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WO2024180169A1 - Moyens et procédés de diagnostic du cancer et/ou d'une maladie inflammatoire aiguë - Google Patents

Moyens et procédés de diagnostic du cancer et/ou d'une maladie inflammatoire aiguë Download PDF

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Publication number
WO2024180169A1
WO2024180169A1 PCT/EP2024/055186 EP2024055186W WO2024180169A1 WO 2024180169 A1 WO2024180169 A1 WO 2024180169A1 EP 2024055186 W EP2024055186 W EP 2024055186W WO 2024180169 A1 WO2024180169 A1 WO 2024180169A1
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sample
poly
peptides
precipitated
cancer
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PCT/EP2024/055186
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English (en)
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Berthold Zwerger
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Carcimun Biotech Gmbh
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Publication of WO2024180169A1 publication Critical patent/WO2024180169A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • 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.
  • the invention relates to a method for assessing the malignancy level of a cancer in a subject.
  • 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).
  • CT computed tomography
  • 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.
  • 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.
  • chronic inflammation may evolve and can lead to further complications, including metabolic diseases, such as coronary heart disease, type 2 diabetes, and rheumatoid arthritis.
  • metabolic diseases such as coronary heart disease, type 2 diabetes, and rheumatoid arthritis.
  • inflammation has more recently also been implicated in the development and progression of cancer (Greten FR, Grivennikov SI.
  • 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:
  • (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
  • 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.
  • 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.
  • 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.
  • 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.
  • cancer 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
  • the cancer is selected from the group of solid cancers. In other embodiments, the cancer is selected from the group of liquid cancers.
  • solid cancer refers to cancers in which a plurality of malignant cells is associated with one another, i.e., contiguous and localized within a confined site.
  • 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.
  • 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.
  • the cancer may be selected from any of these specific kinds of cancers.
  • 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).
  • inflammation refers 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).
  • an immune response e.g., pro- inflammatory markers such as chemokines and cytokines, production of plasma haptoglobin
  • symptoms of inflammation e.g., pain, heat, redness and/or edema.
  • inflammation may be acute or chronic.
  • inflammatory disease 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 mell
  • sepsis 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.
  • sepsis as used herein, also encompasses specific forms and complications thereof, such as “severe sepsis” and “septic shock”.
  • 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.
  • inflammatory markers such as C-reactive protein (GRP) and/or procalcitonin (PCT) are well known in the art and their measurement is nowadays routinely performed.
  • GRP C-reactive protein
  • PCT procalcitonin
  • 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).
  • the subject is a mammal.
  • the subject is a human.
  • 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).
  • a body fluid e.g., blood, such as whole blood, plasma or serum
  • a body tissue e.g., a tissue sample obtained by biopsy.
  • sample may be a purely biological sample or a processed form thereof.
  • 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.
  • 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.
  • 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.
  • (poly)peptide 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.
  • 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.
  • 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.
  • an environmental stimulus /.e., a precipitation-inducing condition, as described below
  • 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.
  • 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).
  • a precipitating agent e.g., an acid
  • (poly)peptides comprised in a body sample e.g., blood plasma
  • a body sample e.g., blood plasma
  • 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).
  • 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.
  • the term “assessing the level of precipitated (poly)peptides” 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.
  • 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
  • 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.
  • 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 gel-electrophoresis e.g., polyacrylamide gel-electrophoresis (PAGE)
  • SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis
  • Coomassie-Blue-staining or other staining approaches, e.g., silver staining
  • Western Blotting wherein the obtained band
  • Example 4 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).
  • LC-MS liquid chromatography-mass spectrometry
  • MALDI Matrix Assisted Laser Desorption/lonization
  • DLS Dynamic Light Scattering
  • NMR nuclear magnetic resonance
  • 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.
  • 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.
  • A280nm wavelength
  • 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).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • 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 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”.
  • 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.
  • 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.
  • I R ionizing radiation
  • the invention provides a method for diagnosing cancer and/or an acute inflammatory disease in a subject, the method comprising:
  • 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;
  • 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;
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • ketone 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%.
  • 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%.
  • 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.
  • 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:
  • step (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:
  • the cancer and/or the acute inflammatory disease is classified as being responsive to the candidate treatment;
  • the cancer and/or the acute inflammatory disease is classified as being non-responsive to the candidate treatment.
  • 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.
  • 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).
  • an agent e.g., a candidate therapeutic agent.
  • 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.
  • cancer agent refers to any agent that is prima facie expected suitable for treating a cancer and/or an inflammatory disease (preferably an acute inflammatory disease).
  • inflammatory disease preferably an acute inflammatory disease.
  • responding agents are more specifically referred to herein as “anti-cancer agents” and “anti-inflammatory agents”, respectively.
  • 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.
  • EMA European Medicines Agency
  • FDA Food and Drug Administration
  • 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).
  • 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.
  • 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.
  • 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
  • preventative e.g., preventing development of disease or the incidence of relapse
  • 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.
  • local therapy e.g., surgery or radiation therapy
  • anti-cancer agent includes any agent that may be suitable for the treatment of a cancer.
  • cancer treatment includes 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.
  • 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.
  • Oxestrogen receptor modulators refers to compounds which interfere with or inhibit the binding of oestrogen to the receptor, regardless of mechanism.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Topicisomerase 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, etop-N
  • Angiogenesis inhibitors refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.
  • 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.
  • NSAIDs non
  • 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).
  • 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,
  • 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).
  • angiogenesis inhibitors such as trastuzumab
  • tumour suppressor genes such as p53
  • HMG-CoA reductase inhibitors refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase.
  • HMG-CoA reductase inhibitors 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.
  • 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.
  • 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).
  • FPTase farnesyl-protein transferase
  • GGPTase-l geranylgeranyl-protein transferase type I
  • Rab GGPTase geranylgeranyl-protein transferase type-ll
  • 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.
  • anti-inflammatory agent 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.
  • 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.
  • steroidal compounds including hydrocortisone and the like
  • nonsteroidal anti-inflammatory agents including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin, and the like.
  • the invention relates to a method for assessing the malignancy level of a cancer in a subject, the method comprising:
  • 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.
  • 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.
  • 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).
  • 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 t is the level of the precipitated (poly)peptides for the i th pathological 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
  • a UC N — y fc(5, x z ) nN
  • 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 I th non-pathological reference sample in N
  • 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.
  • the invention also relates to a method for diagnosing cancer and/or an acute inflammatory disease in a subject, the method comprising:
  • 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 i th pathological 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
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • citric acid pKa 3.13, 4.76, 6.4; Fig 10.1
  • the adjustment of the sample pH is provided by adding, to the sample:
  • 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;
  • 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).
  • 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
  • the acid is selected from:
  • the sample comprises or consists of:
  • 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
  • a body tissue preferably a homogenized body tissue, more preferably a cell-free suspension of a homogenized body tissue.
  • the sample comprises or consists of blood.
  • blood encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined.
  • the blood is preferably blood plasma.
  • the sample comprises, consists essentially of, or consists of plasma (blood plasma).
  • 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).
  • 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).
  • 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.
  • an anticoagulant e.g., K3-EDTA or K2-EDTA or other anticoagulant, preferably K2-EDTA
  • centrifugation e.g., 3000 x g / 10 min
  • 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).
  • 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).
  • the drawn blood is preferably exposed immediately to an anticoagulant to preclude coagulation.
  • anticoagulant 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.
  • the sample additionally comprises:
  • an anticoagulant preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin; and/or
  • 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).
  • 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).
  • the anticoagulant is heparin.
  • Heparin is a sulfated glycosaminoglycan and is used clinically for the prevention of blood clots.
  • Non-limiting examples include unfractionated heparin, such as heparin sodium (e.g., heparin sodium USP, available from Scientific Protein Labs of Waunakee, Wl).
  • protease inhibitors refers generally to a compound, substance and/or composition which is capable of inhibiting the action of one or more proteases.
  • 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.
  • 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.
  • 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.
  • protease inhibitors include alpha-2- macroglobulin, soybean or lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin and egg white cystatin.
  • combinations of protease inhibitors may be employed, such as commercially available “protease inhibitor cocktails”, for example, Roche - completeTM Protease Inhibitor Cocktail and SIGMAFASTTM Protease Inhibitor Cocktail Tablets, EDTA-Free.
  • the sample comprises plasma and EDTA or a salt thereof (preferably potassium EDTA, such as di- or tri-potassium EDTA (K2- or K3-EDTA)).
  • EDTA-plasma A corresponding sample is conventionally referred to as “EDTA-plasma” sample.
  • 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.
  • 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.
  • a 26 pl EDTA-plasma sample was diluted with 110 pl of an aqueous solution comprising 0.57% NaCI (corresponding to 98 mM NaCI).
  • 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 (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
  • a salt preferably sodium chloride (NaCI)
  • (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.
  • the sample, prior to being subjected to the precipitationinducing condition is:
  • (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 ,
  • (ii-a) comprises or consists of water
  • (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 .
  • the sample, prior to being subjected to the precipitation-inducing condition is:
  • (i-a) comprises or consists of water
  • (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
  • (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
  • a salt preferably sodium chloride (NaCI)
  • (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.
  • 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).
  • the precipitation-inducing condition in step (b) is provided, or additionally provided, by:
  • salting-out agent is selected from:
  • 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.
  • 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.
  • 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.
  • the at least one salting-out agent is selected from the group of (NH 4 ) 2 SO 4 , alkali-metal chloride salts (preferably selected from KCI and/or CaCh), guanidine thiocyanate, and MnCh, and is most preferably (NH4)2SO4.
  • the salting-out agent preferably (NH4)2SO 4
  • the salting-out agent 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).
  • the one or more other aliphatic alcohol(s) is/are selected from ethanol, isopropanol, and/or methanol, and is most preferably ethanol.
  • 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).
  • 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).
  • the salting-out agent preferably (NH ⁇ SOt
  • 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.
  • the level of the precipitated (poly)peptides is assessed by spectrophotometry.
  • spectrophotometry 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 (/).
  • 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).
  • spectrophotometry 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.
  • nephelometry 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.
  • the solution must be placed into the light path of a photometer in a defined format.
  • Cuvettes i.e., sample containers featuring optical windows
  • 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.
  • a cuvette which has a shorter pathlength ⁇ 1 cm
  • 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.
  • 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).
  • 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.
  • a dilution medium such as an isotonic salt solution
  • the spectrophotometric analysis was conducted using a conventional clinical chemistry analyzer, i.e., the IndikoTM Plus Clinical Chemistry Analyzer (#98640000, Thermo Fisher).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mE milli extinction units
  • the invention also relates to a method for diagnosing cancer and/or an acute inflammatory disease in a subject, the method comprising:
  • (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:
  • 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
  • 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.
  • 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.
  • 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.
  • the inventor 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.
  • 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).
  • step (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
  • 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.
  • 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).
  • the sample and/or the dilution medium additionally comprises:
  • an anticoagulant preferably selected from ethylenediaminetetraacetic acid (EDTA), citrate and heparin or any salt thereof;
  • one or more stabilizing agents preferably selected from albumin, casein, gelatin, collagen, globulin, protamine;
  • a surfactant preferably selected from Tween, preferably Tween 20 or Tween 80, Triton X- 100, and sodium dodecylbenzenesulfonate;
  • a polyethylene glycol (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
  • a polysaccharide preferably a dextran selected from dextran-1 , dextran-10, dextran-20, dextran-30, and dextran-40 dextran.
  • option (i) is particularly preferred.
  • the sample and/or the dilution medium does not additionally comprise any of (i) to (vii).
  • the dilution medium does not comprise any constituents originating or derived from a human.
  • the albumin is a non-human albumin, for example, bovine serum albumin (BSA); and/or the heparin is non-human heparin.
  • BSA bovine serum albumin
  • the albumin is human serum albumin.
  • the milk (poly)peptide(s) and/or the skim milk powder is/are of non-human origin, more preferably from cow milk.
  • the additive is a (poly)peptide, e.g., serum albumin, or other animal product, e.g., milk powder
  • a (poly)peptide e.g., serum albumin, or other animal product, e.g., milk powder
  • 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.
  • 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.
  • a sample preferably a blood sample
  • the subject is diagnosed as being positive for cancer and as having no acute inflammatory disease.
  • 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.
  • 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.
  • 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.
  • an acute inflammation e.g., increased levels of white blood cells (leukocytes)
  • 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.
  • CRP C-reactive protein
  • PCT procalcitonin
  • fibrinogen C-reactive factor
  • leukocytes preferably the one or more inflammatory markers are selected from C-reactive protein (CRP), procalcitonin (PCT), fibrinogen, and leukocytes.
  • 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).
  • 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.
  • CRP 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.
  • 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”.
  • 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.
  • the presence or absence of an acute inflammation is assessed, or additionally assessed, by evaluating the level of procalcitonin (PCT).
  • PCT procalcitonin
  • PCT Procalcitonin
  • Plasma levels of PCT in healthy individuals are typically below 0.1 ng/mL.
  • concentration of ⁇ 0.2 ng/mL is a useful reference range.
  • 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
  • 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
  • PCT level preferably PCT plasma level
  • >0.10 ng/mL is indicative of the presence of an acute inflammation (esp. of sepsis).
  • the method is conducted:
  • microtiter plates also known as micro-well or multiwell plate, for example, a 6-, 12-, 24-, 48-, or 96-well plate
  • analyzed sequentially /.e., consecutively or in parallel.
  • 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.
  • a device preferably, a diagnostic apparatus or diagnostic device
  • BCA Bicinchoninic acid assay
  • Figure 2 A spectrophotometry-based assay to quantify protein precipitation.
  • 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.
  • 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).
  • 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.
  • HCI hydrochloric acid
  • 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.
  • Data from (A-C) are represented as mean ⁇ SD from at least three biological replicates. *P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001 .
  • FIG. 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.
  • FIG. 5 Acetic acid induces reversible precipitation of blood plasma proteins.
  • 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.
  • 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 Youden index J 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.
  • FIG. 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.
  • FIG. 10.1 Citric acid in 0.9% NaCI aq. used in step 6.
  • 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.
  • Figure 10.3 Perchloric acid in 0.9% NaCI aq.
  • Figure 11.1 Ethanol used in step 6.
  • 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.
  • Figure 11.3 Methanol used in step 6.
  • FIG. 12.1 CaCh used in step 6.
  • FIG. 12 Guanidine thiocyanate used in step 6.
  • FIG.3 MnCh used in step 6.
  • 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 (dH 2 O). 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.
  • 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.
  • Human K2-EDTA blood plasma were purchased from ProteoGenex, Inc. (460 Hindry Ave., Unit A).
  • BCA Bicinchoninic acid assay
  • BCA Bicinchoninic acid assay
  • STET PierceTM BCA Protein Assay
  • 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.
  • 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.
  • 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 OrionTM micro-pH-electrode (9810BN, Thermo Fisher). Osmolality of blood plasma was determined using the VAPRO 5600 pressureosmometer according to the supplier’s manual.
  • UV/VIS Spectrophotometric assay to quantify protein precipitation
  • Protein precipitation was quantified using the IndikoTM 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.
  • sensitivity sensitivity
  • SPE specificity
  • ACC accuracy
  • bACC balanced accuracy
  • F1 score F1 score
  • ACC (TP+TN)/(TP+TN+FP+FN)
  • 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.
  • AUCp area under the curve
  • AUCp — Y k(S,Xt)
  • np is the number of subtraction values in P
  • x- is the subtraction values for the i-th sample in P
  • 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.
  • 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.
  • 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.
  • 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.
  • LR likelihood ratios
  • AUC area under the curve
  • 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.
  • 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.
  • AUCN is defined in Material & Methods
  • N are all measurements of a non- pathological reference group with at least 2 reference samples
  • x is the AUCN threshold greater or equal to 0.64.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • dH2O a dilution series of NaOH or vehicle
  • 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.
  • Table 1 Dataset of pathologic and non-pathologic reference groups with results of AUCp and AUCN analyses.
  • Table 2A Results of the threshold (TH) optimization based on subtraction
  • Table 2B Results of the threshold optimization based on ALICp
  • Table 3A Average performance measures for the determination of the minimum required sample size based on ALICp
  • 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
  • Table 3D Standard deviation of performance measures for the determination of the minimum required sample size based on AUCN
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends on.
  • 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
  • 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,
  • 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).
  • a weight of “about 100 grams” can include weights between 90 grams and 110 grams.
  • 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

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Abstract

La présente invention concerne des méthodes de diagnostic du cancer et/ou d'une maladie inflammatoire aiguë chez un sujet. L'invention concerne en outre un procédé d'évaluation de la réactivité d'un cancer et/ou d'une maladie inflammatoire aiguë à un traitement candidat chez un sujet. De plus, l'invention concerne un procédé d'évaluation du niveau de malignité d'un cancer chez un sujet.
PCT/EP2024/055186 2023-03-02 2024-02-29 Moyens et procédés de diagnostic du cancer et/ou d'une maladie inflammatoire aiguë WO2024180169A1 (fr)

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