a reliable discrimination of the samples in a origin dependent manner (see Table 1). It was thereby surprisingly and advantageously found that, when applying the spectrophotometric assay as illustrated in Figure 2B, the absorbance changes detected for pathological and non-pathological samples fall into two separate, well resolved ranges. As apparent from Table 2A, a subsequent calculation with respect to the various performance indicators (e.g., specificity, sensitivity, and accuracy) revealed that, when applying a measured absorbance increase of at least 78 mE as a minimum threshold for assigning a sample as positive for cancer and / or an acute inflammatory disease, an accuracy of > 95% is achieved. This accuracy is basically maintained, in part even superseded up to a threshold of 170 mE. When applying thresholds beyond 170 mE, the calculated specificity remains high, yet the sensitivity slightly starts to decline. Highest accuracies (99.44%) were calculated for thresholds within the range of between 105 and 134 mE. These findings demonstrate that an accurate discrimination between pathological and non-pathological samples, and hence a reliable diagnosis, will be feasible solely based on an assessment of a sample by conducting the herein disclosed spectrophotometric assay, and comparing the measured change of absorbance to the above- mentioned empirically determined thresholds.

[0188] Hence, in preferred embodiments of the latter embodiments, in the method for diagnosing cancer and / or an acute inflammatory disease in a subject, if the change of absorbance is an absorbance increase in the range of between, with increasing preference, 78-170 milli extinction units (mE), 80-160 mE, 83-155 mE, 88-150 mE, 93-145 mE, 98-140 mE, most preferably 105-134 mE, preferably when measured in a 1 cm-pathlength cuvette or when normalized to a measurement in a 1 cm-pathlength cuvette, the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease. Since the above preferred embodiments of the latter embodiments of the method according to the first aspect of the invention have its own read-out for diagnosing a subject as being positive for cancer and / or an acute inflammatory disease (based on the level of precipitated (poly)peptides) they may also be formulated as independent embodiment. Accordingly, the invention also relates to a method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:

[0189] (a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and

[0190] (b) assessing the level of the precipitated (poly)peptides in the sample relative to:

[0191] (b-i) the level 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 an acute inflammatory disease;

[0192] (b-ii) the level of precipitated (poly)peptides in at least one correspondingly treated reference sample obtained from one or more reference subjects known to be negative for cancer and an acute inflammatory disease; and / or

[0193] (b-iii) a predetermined standard which has been determined based on (b-i) and / or (b-ii); wherein the level of the precipitated (poly)peptides is assessed by spectrophotometrically measuring a change of absorbance, whereby a determined increase of the absorbance is indicative of an increase or decrease, respectively, of the level of precipitated (poly)peptides in the sample, and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if the change of absorbance is an absorbance increase in the range of between, with increasing preference, 78-170 milli extinction units (mE), 80-160 mE, 83-155 mE, 88-150 mE, 93-145 mE, 98-140 mE, most preferably 105-134 mE, preferably when measured in a 1 cm-pathlength cuvette or when normalized to a measurement in a 1 cm-pathlength cuvette; and wherein preferably:

[0194] - the precipitation-inducing condition in (a) is provided by addition of at least one acid having a pKa in the range of between 3.5 and 6.0, preferably between 4.1 and 5.5, and more preferably a pKa of 4.75±0.05; and 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 % NaCI, which is added to the sample to result in a sample pH of between, with increasing preference, pH 3.8 and pH 4.6, pH 4.0 and pH 4.4, pH 4.1 and 4.3, and most preferably pH 4.2±0.05; and / or

[0195] - the change of absorbance is measured at a wavelength in the range of between, with increasing preference, 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, 335 nm and 345 nm and most preferably at 340 nm.

[0196] It is hence understood by the skilled artisan, that the above-referred absorbance ranges which are employed as a diagnostic decision threshold correspond to predetermined standards (as mentioned in the method according to the first aspect of the invention) which have been determined from correspondingly treated reference samples of pathological or non-pathological origin.

[0197] In preferred embodiments, the level of the precipitated (poly)peptides is assessed in step (b) from the remaining soluble fraction of the sample, preferably after removal of the precipitated (poly)peptides.

[0198] When conducting the herein disclosed methods, the inventor typically implemented one or more incubation steps, wherein the sample was allowed to equilibrate prior to its analysis. More specifically, the inventor typically incubated the sample before subjecting it to a precipitationinducing condition at a temperature of 37°C, e.g., for a duration of about 300 s (see Fig. 2B, step 4). Such an equilibration step may particularly be employed in cases where the sample, prior to the provision of the precipitation-inducing condition, is diluted with a dilution medium as described herein.

[0199] Moreover, as is apparent from Figure 4, the inventor found that the increase of the absorbance caused by the provision of the precipitation-inducing condition can be detected immediately from the time point onwards at which the precipitation-inducing condition was applied to the sample. It was further found that this increase, although starting to build up immediately, evolves for a couple of seconds wherein the absorbance increases further until reaching a plateau at which the absorbance remains nearly constant (for hours). Therefore, the sample - after the provision of the precipitation-inducing condition (see Fig. 2B, step 6)- was typically incubated at 37 °C for 300 s before being analysed, e.g., by spectrophotometry (see Fig. 2B, step 7).

[0200] Thus, in particularly preferred embodiments,

[0201] (i) the sample, prior to being subjected to the precipitation-inducing condition in step (a), is incubated at a temperature of between 2 °C and 42 °C, preferably at 37 ± 2 °C, for a duration of at least 10 s, preferably for a duration in the range of between, with increasing preference, 10 s and 5000 s, 20 s and 4000 s, 30 s and 3000 s, 40 s and 2000 s, 50 s and 1000 s, 60 s and 800 s, 80 s and 600 s, 100 s and 500 s, 200 s and 400 s, 250 s and 350 s, preferably between 275 s and 325 s; most preferably for 300 s; and / or

[0202] (ii) the sample, after being subjected to the precipitation-inducing condition in step (a), and prior to step (b), is incubated at a temperature of between 2 °C and 42 °C, preferably at 37 ± 2 °C, for a duration of at least 60 s, preferably for a duration in the range of between, with increasing preference, 60 s and 600 s, 100 s and 500 s, 200 s and 400 s, 250 s and 350 s, 280 s and 320 s; most preferably for 300 s. In other (or even more preferred) embodiments, the measurement of the absorbance is conducted within a time period of between, with increasing preference, 60 s and 600 s, 100 s and 500 s, 200 s and 400 s, 250 s and 350 s, 280 s and 320 s, most preferably for 300 s after the provision of the precipitation-inducing condition in step (a).

[0203] In preferred embodiments, the sample and / or the dilution medium additionally comprises:

[0204] (i) an anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin or any salt thereof;

[0205] (ii) a proteinase inhibitor;

[0206] (iii) one or more stabilizing agents, preferably selected from albumin, casein, gelatin, collagen, globulin, protamine;

[0207] (iv) skim milk powder;

[0208] (v) a surfactant; preferably selected from Tween, preferably Tween 20 or Tween 80, Triton X- 100, and sodium dodecylbenzenesulfonate;

[0209] (vi) a polyethylene glycol (PEG); wherein preferably the PEG has a molecular weight of between 1 ,000 and 20,000 Da and / or is selected from PEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, and PEG20000; and / or

[0210] (vii) a polysaccharide; preferably a dextran selected from dextran-1 , dextran-10, dextran-20, dextran-30, and dextran-40 dextran.

[0211] Among the above options (i) to (vii), option (i) is particularly preferred. In alternative preferred embodiments, the sample and / or the dilution medium does not additionally comprise any of (i) to (vii).

[0212] In preferred embodiments, in particular when the sample is a human sample, the dilution medium does not comprise any constituents originating or derived from a human. Thus, in preferred embodiments, the albumin is a non-human albumin, for example, bovine serum albumin (BSA); and / or the heparin is non-human heparin. However, in other preferred embodiments, the albumin is human serum albumin. In further preferred embodiments, the milk (poly)peptide(s) and / or the skim milk powder is / are of non-human origin, more preferably from cow milk.

[0213] In connection with the above embodiments, it is understood that where the additive is a (poly)peptide, e.g., serum albumin, or other animal product, e.g., milk powder, that said additive does not originate from the same subject and / or species from which the sample has been obtained; and does also not originate from a subject and / or species which, at the time when said additive was obtained, had an acute inflammation and / or a cancer. Standardized albumin preparations such as bovine or human serum albumin, milk proteins or milk powder are commercially available and may be purchased and employed for the herein disclosed purposes.

[0214] In preferred embodiments of the methods according to the first, second, and / or any further aspect of the invention, the method(s) further comprise(s) assessing a sample, preferably a blood sample, obtained from the subject for the presence or absence of an acute inflammation, wherein, in the instance of a subject for which an indication to have cancer and / or an acute inflammatory disease is determined, a determined absence of an acute inflammation indicates that the subject has cancer and no acute inflammatory disease. Thus, in the latter scenario, the subject is diagnosed as being positive for cancer and as having no acute inflammatory disease.

[0215] Such an additional evaluation for the presence of an acute inflammation may be suitably conducted by various conventional methods for assessing the presence of an acute inflammation, such as described below, and a respective evaluation is in particular contemplated in cases where the outcome of the method(s) according to the first, second and / or further aspect of the invention points towards the presence of a cancer and / or an acute inflammatory disease.

[0216] It might thus be of interest to conduct a further analysis to verify whether this finding is caused by a presence of an acute inflammation. Only in case the latter condition can be ruled out, a clear indication towards the presence of cancer is provided. On the other hand, in cases where the outcome of the method according to the first, second, and / or any further aspect of the invention provides an indication to the presence of cancer and / or an acute inflammatory disease, and where the presence of an acute inflammation is confirmed by such an additional evaluation, the possibility of a simultaneous presence of cancer besides an acute inflammation cannot be excluded.

[0217] It will be readily understood by those skilled in the art that the sample may, if convenient and applicable dependent on the individual method employed for assessing the presence of an acute inflammation, pertain to the same sample, or a portion or fraction thereof, as the sample obtained from the subject and employed in step (a) of the method according to the first, second and / or further aspect of the invention, or that the sample may be a distinct, further sample obtained from the subject. For example, if the sample employed for the initial analysis conducted in connection with the method according to the first, second and / or further aspect of the invention is a cell-free sample (e.g., a blood plasma sample), and the inflammation were to be assessed through assessment of the level of certain cells known to be indicative of the presence of an acute inflammation (e.g., increased levels of white blood cells (leukocytes)), then a further sample needs to be obtained from the subject. In practice, it may be convenient to initially obtain a whole blood sample from the subject, and to prepare a plasma sample from only a portion thereof, whereas the remaining whole blood sample may then be employed for the analysis of the presence of absence of an acute inflammation.

[0218] In preferred embodiments of the latter embodiments, the presence or absence of an acute inflammation is assessed by evaluating the level(s) of one or more inflammatory markers; wherein preferably the one or more inflammatory markers are selected from C-reactive protein (CRP), procalcitonin (PCT), fibrinogen, and leukocytes.

[0219] In preferred embodiments, the presence or absence of an acute inflammation is assessed by evaluating the levels of, with increasing preference, at least one, at least two, at least three, all four of the above-referred inflammatory markers, or by additionally evaluating the level(s) of any further known inflammatory marker(s).

[0220] In particularly preferred embodiments, the presence or absence of an acute inflammation is assessed by evaluating the level of CRP. The level of CRP is generally determined in a sample, wherein the sample is preferably a blood sample or blood-derived sample (e.g. plasma or serum), and most preferably plasma.

[0221] “C-reactive protein” (CRP) is a liver-produced glycoprotein characterized by precipitation with pneumococcal C-polysaccharide. This protein is not normally produced. In the presence of acute inflammation, body tissue is destroyed causing the release of interleukins 1 and 6 that stimulate the production of this protein and cause a rapid increase in CRP levels (hence this protein also known as reactive protein of the acute phase). Once the acute inflammation is over, CRP rapidly disappears. Therefore, CRP is considered as a marker reflecting the activation of the systemic inflammatory response. CRP is non-specific and its levels are elevated in all acute inflammatory conditions. Typically, CRP will increase within 6 hours of acute inflammation allowing an early inflammation early. The skilled artisan will be aware that two types of CRP tests are routinely used in the 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 their distinct sensitivities, dependent on which test is applied, different levels of detected CRP are commonly considered indicative of the presence or absence of an acute inflammation. As used herein, dependent on which test is applied for detection, the respective CRP level is either referred to as “standard CRP level” or “high-sensitivity CRP (hs-CRP) level”.

[0222] Accordingly, in preferred embodiments:

[0223] - a determined standard CRP level of up to 5 mg / dl, a determined hsCRP level of up to 0.5 mg / dl, a determined fibrinogen level of up to 400 mg / dl, and / or a determined leukocyte count of up to 10000 per microliter is indicative of the absence of an acute inflammation; and / or - a determined CRP level of above 5 mg / dL, a determined hsCRP level of above 0.5 mg / dl, a determined fibrinogen level of above 400 mg / dl, and / or a determined leukocyte count of more than 10000 per microliter is indicative of the presence of an acute inflammation.

[0224] In alternative or even more preferred embodiments, the presence or absence of an acute inflammation is assessed, or additionally assessed, by evaluating the level of procalcitonin (PCT).

[0225] Procalcitonin (PCT) is a well-established biomarker for the diagnosis of an acute inflammation, and especially for the early diagnosis of sepsis where PCT level is known to be reflective of the severity of bacterial infection and is in particular used to monitor progression of infection into sepsis, severe sepsis, or septic shock (see, e.g., Meisner M. Ann Lab Med. 2014 Jul;34(4):263- 73). Plasma levels of PCT in healthy individuals are typically below 0.1 ng / mL. To exclude sepsis and acute inflammation, a concentration of <0.2 ng / mL is a useful reference range. To diagnose sepsis and acute inflammation with a high likelihood, a concentration of >10 ng / mL is a useful reference range. The level of PCT is generally determined in a sample, wherein the sample is preferably a blood sample or blood-derived sample (e.g. plasma or serum), most preferably plasma

[0226] Accordingly, in preferred embodiments:

[0227] - a determined PCT level (preferably PCT plasma level) of <0.2 ng / mL (preferably below 0.1 ng / mL) is indicative of the absence of an acute inflammation (esp. of sepsis); and / or

[0228] - a determined PCT level (preferably PCT plasma level) of >0.10 ng / mL is indicative of the presence of an acute inflammation (esp. of sepsis).

[0229] In preferred embodiments of the methods according to the first, second, third, fourth and / or any further aspect of the invention, the method is conducted:

[0230] (i) on a (bio-)chip;

[0231] (ii) in a miniaturized format, e.g., using sample volumes ranging from 1 picoliter to 100 microliters.

[0232] (iii) in an automated or semi-automated manner;

[0233] (iv) using a microfluidic device / system; and / or

[0234] (v) in high-throughput and / or multiplexed format, wherein multiple samples are placed in separate wells of one or several microtiter plates (also known as micro-well or multiwell plate, for example, a 6-, 12-, 24-, 48-, or 96-well plate) and analyzed sequentially ( / .e., consecutively) or in parallel.

[0235] A miniaturized format of the methods according to the invention is shown in Example 6. In a further aspect, the invention provides a system, an apparatus, or a device (preferably, a diagnostic apparatus or diagnostic device) configured for performing the methods according to the first, second, third, fourth, fifth, or any further herein disclosed aspect of the invention, preferably in an automated or semi-automated manner.

[0236] The Figures show:

[0237] Figure 1 : Environmental stimuli differentially affect blood plasma proteins from pathological origin. Blood plasma derived from pathological (n = 5 or 8) and non-pathological (n = 4 or 7) sources were exposed to extreme stimuli known to compromise protein structure. The precipitated protein was recovered by centrifugation and the protein amount present in the pellet (“Pellef’) and in the supernatant (“S / V”) was determined by a Bicinchoninic acid assay (BCA). Blood plasma was exposed to A) -0.2% acetic acid (“ACID") for 5 min at 37°C; B) 70°C (“HEAT’) for 1 min; C) -13% ammonium sulfate (“SALT’) for 5 min at room temperature; D) -18.5% of acetone (“Ketone”); or E) -18.5% of ethanol (“Alcohol”) for 5 min at 4°C. A two-sided independent student’s t test was performed. Data from (A-E) are represented as mean ± SD from at least four biological replicates. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0238] Figure 2: A spectrophotometry-based assay to quantify protein precipitation. A) Graphical 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 the absorbance at 340 nm, the wavelength of light scattering. It is of note that the addition of the acetic acid in step 6 initiates the precipitation. In step 7 the precipitation can either be measured kinetically, e.g., over a time of 1 h (see Fig. 4) or once after a period of time allowing the precipitate to form (e.g., 300 s as illustrated by the appended examples). C) Lyophilized plasma was reconstituted to contain indicated amounts of protein and precipitation was conducted as outlined in (B). Absorbance was determined 5 min after exposure to acetic acid. Each data point represents the mean of two technical replicates. The coefficient of determination (r2) is indicative of a linear relationship between the amount of blood plasma protein and the background subtracted absorbance.

[0239] Figure 3: Key determinants of acetic acid-induced protein precipitation. A) Blood plasma proteins from pathological and non-pathological (n = 3) subjects were precipitated and quantified as described in Fig. 2. As indicated, the concentration of the added acetic acid increased, while the pH of the precipitating agent decreased. This shows that it is possible to optimize the precipitating conditions such that blood plasma can be distinguished based on its pathological state. A two- sided independent Student’s t-test with Benjamini-Hochberg false discovery (FDR) correction was performed to compare pathological vs. non-pathological samples for every indicated concentration of acetic acid. B) The experiment was conducted as in A), but acetic acid was substituted for hydrochloric acid (HCI) as the precipitating agent. The x-axis indicates the pH of HCI before it was mixed with the plasma showing that lowering the pH is not enough to trigger robust precipitation. A two-sided independent Student’s t-test with Benjamini-Hochberg FDR correction was performed to compare pathological (n = 3) vs. non-pathological (n = 3) samples for every indicated pH value. C) The experiment was conducted as in A), but acetic acid was dissolved in indicated amounts of sodium chloride (NaCI). The pH decreased with an increasing amount of salt as indicated. A two-sided independent Student’s t-test with Benjamini-Hochberg FDR correction was performed to compare pathological (n = 4) vs. non-pathological (n = 3) samples for every indicated concentration of NaCI. Data from (A-C) are represented as mean ± SD from at least three biological replicates. *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0240] Figure 4: Acetic acid triggers precipitation by a self-propelling chain reaction in blood plasma. The experiment was conducted as described in Fig. 2. Precipitation was monitored over the course of 1 hour. The addition of acetic acid triggered rapid precipitation of pathological samples, while non-pathological ones were much less affected. A two-way ANOVA with repeated measures and Sidak’s posthoc test for multiple comparison correction was performed. Data are represented as means ± SD of 5 biological replicates. *P < 0.05, **P < 0.01 , ***P < 0.001.

[0241] Figure 5: Acetic acid induces reversible precipitation of blood plasma proteins. A) Workflow of the assay setup. In contrast to the default assay (see Fig. 2B), acetic acid was present 1 minute before the reaction was further manipulated by the addition of 16 mM NaOH or vehicle (dH2O). B-E) Two pathological (B, C) or two non-pathological (D, E) samples were exposed to the workflow as described in 5A. Acetic acid-induced rapid precipitation over the course of 11 minutes in the pathological samples but the reaction was almost absent in the non-pathological ones. The addition of 16 mM NaOH, which balanced the pH to 7.4, led to a complete reversal of the reaction while NaOH had no effect on acetone (18.5%) induced precipitation. 5B). The instant reversal of acetic acid-induced protein precipitation by the addition of NaOH. This demonstrates that acetic acid-induced protein precipitation is pH dependent. All biological replicates are shown (B-E). Data points connected by a line represent repeated measures from the same biological replicate.

[0242] Figure 6: Pathological blood plasma proteins exhibit compromised structural stability. The experiment was conducted as described in Fig. 2A. To assess the level of precipitated proteins, the absorbance was determined 5 minutes after the addition of acetic acid. A-B) The blood plasma of pathological (n = 3) or non-pathological (n = 3) origin was pre-incubated for 5 min at 37°C or 50°C before the addition of acetic acid. Non-pathological samples became susceptible to acetic acid-induced precipitation after incubation at 50°C. This shows that pathological samples are comprised of structurally different proteins that are prone to precipitation. To compare samples incubated at 37°C vs 50°C, a two-tailed paired t test was conducted. C-D) Pathological (n = 3) or non-pathological (n = 3) samples were left untreated, or centrifuged multiple times (10x) through a 3 kDa cut-off filter to exchange non-protein bound substances (= plasma matrix). The blood plasma matrix was substituted to 0.9% NaCI (“exchange”) or the separated matrix was reintroduced (“mock exchange”). A one-way ANOVA with repeated measures and Tukey’s posthoc test for multiple comparisons was performed. All biological replicates are shown (A-E). Data points connected by a line represent repeated measures from the same biological replicate. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0243] Figure 7: Evaluation of clinical performance and threshold optimization. A) Based on a training set of samples being known as being positive (pathologic) or negative for cancer (non-pathologic) and not having an inflammatory disease the optimal clinical measure was evaluated using the receiver operating characteristic (ROC) curve and its area under the ROC curve (AUC) metric, showing that the subtraction value ( / .e., extinction subtracted by the blank value) performs best. The performance of random chance (AUC = 0.5) is presented for illustrative purposes (in black). B) We obtained a reference range for the subtraction value of 0-84.7 based on its distribution for the non-pathologic reference group (n = 241), which could be almost perfectly separated from the pathological reference group (n = 114). The Youden index J, as indicated in (A), was used to determine the optimal subtraction value threshold of 134. D-E) p values were obtained based on the subtraction values by comparing a single subject against the non-pathological reference group using the Wilcoxon signed-rank tests. To evaluate whether a subject is diagnosed as pathological, the thresholds for the p-value and the size of the subset of the respected reference group were optimized for the number of wrong classifications (C) and the balanced accuracy (D). F-l) Likewise, we optimized (as in D-E) the threshold for the AUC comparing a single subject against either the pathological (F-G) or the non-pathologic reference (H-l) group, denoted as AUCp and AUCN, respectively. J-K) We estimated the minimum required sample size for the pathologic and non-pathological reference group using the AUCP (in red) and AUC«(in blue), respectively.

[0244] Figure 8: Acetic acid-induced protein precipitation serves as a test for pan-cancer diagnosis. A total of 555 “blind” samples (for which it was not known from which subject they were obtained) was examined using the workflow as described in Fig. 2 with a 5-minutes endpoint readout. The subjects had the following clinical diagnosis: healthy (n = 214), brain tumors (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), colorectal 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), malignant skin tumors (n = 18), sarcoma (n =5), ovarian cancer (n =14), neuroendocrine tumors (n =14), testicular cancer (n =14), laryngeal carcinoma (n = 14), bone cancer (n = 14), lymphoma (n = 5), cervical carcinoma (n = 14), gastro intestinal stromal tumor (n = 5), squamous cell carcinoma (n = 1), bile duct and gallbladder cancer (n = 5), Peritoneal carcinosis (n = 5), fibrosis (n = 2), lung fibrosis (n = 3), sarcoidosis (n = 4), bronchitis (n = 2). The dashed black line indicates the extinction threshold (120 milli ext. units) above which the subjects are considered to be pathologic. This threshold is within the threshold range as shown in Figure 7C that can be used equally good for distinguishing pathologic from non-pathologic samples.

[0245] Figure 9: A) The protein concentration of K2-EDTA blood plasma derived from pathological (n = 8) or non-pathological origin (n = 8) was determined with BCA. Two-sided independent student’s t-test. B) The osmolality of K2-EDTA blood plasma derived from pathological (n = 10) or non- pathological origin (n = 10) was determined by vapor pressure osmometry. Two-sided independent student’s t-test. C) The osmolality of K2-EDTA blood plasma derived from pathological (n = 10) or non-pathological origin (n = 10) was determined by vapor pressure osmometry. Two-sided independent student’s t-test. D). The experiment was conducted as described in Fig. 2B. The absorbance at 340 nm was determined for pathological (n = 10) or non- pathological (n = 10) samples before the addition of acetic acid (= blank absorbance). This experiment demonstrates that there is no pre-existing protein aggregate that could account for the observed differences between pathological and non-pathological samples. Two-sided independent student’s t-test. E) The pH of K2-EDTA blood plasma derived from pathological (n = 7) or non-pathological origin (n = 7) was determined after addition of acetic acid. Two-sided independent student’s t-test. Data from (A-E) are represented as mean ± SD of 8-10 biological replicates. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0246] Figure 10: Environmental stimuli differentially affect blood plasma proteins from pathological origin. Blood plasma derived from healthy controls and cancer patients was handled as depicted in Figure 2B. Step 6 was altered to initiate protein precipitation with different acids in varying concentrations. Measurement at 340 nm after 5 min (300 s) and 10 min each. Carcimun levels (y-axis) indicate milli extinction units from the Indiko™ Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are represented as mean ± SD from at least three biological replicates in one or two-sided t-tests. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0247] Figure 10.1 : Citric acid in 0.9% NaCI aq. used in step 6. A) 10% citric acid. B) 5% citric acid. C) Measurement of precipitation kinetics over the course of 12 time points of both 10% and 5% citric acid as precipitating agent. Time points are separated by a 54 second time interval.

[0248] Figure 10.2: Formic acid in 0.9% NaCI aq. A) 0.8% formic acid. B) 0.4% formic acid. C) 0.2% formic acid. D) Measurement of precipitation kinetics over the course of 12 time points of 0.8%, 0.4%, and 0.2% formic acid as precipitating agent. Time points are separated by a 54 second time interval.

[0249] Figure 10.3: Perchloric acid in 0.9% NaCI aq. A) 0.875% perchloric acid. B) Measurement of precipitation kinetics over the course of 12 time points of 0.875% perchloric acid as precipitating agent. Time points are separated by a 54 second time interval. Figure 11 : Environmental stimuli differentially affect blood plasma proteins from pathological origin. Blood plasma derived from healthy controls and cancer patients was handled as depicted in Figure 2B. Step 6 was altered to initiate protein precipitation with different alcohols in varying concentrations. Measurement at 340 nm after 5 and 10 minutes each. Carcimun levels (y-axis) indicate milli extinction units from the Indiko™ Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are represented as mean ± SD from at least three biological replicates in one or two-sided t-tests. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0250] Figure 11.1 : Ethanol used in step 6. A) 75% ethanol. B) 50% ethanol. C) Measurement of precipitation kinetics over the course of 12 time points of both 75% and 50% ethanol as precipitating agent. Time points are separated by a 54 second time interval.

[0251] Figure 11.2: Isopropanol used in step 6. A) 75% isopropanol. B) Measurement of precipitation kinetics over the course of 12 time points of 75% isopropanol as precipitating agent. Time points are separated by a 54 second time interval.

[0252] Figure 11.3: Methanol used in step 6. A) 75% methanol. B) 50% methanol. C) Measurement of precipitation kinetics over the course of 12 time points of both 75% and 50% methanol as precipitating agent. Time points are separated by a 54 second time interval.

[0253] Figure 12: Environmental stimuli differentially affect blood plasma proteins from pathological origin. Blood plasma derived from healthy controls and cancer patients was handled as depicted in Figure 2B. Step 6 was altered to initiate protein precipitation with different salts in varying concentrations. Measurement at 340 nm after 5 and 10 minutes each. Carcimun levels (y-axis) indicate milli extinction units from the Indiko™ Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). Data are represented as mean ± SD from at least three biological replicates in one or two-sided t-tests. *P < 0.05, **P < 0.01 , ***P < 0.001 , ns = non-significant.

[0254] Figure 12.1 : CaCh used in step 6. A) 5% CaCh. B) 2.5% CaCh. C) Measurement of precipitation kinetics over the course of 12 time points of both 5% and 2.5% CaCh as precipitating agent. Time points are separated by a 54 second time interval.

[0255] Figure 12.2: Guanidine thiocyanate used in step 6. A) 10% guanidine thiocyanate. B) Measurement of precipitation kinetics over the course of 12 time points of 10% guanidine thiocyanate as precipitating agent. Time points are separated by a 54 second time interval.

[0256] Figure 12.3: MnCh used in step 6. A) 5% MnCh. B) 2.5% MnCh. C) Measurement of precipitation kinetics over the course of 12 time points of both 5% and 2.5% MnCh as precipitating agent. Time points are separated by a 54 second time interval.

[0257] Figure 13: Densitometric analysis of Coomassie Blue-stained precipitates of plasmas of healthy (N=3) and cancer (N=3) patients after SDS-PAGE with Imaged. Figure 14: Acetic acid induces reversible precipitation of blood plasma proteins. Workflow of the assay setup shown in Figure 5A but expanded by an additional step 10. In contrast to the assay (see Fig. 5A), acetic acid was present 5 minutes (step 7) before the reaction was further manipulated by the addition of a dilution series of NaOH or vehicle (dH2O). When 17 and 12 mM NaOH were added respectively the reaction reversed (similar to results shown in Figure 5B). Addition of 7.5 mM NaOH stops the reaction but does not reverse it. Samples that were stopped with 17 and 12 mM NaOH were further manipulated in step 10 by the addition of 0.4% acetic acid or vehicle (dH2O). It is noteworthy, that the volume of added acetic acid was chosen to reach final concentration of acetic acid in the reaction mixture of approximately 0.15% representing default assay conditions. The precipitation was initiated again (SN1AA & SN2AA) underlining the reversibility of this reaction.

[0258] Figure 15: Miniaturization of the Carcimun Test by NanoDrop readout measurement: Table of a dilution series

[0259] Figure 16: Miniaturization of the Carcimun Test by NanoDrop readout measurement: (a) The NanoDrop readout measured at 340 nm shows a significant difference between samples derived from healthy patients against samples from cancer patients; (b) The test remains robust even when lowering total reaction volume by many folds.

[0260] The following example(s) illustrate the invention:

[0261] Example 1 :

[0262] Materials & Methods

[0263] Blood plasma sampling:

[0264] Human K2-EDTA blood plasma were purchased from ProteoGenex, Inc. (460 Hindry Ave., Unit A

[0265] Inglewood, CA 90301 , USA).

[0266] Bicinchoninic acid assay (BCA):

[0267] To determine the concentration of proteins present in blood plasma, we performed Bicinchoninic acid assay (BCA) using the Pierce™ BCA Protein Assay (#23227, Thermo Fisher) according to the manufacturer’s instructions. Briefly, blood plasma was diluted 1 :100 in STET (50 mM Tris, pH 7.5, 150 mM NaCI, 2 mM EDTA, 1 % NP40) lysis buffer. Bovine serum albumin was serially diluted to establish a standard curve ranging from 0 - 2 mg / mL. The standards and samples were incubated in Kit reagent A and B for 30 min at 37°C. The spectrophotometric absorbance was determined at 562 nm using the Tecan Infinite M200 Nanoquant.

[0268] Plasma protein precipitation:

[0269] To quantify protein precipitation using the BCA assay, blood plasma was exposed to various stimuli known to cause protein precipitation. All samples were diluted in a similar manner before triggering precipitation. 110 ul of 0.57% NaCI was added to 26 ul of K2-EDTA blood plasma and incubated for 30 min at 37°C in 1.5 mL Eppendorf tubes. For heat-induced protein precipitation, the samples were incubated for 1 min at 70°C. Ketone- and alcohol-induced precipitation was triggered by addition of 80 ul of 50% ice-cold acetone or ethanol for 5 minutes at 4°C. Salt-induced precipitation was done by adding of 80 ul of 35% ammonium-sulfate for 5 minutes at room temperature. The precipitation with acid was performed at 37°C by adding 80 ul of 0.4% acetic acid dissolved in 0.81% NaCI for 5 min. After incubation with the precipitating agent, the samples were immediately centrifuged at 21.000 x g for 1 min at 4°C. The supernatant was transferred to a fresh 1.5 mL Eppendorf tube. The supernatant was immediately diluted 1 :200 in STET buffer and stored at 4°C to delimit forthgoing precipitation. The protein pellets were resuspended in STET buffer containing 1 % SDS and sonicated until completely dissolved. For reverting acid- induced precipitation, 5 mM NaOH was added to the STET buffer. The protein concentration was determined as described using the BCA assay.

[0270] Determination of the pH and osmolality of blood samples.

[0271] The pH of K2-EDTA blood plasma samples was determined at room-temperature (21°C) using the Seven pH-meter (Mettler Toledo) equipped with the Orion™ micro-pH-electrode (9810BN, Thermo Fisher). Osmolality of blood plasma was determined using the VAPRO 5600 pressureosmometer according to the supplier’s manual.

[0272] UV / VIS Spectrophotometric assay to quantify protein precipitation:

[0273] Protein precipitation was quantified using the Indiko™ Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher). A seven-step protocol was programmed using the Indiko’s default software (see Fig. 2B). Briefly, the blood plasma sample is diluted with approx. 0.57% (w / v) NaCI to reach a final volume of 136 ul with a NaCI concentration of approx. 0.63%. The sample is then incubated for 5 min at 37°C in a cuvette with 0.7 cm pathlength. The spectrophotometric absorbance at 340 nm of the diluted sample was determined and this served as the blank value. Thereafter, 80 ul of 0.4% (v / v) acetic acid containing 0.81% (w / v) NaCI was added to the diluted sample. The absorbance at 340 nm was determined either kinetically, e.g., over a time of 1 h (see Fig. 4) or once after a period of time allowing the precipitate to form (e.g., 300 s as illustrated herein). The blank value was subtracted as background. Alterations in the concentration of acetic acid and salts are indicated in the legends of respective figures.

[0274] For experiments using reagents intended to stop the acid-induced precipitation reaction, NaOH was added to the reaction 60 sec after addition of 0.4% acetic acid or 50% acetone.

[0275] Statistics:

[0276] The statistical tests used in the analyses are indicated within the respective figure legend. The statistics were calculated by GraphPad 9 software. For threshold optimization (see Fig. 7) data processing, data visualization, and statistical analysis were performed in Python 3.8 using pandas 1.1.4, matplotlib 3.3.4, seaborn 0.11 , scipy 1.6.2, and statsmodels 0.12.2.

[0277] Evaluation measures and threshold optimization:

[0278] To evaluate the quality of our assay, five performance measures — sensitivity (SEN), specificity (SPE), accuracy (ACC), balanced accuracy (bACC), and F1 score (F1) — were used, defined as follows:

[0279] SEN=TP / (TP+FN)

[0280] SPE=TN / (TN+FP)

[0281] ACC=(TP+TN) / (TP+TN+FP+FN) bACC=(SEN+SPC) / 2=(TP / (TP+FN)+TN / (TN+FP)) / 2

[0282] F1=2TP / (2TP+FP+FN) where TP, FP, TN, FN are the number of true positives, false positives, true negatives, and false negatives, respectively. Sensitivity and specificity are also called true positive rate (TPR) and the true negative rate (TNR), respectively.

[0283] Given a subtraction value S ( / .e., measured UV extinction subtracted by blank) for a tested sample, the area under the curve (AUC) was applied to compare S against a set of subtraction values from a positive / pathological reference group P, denoted as AUCp. The AUCp was computed as: rip

[0284] AUCp = — Y k(S,Xt)

[0285] Tip i where np is the number of subtraction values in P, x-, is the subtraction values for the i-th sample in P, and the indicator function k for comparing S against x, is defined as: 0 if S < xtk(S, Xi) = 0.5 ifS = Xi

[0286] 1 1 if S > Xi

[0287] Alternatively, the AUG can be applied to compare S against a set of subtraction values from a non-pathological reference group N, denoted as AL / Cwand computed by: where n«is the number of subtraction values in N, x-, is the subtraction values for the i-th sample in N, and the indicator function k is defined as before. Please note that outliers ( / .e., 3 standard deviations away from the average) were removed from the negative reference group to increase consistency.

[0288] Example 2:

[0289] Results & Discussion

[0290] Distinct conformational states of proteins are a hallmark of many pathological conditions because protein conformation is key to a protein’s biological function. Protein stability, which is the ability to maintain a conformation under a given set of conditions, is therefore crucial. There are different ways to experimentally interrogate and quantify protein stability, but the most common way is to make use of the fact that conformational changes are accompanied by altered protein solubility, which is influenced by a protein’s size, charge, and three-dimensional structure. The three- dimensional structure, however, is mainly determined by its primary, secondary, tertiary, and quaternary structure, but a protein’s conformation is also affected by environmental factors, such as pH, temperature, and the presence of ligands or other molecules. Because pathological conditions have been found to be reflected by changes in the composition of blood plasma (Geyer, P.E. et al., Cell systems (2016) 2:185-195), it is explored herein whether altered blood plasma composition could affect the solubility of plasma proteins.

[0291] To test this, K2-EDTA blood plasma from four individuals with no reported pathological condition and five pathological specimens were exposed to conditions known to affect protein solubility (Fig. 1) and the amount of insoluble protein was quantified after recovery of the precipitated material (1 min 14.000 x g) using a Bicinchoninic acid assay. As shown in Figure 1A, the presence of acetic acid (-0.15%) rendered about 2% of the input material insoluble in the pathological samples, but only about 1.5% precipitated from non-pathological samples. Consequently, the remaining protein in the supernatant was lower in the pathological samples than in the non- pathological ones, although the detected difference was not significant, which is most likely due to the specific assay conditions applied in the BCA assay. Similar results could be observed when other protein precipitation strategies were employed (Fig. 1 B-E). When using heat to denature proteins, the samples were exposed to 70°C for 1 min, which led to the precipitation of about 7% from pathological samples, but healthy ones were less affected (~4%, Fig. 1 B). Similar differences were observed when we used ethanol, ammonium sulfate, and acetone to induce precipitation (Fig. 1C,E). Interestingly, the presence of ethanol or ammonium sulfate affected a much larger fraction (up to 30%) of the plasma proteins (Fig. 1 C, E), whereas acetone affected less than 10% of the total proteins (Fig. 1 D). It is noteworthy, that only a subset of the proteome was affected with either of the chosen precipitation condition. However, it was astonishing that in particular acetic acid, which affected only around 1-2% of the total proteins at a concentration of about 0.15% allowed for a robust and statistically significant distinction between pathological and non-pathological samples already based on a small number of samples. Notably, because blood plasma protein concentration differs among individuals, we normalized the precipitated proteins to the total amount of protein before precipitation was induced (Fig. 1 , Fig. 9).

[0292] From these results, we concluded that protein stability is altered by the presence of a pathological condition and that the phenomenon of differential protein precipitation could be leveraged to distinguish samples derived from individuals with a pathological condition from those that had no medical condition. Further, we hypothesized that precipitation conditions optimized to the degree that they affect only a small fraction of the plasma proteome and still allow for the distinction of samples with a certain pathology could be an interesting approach for diagnostics. Therefore, we decided to investigate protein precipitation under mildly acetic conditions using acetic acid as the denaturing agent.

[0293] Next, we set out to further characterize and optimize acetic acid-induced protein precipitation as this would potentially inform about the nature of the underlying molecular mechanism. A common way to observe protein precipitation is the measurement of the spectrophotometric absorbance at 340 nm. To do so, we set up an assay using the Indiko™ Plus Clinical Chemistry Analyzer (Thermo Fisher) to specifically track protein precipitation (Fig. 2A, B). Lyophilized blood plasma were serially diluted to determine the optimal plasma protein concentration required for the detection of protein precipitation at 340 nm. As shown in Fig 2C, the assay was able to establish a linear relationship between absorbance and input protein when 0.4% (v / v) acetic acid was added to a pre-diluted plasma sample for 5 minutes. In the range of 100 -4500 pg of input protein, the assay showed near-perfect linearity.

[0294] Having established that blood plasma derived from a pathological background when exposed to extreme stimuli exhibits a higher propensity for undergoing precipitation compared to blood plasma from a non-pathological background and in particular that the assay was able to elicit a robust and linear effect on blood plasma proteins, we sought to investigate the key 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, we gradually increased the concentration of acetic acid that was added to the pre-diluted protein sample for 5 minutes at 37°C. As expected, the proteins precipitated with increasing amounts of acetic acid (Fig. 3A). However, it was astonishing that there was hardly any precipitation observable at concentrations below 0.2%. At higher concentrations (1 .6%) proteins were precipitating markedly, but it was not anymore possible to distinguish the origin (pathologic vs. non-pathologic) of the blood plasma. The most pronounced difference could be observed at 0.4%, which accounts for a 0.15% final concentration (Fig. 3A). Having determined the optimal concentration of acetic acid we wondered if this was triggered by the acidic environment or if this was due to a specific effect elicited by acetic acid itself. We used the strong acid hydrochloric acid (HCI) to trigger precipitation. Surprisingly, even when we added HCI at pH 1.5, there was no robust precipitation being triggered, although the pathological samples reacted stronger to the pH shift than the non- pathological ones (Fig 3B). This is good evidence to claim that pH is not the only determinant of plasma origin-dependent protein precipitation. Next, we tested the effects of salt on protein stability, since elevated salt levels are known to lower the pH of an aqueous solution. Hence, we expected that the precipitation is increasing upon exposure to ascending amounts of salt. Surprisingly, precipitation of both pathological and non-pathological plasma proteins was decreased with intermediate amounts of sodium chloride, but strongly elevated when no salt or very high amounts of salt was present (Fig. 3C). It is tempting to speculate that we observed a so-called “salting-in” effect, meaning that sodium chloride increased protein stability at low concentrations, as it is known for salts of the Hofmeister series that are neither chao- nor cosmotropic (Gregory, K.P. et al., Physical chemistry chemical physics: PCCP (2022).24, 12682- 12718), such as sodium chloride. In line with this idea, at high concentrations (in this case at 9%), salts can “salt-out” proteins and in combination with the decreasing pH trigger protein precipitation. Interestingly, physiological NaCI levels (-0.9%) impacted the precipitation behavior of healthy and pathological samples much less than lower or higher concentrations. We concluded that certain precipitation conditions are required to distinguish blood plasma in an origin-dependent manner. Optimal conditions could be achieved by manipulating several key determinants of acetic acid-induced protein precipitation such as the concentration of acid, salt, and its resulting pH. It is noteworthy that protein solubility could also be affected by the body’s electrolyte-water balance, which is interconnected with the concentration of salt and the pH of blood (Auton, M. et al. (2011). Biophysical chemistry 159, 90-99). The normal human reference range of plasma osmolality is between 275-300 mOsm / kg. To test whether the structural instability of blood plasma derived proteins stems from altered pH or the body’s intrinsic water balance, we experimentally determined the pH and osmolality of K2-EDTA plasma and found no significant difference (Fig. 9B,C).

[0295] Next, we wanted to map the precipitation reaction kinetically, because protein precipitation is known to start at certain hot spots, but then quickly extends to its surroundings causing unaffected proteins in close proximity to precipitate themselves. In line with this, we hypothesized that the distinct precipitation behavior of blood plasma could be explained by the presence of so-called “seeds”, which are tiny protein aggregates that could serve as a hub for protein precipitation. To address this question, we performed a one-hour time course experiment and found acetic acid triggering a reaction that increased rapidly in the first 10 minutes but then flattened and remained almost completely stable (Fig. 4) in both the pathological and non-pathological samples. Strikingly, the speed and the extent by which pathological samples reacted were significantly different from the non-pathological ones. Importantly, we did not observe a higher baseline absorbance at 340 nm before acetic acid was added (Fig. 9D). This shows that the observed differences were unlikely from the presence of aggregation seeds that are present in the pathological samples but are absent or much less frequent in the non-pathological ones. To substantiate this notion, we wanted to find out whether acetic acid caused proteins to be transiently insoluble or led to irreversible denaturation and subsequent aggregation. Indeed, it is known that acetic acid causes proteins to transiently precipitate when used at low concentrations. Hence, if aggregation seeds were present that required to be triggered by a denaturing agent, the reaction would be expected to be self-driven as the levels of aggregated proteins rise. On the contrary, if this phenomenon was not due to a self-driven chain reaction caused by pre-existing seeds, the reaction should be reversible when acetic acid is neutralized even a long time after it was triggered. To test this, we included the addition of NaOH to stop the precipitation reaction (Fig. 5A). We leveraged the fact that at neutral pH, acetic acid loses its acidic properties because the majority of protons dissociate. As expected, pH neutralization caused the precipitation to be completely reversed, but acetone-induced precipitation did not (Fig. 5B-E). This shows that acetic acid induces a chain reaction that is depending on its acidic form. We concluded that intrinsic structural instability rather than pre-existing aggregation seeds was the reason for this effect. However, since blood plasma is a complex mix of thousands of constituents, we wondered whether the structural instability was due to a protein intrinsic or exogenous phenomenon. To test this, we mildly heated (5 min, 50°C) the blood plasma before exposing it to acetic acid. Strikingly, the pathological samples did not change their precipitation behavior, whereas non-pathological samples precipitated like pathological ones. This hints towards a conformational change of pathological proteins rendering them susceptible to precipitation. To further substantiate the notion that the observed effect is dependent on the proteins’ conformation, we exchanged the plasma matrix with 0.9% NaCI by multi-step centrifugation through a 3-kDa cut-off column. The precipitation behavior remained unchanged in each condition (Fig. 6C,D). Most importantly, the differential precipitation of pathological vs. non-pathological was conserved showing that the observed phenomenon is due to a protein intrinsic effect that impacts the structural integrity of plasma proteins.

[0296] Having developed an assay and the causative basis for the distinction of pathological and non- pathological samples, we opted to increase the sample size markedly in order to establish a threshold that will allow to discriminate pathological and non-pathological precipitation behavior.

[0297] To optimize the clinical performance of our assay, we assessed the measured UV extinction, this extinction subtracted by the blank value (hereafter referred to as ‘subtraction’), and the ratio between the extinction and the blank value. Given a positive / pathological reference group P (n = 114) and a negative / non-pathological reference group N (n = 241), we obtained for these three measures the Receiver Operating Characteristic (ROC) curves (Sciences & Kumar, 2011) (Fig. 7A). The ROC curves indicate a preference for the subtraction (AUC=0.999) over the ratio (AUC=0.997) and the extinction (AUC=0.948). Next, we defined the clinical reference range (Haggstrdm, 2014) for our assay using the subtraction values of the non-pathological reference group / V (mean = 42.3 ± 24.5). Therefore, we removed two outliers from / V with three standard deviations (SD) higher than the average subtraction value. The reference range was then defined without the outliers as mean ± 2SD (0-84.7; n = 239, mean = 41 ,3±21.7), so 228 out of 241 non- pathological samples fall into it (95%, as required for clinical tests). The reference range is visualized in Fig. 7B.

[0298] We aimed to assess whether a tested subject can be diagnosed as having cancer and / or an acute inflammatory disease (hereafter referred to as ‘diagnosed as pathologic’) based on S, P, and N, which are the subtraction values for the tested subject, the pathological reference group, and the non-pathological reference group, respectively. Therefore, we optimized the subtraction threshold using the Youden index J (Fluss, Faraggi, & Reiser, 2005; Schisterman, Perkins, Liu, & Bondell, 2005). Given the sensitivity (SEN, true positive rate) and the specificity (SPE, true negative rate) of a test, the Youden index is defined as J=SEN+SEP-1 (see Example 1 , Material & Methods). We computed J over a range of subtraction thresholds and obtained an optimum threshold of 134 for the maximum J = 0.99, as indicated in the ROC curve (Fig. 7B). This value was within the range of the optimal specificity threshold (105) and sensitivity threshold (134), defined as the highest value observed in N (without outliers) and the lowest value observed in P, respectively (Fig. 7C). Consequently, newly tested subjects are diagnosed positive with S > x, where x ranges between a subtraction value of 105 and 134. As this threshold depends on the measurements of the reference group and will be altered if the assay setup assay is changed, we tested three threshold-free approaches (Yuan, Su, & Zhu, 2015).

[0299] First, we attempted an approach from Bayesian statistics called likelihood ratios (LR) (Fierz & Bossuyt, 2021), which expresses the probability that a test result is positive or negative. However, the LR is only a general estimation of the chance that a subject is diagnosed as pathological or non-pathological and does not consider the actual assay readout. Thus, a direct application to evaluate the assay readout of a subject is not suitable.

[0300] Second, as a simple alternative, we determined whether S ( / .e., subtraction value measured for a subject) is significantly greater than the values in / V ( / .e., subtraction values of non-pathological reference group or healthy controls) using the Wilcoxon signed-rank test. We analyzed the applicability of this strategy by obtaining the number of wrongly classified subjects from our two reference groups (n = 355) using a p-value threshold >= 0.05, which yielded 110 wrong classifications (Fig. 7D). Since the p-value depends on the underlying reference group, we tested subsets of N — given as a percentage of non-pathological subjects with highest subtraction values — in conjunction with different commonly used p-value thresholds (0.05, 0.01 , 0.001 , 10’4, 10’5, 10’6, 10’7). Our optimization shows that using only 5% of the total reference group and a p- value threshold of 0.01 or 0.001 results in only two false positives or a balanced accuracy of 99.6% (Fig. 7E), a performance measure used for unbalanced data (see Example 1). Although this indicates a nearly perfect discriminative power, the downside of this approach is that the p-value threshold replaces the subtraction threshold and that the size of the reference group must be optimized additionally, which also depends on the assay setup. Because of this, we gauged this approach as not being feasible as a general performance measure for our assay.

[0301] Third, to overcome potential problems of the classical statistical approaches such the above statistical approach of the Wilcoxon 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 a reference group. To avoid confusion with the AUC used above in connection with optimizing the subtraction threshold, we will refer to the AUC for this single-value comparison as AUCp or AUCN, depending on whether P or N is used as reference group (see Example 1). We will describe the AUCp in the following, but the AUCN works analogously. The AUCp ranges between 0 and 1 , indicating the extreme cases where all subtraction values in P are either higher or lower than S, respectively. An AUCp of 0.5 reflects that 50% of the subtraction values in P are higher and 50% of them are lower than S. The results of the AUCp are consistent with non-parametrical tests such as the Wilcoxon signed-rank test (Mason & Graham, 2002). Additionally, it is easier to interpret and more robust because it varies between 0 and 1 , regardless of the assay setup. Therefore, a newly tested subject is diagnosed as pathological if its AUCp exceeds a certain threshold. To optimize this A U Cp threshold, we used the entire set P (n = 114) as pathologic reference group, computed the AUCp for each value in P and N, and calculated various performance measures, such as accuracy (see Example 1 , Material & Methods), using different AUCp threshold. Likewise, the entire set N (n = 241) was used to optimize the AUCN threshold. Our optimization of the AUCp (Fig. 7F,G) produced similar results as our statistical approach (Fig. 7D,E) and supports the theoretically optimal AUCp threshold of 0.

[0302] In consequence, we suggest that newly tested subjects are diagnosed as pathological with an AUCp > 0, meaning that their subtraction value is within the distribution of subtraction values of the pathological reference group. Likewise, the optimization of AUCN (Fig. 7H-I) suggests an optimal threshold of 1. Thus, newly tested subjects are diagnosed as pathological with an AUCN = 1 , meaning that their subtraction value is greater than all subtraction values in the non- pathological reference group. Our data, however, also demonstrate that even less strict thresholds for AUCp (>=0.77) and AUCN (>=0.64) are sufficient to achieve an accuracy of 75%. See Table 1 for the AUCp and AUCN values for both reference groups. See Tables 2A,B,C for a detailed overview of the threshold optimization results for subtraction, AUCp, and AUCN.

[0303] Determination of the required minimum sizes of the pathological and non-patholoqical reference groups

[0304] Finally, we assessed the minimum size required for the reference group to achieve good performance using the AUCp or the AUCN. We, therefore, performed a train-test-set splitting by randomly selecting 50 subtraction values each for the pathological and non-pathological reference groups. These 100 values were then used as the training sets, while the remaining 255 values were used for testing (64 values from P and 191 from N). We randomly sampled n values for the pathological and non-pathological training set, starting with n=1 and increasing to n=50. For each n, we used the training subset of subtraction values as the reference group to compute the AL / Cwand the AL / Cp for all samples in the test set. By using our proposed threshold (AUCp> 0 and AUCN= 1), we evaluated different prediction metrics as before. To ensure statistically robust results, we repeated the random sampling for each n 10 times and averaged the results. We also repeated the train-test-set splitting 10 times and calculated the average value with standard deviation for each n, which were used to determine the minimum required sample size. The results indicate that a minimum sample size of 2 is required for the non-pathologic and pathologic reference group to achieve an accuracy of 75% using the AUCN and AUCp, respectively (Fig. 7J). To achieve an accuracy of 95%, a minimum sample size of 4 is sufficient for the pathologic reference group, while a minimum sample size of 22 is required for the non-pathological reference group (Fig. 7K). See Table 3A-D for more details.

[0305] Based on our results, we recommend the following application: a subject is diagnosed as having an increased risk for having cancer and / or an acute inflammatory disease if: • a measurement S of a test sample of the respective subject results in an AUCp(S, P) > x, where AUCp is defined in Material & Methods, P are all measurements of a pathological reference group with at least 2 reference samples, and x is the AUCp threshold in a range between >0 and 0.77.

[0306] • a measurement S of a test sample of the respective subject results in an AUCN(S, / V) >= x, where AUCN is defined in Material & Methods, N are all measurements of a non- pathological reference group with at least 2 reference samples, and x is the AUCN threshold greater or equal to 0.64.

[0307] In general, it is noteworthy that the reference range and the optimized thresholds depend on the assay setup and the reference group of pathological and non-pathological samples. Thus, they must be adjusted before any clinical application. In contrast, the threshold-free measure of AUCp and AUCN are independent of the assay readout and, therefore, enables an interpretation between different assay platforms (Fierz & Bossuyt, 2021).

[0308] Having established a threshold to distinguish between pathological and non-pathological samples, we wanted to leverage this tool to streamline pathological samples. To this end, we obtained blood plasma samples from individuals with a defined medical condition and determined their precipitation behavior (Fig. 8). Acetic acid-induced precipitation was potent in discerning samples originating from patients with malignant tumors from those without any reported (non- pathological) medical condition. Notably, samples derived from non-cancerous subjects (e.g., bronchitis) or benign conditions (e.g., sarcoidosis) yielded similar results as non-pathological samples, derived from subjects commonly considered to be healthy.

[0309] Taken together, we have established a protocol that leverages the ability of mildly acidic conditions to differentially precipitate plasma proteins. This phenomenon could be used to interrogate the structural stability of blood plasma-derived proteins and allowed for an originbased distinction of blood plasma samples. Most importantly, our protocol was successfully applied for the detection of 27 different cancer types, while other common medical conditions behaved like samples from healthy individuals. The herein disclosed results demonstrate that differential protein precipitation has the capacity for being utilized as a highly sensitive tool for pan-cancer detection as well as acute inflammatory disease.

[0310] Example 3: Testing of further precipitation-inducing conditions

[0311] As shown in Figure 2 A,B,C, it was possible to set up an assay using the Indiko™ Plus Clinical Chemistry Analyzer (Thermo Fisher) to specifically track protein precipitation. Next, different acids, alcohols and salts were used as precipitating agents in step 6 (Fig. 2B). • As depicted in Figure 10.1 , citric acid is suitable to induce distinguishable protein precipitation when comparing healthy vs. cancer patients. This accounts also for dilution ranges of formic acid and perchloric acid (see Figs. 10.2 & 10.3).

[0312] • To test if alcohols could be used as precipitating agents, dilution series of ethanol, isopropanol and methanol were tested in step 6. As shown in Figures 11.1.-11.3, each of the tested alcohols was also suitable to induce protein precipitation in a distinguishable manner when comparing healthy vs. cancerous samples.

[0313] • This procedure was repeated with various salts to induce protein precipitation in step 6. CaCh, guanidine thiocyanate and MnCh were used as ‘salting out’ agents (see Figs. 12.1- 12.3). Conclusively, it is suitable to use different precipitation methods instead of the default protocol applying acetic acid in saline. As used herein, the term "saline", alternatively also referred to herein as “0.9% NaCI aq.” or “0.9% saline”, refers to an aqueous solution containing 0.9% (w / v) NaCI. Nevertheless, a threshold optimization is preferred for each compound.

[0314] Figures 10-12 show both, 5 min endpoint measurement, as well as 10 min endpoint measurement. A kinetic measurement consisting of 12 measurements separated by 54 sec intervals is provided along with above mentioned endpoint measurements.

[0315] Having shown, that BCA (Fig. 1) & the Indiko™ Plus analyzer (Fig. 2C) are suitable to quantify precipitates, we wanted to show that this was possible with another standard protein quantification procedure. As shown in Fig. 13, precipitates (initiated by standards method) and their corresponding plasmas from 3 patients each (healthy vs. cancer) were diluted with gel loading dye, before an SDS-PAGE was run. The gel was then stained with Coomassie Brilliant Blue. Total protein levels (meaning every lane) were quantified via densitometric analysis using the Imaged software. As shown in Fig. 13, there is a significant difference between healthy and cancer samples.

[0316] Example 4: Reversibility of the precipitation

[0317] Further, we could show that acetic acid in the presence of 0.9% (w / v) NaCI induces reversible precipitation of blood plasma proteins (Fig.14). The workflow of the assay setup is shown in Figure 5A but was expanded by an additional step 10. In contrast to the assay (see Fig. 5A), acetic acid was present 5 minutes (step 7) before the reaction was further manipulated by the addition of a dilution series of NaOH or vehicle (dH2O). When 17 and 12 mM NaOH were added respectively the reaction reversed (similar results shown in Figure 5B). Addition of 7.5 mM NaOH stops the reaction but does not reverse it. Samples that were stopped with 17 and 12 mM NaOH were further manipulated in step 10 by the addition of 4% acetic acid or vehicle (dH2O). It is noteworthy, that the volume of added acetic acid was chosen to reach final concentration of acetic acid in the reaction mixture of approximately 0.15% v / v] representing default assay conditions. The precipitation was initiated again (SN1AA & SN2AA) underlining the reversibility of this reaction.

[0318] Example 5: Chemosensitivity

[0319] The below Table 4 shows that by the Carcimun values as obtained by the herein described method correlated with the clinical categorization of the cancer during chemotherapy. Remitting and stable tumors show significantly lower Carcimun values as compared to progressing and fast progressing tumors.

[0320] Example 6: Miniaturization of the Carcimun Test

[0321] In order to show that the carcimun test can be miniaturized, the default protocol (Figure 2b) of the carcimun assay was conducted using healthy (N=3) and cancer (N=3) patient samples, but readout was performed using a NanoDrop spectrometer (NanoQuant infinite M200pro, Tecan Trading AG, Switzerland) with 1 pl of the reaction sample. The reaction was performed in 1.5 ml reaction tubes on a table-top shaker at 37° C. As shown in Figure 16a, the NanoDrop readout measured at 340 nm shows a significant difference between samples derived from healthy patients against samples from cancer patients. A table of a dilution series is shown in Figure 15.

[0322] Next, the 4th dilution (Figure 15) was conducted (same concentrations & steps, but lower total reaction volumes) to show that the volume of the reaction mixture could be scaled down. For this purpose, two cancer samples were compared with to healthy controls. As shown in Figure 16b, the test remains robust even when lowering total reaction volume by many folds.

[0323] These results underline the fact that it is possible to scale down the test protocol shown in Figure 2b. Readout of the test assay can be performed using a NanoDrop spectrometer and the only limiting factor is the pipette-able volume of each compound in every step, which can be overcome by automated systems. Further References

[0324] 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

[0325] 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

[0326] Haggstrdm, M. (2014). Establishment and clinical use of reference ranges. WikiJournal of Medicine, 1(1), 1-7. https: / / doi.org / 10.15347 / wjm / 2014.003

[0327] 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.

[0328] 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

[0329] 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

[0330] 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

[0331] Table 1 : Dataset of pathologic and non-pathologic reference groups with results of AUCp and AUCN analyses.

[0332] Table 2A: Results of the threshold (TH) optimization based on subtraction Table 2B: Results of the threshold optimization based on ALICp

[0333]

[0334] Table 2C: Results of the threshold optimization based on AUCw

[0335] Table 3A: Average performance measures for the determination of the minimum required sample size based on ALICp

[0336] Table 3B: Standard deviation of the performance measures for the determination of the minimum required sample size based on AUCp Table 3C: Average performance measures for the determination of the minimum required sample size based on AUCN

[0337] Table 3D: Standard deviation of performance measures for the determination of the minimum required sample size based on AUCN

[0338] It is understood that the definitions and embodiments as described above in connection with the first aspect of the invention also apply, in as far as possible, mutatis mutandis to the second, third, fourth, fifth and sixth aspects of the present invention.

[0339] The invention is herein described, by way of example only, with reference to the accompanying drawings for purposes of illustrative discussion of the preferred embodiments of the present invention.

[0340] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the patent specification, including definitions, will prevail.

[0341] Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends on. For example, in case of an independent claim 1 reciting 3 alternatives A, B, and C, a dependent claim 2 reciting 3 alternatives D, E, and F and a claim 3 dependent on claims 1 and 2 and reciting 3 alternatives G, H, and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A,

[0342] 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,

[0343] 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 specifically mentioned otherwise.

[0344] Similarly, also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2, and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2, and 1 , of claims 4, 3, and 1 , as well as of claims 4, 3, 2, and 1 is clearly and unambiguously disclosed.

[0345] The above considerations apply mutatis mutandis to all appended claims.

[0346] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

[0347] Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary' skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.

[0348] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The terms “method” and “process” are used interchangeably herein.

[0349] The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a cell” can mean “one or more cells”) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter ( / .e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values ( / .e., “about 1 , 2 and 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. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof

[0350] (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

[0351] Certain embodiments of the technology are set forth in the claim(s) that follow(s).

Claims

CLAIMS1. A method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:(a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and(b) assessing the level of the precipitated (poly)peptides in the sample relative to:(b-i) the level 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 an acute inflammatory disease;(b-ii) the level of precipitated (poly)peptides in at least one correspondingly treated reference sample obtained from one or more reference subjects known to be negative for cancer and an acute inflammatory disease; and / or(b-iii) a predetermined standard which has been determined based on (b-i) and / or (b- ii); whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if:- the level of the precipitated (poly)peptides in the sample is at least, with increasing preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, most preferably at least 100% of the level of the precipitated (poly)peptides in the reference sample (b-i), or as compared to a respective predetermined standard; and / or- the level of the precipitated (poly)peptides in the sample exceeds the level of the precipitated (poly)peptides in the reference sample (b-ii), or as compared to a respective predetermined standard, by at least, with increasing preference, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, most preferably by at least 30%.

2. A method for diagnosing cancer and / or an acute inflammatory disease in a subject, the method comprising:(a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample, wherein the precipitationinducing condition is provided by:(i) adjusting the pH of the sample to a value in the range of between, with increasing preference, 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, pH 4.1 and 4.3, most preferably pH 4.2±0.05; preferably by addition of an acid, or an aqueous solution comprising the acid, wherein the acid has a pKain the range of between, with increasing preference,3.0 and 7.0, 3.5 and 6.5, 3.8 and 5.8, 4.1 and 5.5, and most preferably has a pKaof 4.75±0.05; wherein preferably the acid is an organic acid, wherein the organic acid is preferably a carboxylic acid, more preferably a monocarboxylic acid, most preferably acetic acid;(ii) an increase of the sample temperature;(iii) addition of at least one salting-out agent;(iv) addition of one or more aliphatic alcohol(s), preferably alkanol(s), and most preferably ethanol; and / or(vi) addition of a ketone, preferably acetone; and(b) determining the fraction of the precipitated (poly)peptides from the total amount of (poly)peptides in the sample; whereby:- in the instance of the precipitation being induced by lowering of the sample pH, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, more preferably more than 2.1 % of the total amount of (poly)peptides in the sample;- in the instance of the precipitation being induced by an increase of the sample temperature, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 5%, 5.5%, 6%, 6.5%, more preferably more than 7% of the total amount of (poly)peptides in the sample;- in the instance of the precipitation being induced by addition of at least one salting-out agent, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, more preferably more than 2.1 % of the total amount of (poly)peptides in the sample;- in the instance of the precipitation being induced by addition of ethanol and / or other aliphatic alcohol, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 4%, 6%, 8%, 10%, 11%, more preferably more than 12% of the total amount of (poly)peptides in the sample;- in the instance of the precipitation being induced by addition of a ketone, if the determined fraction of the precipitated (poly)peptides corresponds to more than, with increasing preference, 1 %, 2%, 3%, 4%, more preferably more than 5% of the total amount of (poly)peptides in the sample; the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease.

3. A method for evaluating the responsiveness of a cancer and / or an acute inflammatory disease to a candidate treatment in a subject, the method comprising:(a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample, wherein said sample has been obtained at a time point after which the candidate treatment has been initiated;(b) assessing the level of the 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 of step (a) was obtained from the subject, wherein the reference sample has been subjected to a corresponding precipitation-inducing condition as in step (a); and whereby:- if the level of the precipitated (poly)peptides in the sample is equal to or lower than the level of the precipitated (poly)peptides in reference sample, the cancer and / or the acute inflammatory disease is classified as being responsive to the candidate treatment; and / or- if the level of the precipitated (poly)peptides in the sample exceeds the level of the precipitated (poly)peptides in the reference sample, the cancer and / or the acute inflammatory disease is classified as being non-responsive to the candidate treatment.

4. A method for assessing the malignancy level of a cancer in a subject, the method comprising:(a) subjecting a sample obtained from the subject to a condition inducing precipitation of one or more (poly)peptides comprised in the sample; and(b) assessing the level of the precipitated (poly)peptides in the sample relative to:(i) at least one correspondingly treated reference sample obtained from one or more reference subjects having a cancer with a known malignancy level; and / or(ii) a predetermined standard that has optionally been obtained based on one or more reference sample(s) according to (b)(i); wherein a higher, lower, or substantially same level of the precipitate in the sample relative to the level of precipitate in the reference sample provides an indication that the cancer in the subject has a higher, lower, or substantially same malignancy, respectively, relative to the cancer of the reference subject or the predetermined standard.

5. The method of claim 1 , wherein in step (b) the level of the precipitated (poly)peptides in the sample is assessed based on a statistical comparison relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be positive for cancer and / or an acute inflammatory disease (pathological reference samples) and / or relative to the level of precipitated (poly)peptides in a group of correspondingly treated reference samples, wherein each reference sample has been obtained from a reference subject known to be negative for cancer and an acute inflammatory disease (non-pathological reference samples), wherein the statistical comparison is conducted by:(a) an area under the curve (AUC) calculation; wherein preferably:(a-1) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of pathological reference samples (P) by calculating an ALICp value as defined by formula (I):wherein np is the number of reference samples in the group of pathological reference samples P, x;is the level of the precipitated (poly)peptides for the ithpathological reference sample in P, and k is the indicator function for comparing S against x, as defined by formula (II):and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an AUCp value is calculated in the range of between, with increasing preference, with increasing preference, >0.75 and 1.0; >0.7 and 1.0, >0.6 and 1.0, >0.5 and 1.0, >0.4 and 1.0, >0.3 and 1.0, >0.2 and 1.0, >0.001 and 1.0, and most preferably between >0 and 1.0; and / or(a-2) the level of the precipitated (poly)peptides in the sample (S) is compared with the level of the precipitated (poly)peptides in the group of non-pathological reference samples ( / V) by calculating an AUCw value as defined by formula (III):whereis the number of samples in the group non-pathological reference samples l\l, x> is the level of the precipitated (poly)peptides for the Ithnon- pathological reference sample in N, and k is the indicator function for comparing S against x, as defined by formula (II):and whereby the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease if for S an AUCw value is calculated in the range of between, with increasing preference, 0.65 and 1 .0, 0.70 and 1.0 0.75 and 1 .0, 0.80 and 1.0, 0.85 and1 .0, 0.95 and 1 .0, 0.96 and 1.0, 0.97 and 1.0, 0.98 and 1.0, 0.99 and 1.0, most preferably of 1.0.

6. The method of any one of claims 1 and 3 to 5, wherein the precipitation-inducing condition is provided by an alteration of the sample pH, preferably by adjusting the sample pH to a value in the range of between, with increasing preference, 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, pH 4.1 and 4.3, most preferably pH 4.2±0.05.

7. The method of claim 6, wherein the adjustment of the sample pH is provided by adding, to the sample:(a) at least one acid having a pKa in the range of between, with increasing preference, 2.0 and -10.0, 3.0 and 7.

0. 3.5 and 6.5, 3.8 and 5.8, 4.1 and 5.5, and most preferably a pKa of 4.76±0.05; wherein preferably the at least one acid is an organic acid, wherein preferably the organic acid is:(i) a carboxylic acid, more preferably a (i-1) monocarboxylic acid, preferably selected from the group consisting of formic acid, acetic acid, benzoic acid, propionic acid and butyric acid, or (i-2) a di- or tri-carboxylic acid, preferably citric acid;(ii) barbituric acid; and / or(iii) perchloric acid; and / or(b) an aqueous solution comprising at least one acid as defined in (a); wherein preferably:- the aqueous solution comprises the at least one acid at a total concentration in the range of between, with increasing preference, 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%, 0.35 vol% and 0.45 vol%, most preferably at a concentration of 0.4 vol%;- the aqueous solution has a pH in the range of between, with increasing preference, pH 2 and pH 5, pH 2 and pH 4, pH 2.5 and pH 3.5, pH 2.8 and pH 3.2, pH 2.9 and 3.1 , and most preferably to a value of pH 2.97±0.05;- the aqueous solution is added to the sample at a volume-to-volume ratio of aqueous solution to sample of between, with increasing preference, 0.3:1 and 0.9:1 , 0.4:1 and 0.8:1 , 0.5:1 and 0.7:1 , most preferably at a concentration of 0.59:1 ; and / or- the aqueous solution additionally comprises sodium chloride at a concentration in the range of between, with increasing preference, 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), 0.7% (w / v) and 0.9% (w / v), most preferably at a concentration of 0.81 % (w / v).

8. The method of any one of claims 1 to 7, wherein the sample comprises or consists of:(a) a body fluid, preferably selected from blood (preferably plasma), saliva, mucus, sputum, vomitus, sweat, tear, urine, semen, vaginal fluid, feces, and exudate, or any mixture thereof; and / or(b) a body tissue, preferably a homogenized body tissue, more preferably a cell-free suspension of a homogenized body tissue.

9. The method of any one of claims 1 to 8, wherein the sample, prior to being subjected to the precipitation-inducing condition, is diluted with a dilution medium, wherein the dilution medium preferably:(i) is an aqueous solution comprising a salt, preferably sodium chloride, at a concentration in the range of between, with increasing preference, 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, and most preferably 98 mM; and / or(ii) is added to the sample at a volume-to-volume(v / v)-ratio of dilution medium to sample of between, with increasing preference, 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 , and most preferably at a v / v-ratio of dilution medium to sample of 4.2:1.

10. The method of any one of claims 1 to 9, wherein the precipitation-inducing condition is provided, or additionally provided, by:(i) an increase of the sample temperature;(ii) addition of at least one salting-out agent to the sample; wherein preferably the saltingout agent is selected from:(ii-a) a salt comprising a cation selected from Ca2+, (CH3)4N+, Cs+, Rb+, NH4+, K+, Na+, Mn2+, and Li+; and an anion selected from OH", SO42", HPO42", CHsCOO" , C3HSO(COO)33”, CO32", CIO3", BrOs", thiocyanate (SON"), and Cl"; more preferably KCI and / or, (NH4)2SO4; and / or(ii-b) an alkali metal sulfate; preferably U2SO4, Na2SO4, K2SO4, Rb2SO4, and / or CS2SO4;(iii) addition of one or more other aliphatic alcohol(s), preferably alkanol(s), most preferably ethanol;(iv) concentration of the sample by evaporation;(v) addition of a ketone, preferably acetone; and / or(vi) any combination of any of (i) to (v).

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

12. The method of claim 11 , wherein in step (b) the level of the precipitated (poly)peptides is assessed by spectrophotometrically measuring a change of absorbance, whereby a determined increase or decrease of the absorbance is indicative of an increase or decrease, respectively, of the level of precipitated (poly)peptides in the sample; wherein preferably the absorbance is measured at a wavelength in the range of between, with increasing preference, 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, 335 nm and 345 nm and most preferably at 340 nm.

13. The method of claim 12, wherein, in the method for diagnosing cancer and / or an acute inflammatory disease in a subject, if the change of absorbance is an absorbance increase in the range of between, with increasing preference, 78-170 milli extinction units (mE), 80- 160 mE, 83-155 mE, 88-150 mE, 93-145 mE, 98-140 mE, most preferably 105-134 mE, preferably when measured in a 1 cm-pathlength cuvette or when normalized to a measurement in a 1 cm-pathlength cuvette, the subject is diagnosed as being positive for cancer and / or an acute inflammatory disease.

14. The method of any one of claims 1 to 13, wherein in step (b) the level of the precipitated (poly)peptides is reversely assessed from the remaining soluble fraction of the sample, preferably after removal of the precipitated (poly)peptides.

15. The method of any one of claims 1 to 14, wherein:(i) the sample, prior to being subjected to the precipitation-inducing condition in step (a), is incubated at a temperature of between 2 °C and 42 °C, preferably at 37 ± 2 °C, for a duration of at least 10 s, preferably for a duration in the range of between, with increasing preference, 10 s and 5000 s, 20 s and 4000 s, 30 s and 3000 s, 40 s and 2000 s, 50 s and 1000 s, 60 s and 800 s, 80 s and 600 s, 100 s and 500 s, 200 s and 400 s, 250 s and 350 s, preferably between 275 s and 325 s; most preferably for 300 s; and / or(ii) the sample, after being subjected to the precipitation-inducing condition in step (a) and prior to step (b) is incubated at a temperature of between 2 °C and 42 °C,preferably at 37 ± 2 °C, for a duration of at least 60 s, preferably for a duration in the range of between, with increasing preference, 60 s and 600 s, 100 s and 500 s, 200 s and 400 s, 250 s and 350 s, 280 s and 320 s; most preferably for 300 s.

16. The method of any one of claims 1 to 15, wherein the sample and / or the dilution medium additionally comprises:(i) an anticoagulant; preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin or any salt thereof;(ii) a proteinase inhibitor;(iii) one or more stabilizing agents, preferably selected from albumin, casein, gelatin, collagen, globulin, protamine;(iv) skim milk powder;(v) a surfactant; preferably selected from Tween, preferably Tween 20 or Tween 80, Triton X-100, and sodium dodecylbenzenesulfonate;(vi) a polyethylene glycol (PEG); wherein preferably the PEG has a molecular weight of between 1 ,000 and 20.000 Da and / or is selected from PEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000. PEG15000, PEG20000; and / or(vii) a polysaccharide; preferably a dextran selected from dextran-1, dextran-10, dextran- 20, dextran-30, and dextran-40 dextran.

17. The method of any one of claims 1, 2 and 5 to 16, further comprising assessing a sample, preferably a blood sample, obtained from the subject for the presence or absence of an acute inflammation, wherein, in the instance of a subject for which an indication to have cancer and / or an acute inflammatory disease is determined, a determined absence of an acute inflammation indicates that the subject has cancer and no acute inflammatory disease.

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

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