WO2020043864A1 - Method of predicting stage of ulcerative colitis - Google Patents

Method of predicting stage of ulcerative colitis Download PDF

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Publication number
WO2020043864A1
WO2020043864A1 PCT/EP2019/073173 EP2019073173W WO2020043864A1 WO 2020043864 A1 WO2020043864 A1 WO 2020043864A1 EP 2019073173 W EP2019073173 W EP 2019073173W WO 2020043864 A1 WO2020043864 A1 WO 2020043864A1
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Prior art keywords
seq
nos
polypeptides
ulcerative colitis
control sample
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PCT/EP2019/073173
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French (fr)
Inventor
Terkel Hansen
Armin SCHNIERS
Jon FLORHOLMEN
Guro FORSDAHL
Rasmus GOLL
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Universitetet I Tromsø - Norges Arktiske Universitet
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Publication of WO2020043864A1 publication Critical patent/WO2020043864A1/en

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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
    • G01N33/68—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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention relates generally to the field of inflammatory bowel disease (IBD), and in particular ulcerative colitis (UC).
  • IBD inflammatory bowel disease
  • UC ulcerative colitis
  • the present invention provides methods for predicting clinical course of UC in a patient. By being able to predict the clinical course of UC, it is now possible to conclude on a therapeutic approach adapted to reflect the actual clinical picture of the patient at an early stage. Such a diagnostic test will create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
  • IBD Crohn’s disease
  • UC Crohn’s disease
  • IBD Crohn’s disease
  • UC Crohn’s disease
  • the immune response is dysregulated in IBD such as in other autoimmune diseases (Mucosal immunology 2014, 7, 1139-1150).
  • the cause of the disease is unknown, but it is generally accepted that IBD represents a dysregulation of the intestinal immune tolerance to microbiological and/or nutritional antigens. This dysregulation results from a combination of environmental and genetic factors, in which dysfunctional gene products may initiate inflammation via signaling pathways that have been defined to varying degrees, including innate, adaptive and T-regulatory immunity systems.
  • UC ulcerative colitis
  • UC ulcerative colitis
  • CD decisions about drug therapy are dependent on both location and behavior of the disease.
  • the medication in CD usually includes aminosalicylates and antibiotics to treat mild mucosal disease, corticosteroids to moderate disease, and biological molecules to treat fistulizing disease.
  • aminosalicylates, azathioprine, mercaptopurine, methotrexate, metronidazole, and associations can be used as maintenance therapies.
  • these drugs can generate several side effects. Moreover, these therapies do not achieve clinical remission and they can lead to the onset of other conditions such as renal impairment. At the same time the classical treatments are widely used, new therapeutic strategies are under development in the attempt of improving the patient’s life quality.
  • the new therapies aim to reduce the side effects and to treat patients who do not respond satisfactorily to conventional therapies. These involve the manipulation of the microbiome using antibiotics, probiotics, prebiotics, diet, and fecal microbiota transplantation.
  • the goal of medical treatment of IBD, and UC in particular, is to rapidly induce remission while at the same time preventing complications of the disease itself and its treatment.
  • the choice of treatment depends on a number of factors such as severity, localization and the course of the disease.
  • WO2016/049772 discloses protein biomarkers and methods for their use in diagnosing IBD, UC and CD. There is also a disclosure of methods for diagnosing the severity of UC disease, but there is no mentioning of a method suitable to predict the clinical course of UC.
  • Predictors of clinical outcome will create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
  • the present invention has solved the above need by providing a method for predicting clinical course of UC in a patient. By being able to predict the clinical course of UC, it is now possible to conclude on a therapeutic approach which at an early stage is adapted to reflect the actual clinical picture.
  • a first aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
  • each of said at least ten polypeptides comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896;
  • step b) comparing the results obtained in step a) with
  • the results obtained in step a) are compared with i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 4 months after the first control sample was taken; and/or
  • the results obtained in step a) are compared with
  • the results obtained in step a) are compared with
  • second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after the second control sample was taken; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after the third control sample was taken.
  • the results obtained in step a) are compared with
  • the results obtained in step a) are compared with
  • the results obtained in step a) are compared with:
  • step a) the results obtained in step a) are compared with:
  • second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after onset of disease and the second control sample was taken at onset of disease; and/or
  • step a) the results obtained in step a) are compared with:
  • second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 24 months after onset of disease and the second control sample was taken at onset of disease; and/or
  • the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the first control sample;
  • the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the second control sample;
  • the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the third control sample.
  • onset of disease is the first time the subject experiences symptoms of ulcerative colitis.
  • onset of disease is when the subject experiences symptoms of ulcerative colitis.
  • onset of disease is at an early stage of ulcerative colitis, such as the first time the subject experiences symptoms of ulcerative colitis.
  • the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis.
  • the subject to be diagnosed is diagnosed:
  • the first control subject has been diagnosed with moderate ulcerative colitis within 12 months, such as within 24 months, from the onset of disease;
  • the second control subject has been diagnosed with severe ulcerative colitis within 12 months, such as within 24 months, from the onset of disease;
  • the third control subject has been diagnosed with mild ulcerative colitis within 12 months, such as within 24 months, from the onset of disease.
  • the first control subject has been diagnosed with moderate ulcerative colitis within 5 years from the onset of disease;
  • the second control subject has been diagnosed with severe ulcerative colitis within 5 years from the onset of disease.
  • the third control subject has been diagnosed with mild ulcerative colitis within 5 years from the onset of disease.
  • the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
  • the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896 with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others.
  • the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896 with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides.
  • the top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least one of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least two of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least three of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least four of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least five of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least six of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least seven of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least eight of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least nine of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • each and all of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • one of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27, 213, 617, 894, 903, 1011, 1513, 1920, 1987 or 2412 respectively.
  • sequence identity such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27, 213, 617, 894, 903, 1011, 1513, 1920, 1987 or 2412 respectively.
  • a first polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
  • a second polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
  • a third polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 617;
  • a fourth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 894;
  • a fifth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 903;
  • a sixth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 101 1;
  • a seventh polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1513;
  • a eight polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1920;
  • a ninth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1987;
  • a tenth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 2412.
  • each of said at least ten polypeptides comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 2896.
  • each of said at least ten polypeptides consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1 to 2896.
  • step b) is performed using software-based statistical and bioinformatics data analysis.
  • the biological sample, first control sample, second control sample and third control sample are selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and biopsy, such as colon biopsy.
  • first control sample is colon biopsy
  • second control sample and third control sample are colon biopsy
  • the colon biopsy is subjected to sample preparation prior to step a).
  • such a sample preparation comprises the following steps:
  • step ii) subjecting the product of step i) to homogenization.
  • the biological sample, first control sample, second control sample and third control sample are a colon biopsy; and the colon biopsy is subjected to sample preparation prior to step a); the sample preparation comprising the following steps:
  • step ii) subjecting the product of step i) to homogenization
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate,
  • cetyltrimethyl ammonium N-methyldioctylammine or any combination thereof.
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate. Even more preferably, the lysis buffer comprises sodium deoxycholate, urea and triethylammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM
  • the product of step i) and/or product of step ii) is subjected to enzymatic digestion.
  • the enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
  • step i), product of step ii) and/or the product of step iii) is preferably subjected to treatment suitable for reducing and alkylating cysteines.
  • the levels of the at least ten polypeptides or fragments thereof are detected and quantified by mass spectroscopy, preferably liquid chromatography mass spectroscopy (LC-MS), especially high performance liquid chromatography mass spectroscopy (HPLC-MS) or reverse phase liquid chromatography mass spectroscopy (RPLC-MS); electro- spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC-MS), atmospheric pressure chemical ionization mass spectroscopy (APCI-MS), capillary electrophoresis mass spectroscopy (CE-MS), tandem mass spectroscopy (MS-MS); or any combination thereof.
  • mass spectroscopy preferably liquid chromatography mass spectroscopy (LC-MS), especially high performance liquid chromatography mass spectroscopy (HPLC-MS) or reverse phase liquid chromatography mass spectroscopy (RPLC-MS); electro- spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC-MS), atmospheric pressure chemical ionization mass
  • levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
  • the levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
  • the length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10- 55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
  • the length of each of said fragments is in the range 5-70 amino acids long, such as in the range 10-70.
  • the comparison is performed using software-based statistical and bioinformatics data analysis.
  • the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo.
  • An alternative aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
  • step b) comparing the results obtained in step a) with reference values of levels of said at least one polypeptide or a fragment thereof from subjects suffering from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis respectively;
  • the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from mild ulcerative colitis;
  • the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from moderate ulcerative colitis;
  • the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from severe ulcerative colitis.
  • a preferred embodiment according to the alternative aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
  • polypeptides comprising an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs:
  • step b) comparing the results obtained in step a) with reference values of levels of said at least ten polypeptides or fragments thereof from subjects suffering from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis respectively;
  • the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from mild ulcerative colitis;
  • the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from moderate ulcerative colitis;
  • the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from severe ulcerative colitis.
  • the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
  • the at least one polypeptide or each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896; with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others.
  • the at least one polypeptide or each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896.
  • each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896; with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides.
  • the top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • the at least one polypeptide comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least one of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least two of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least three of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least four of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least five of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least six of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least seven of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least eight of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least nine of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • each and all of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • SEQ ID NOs: 617 amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 2
  • a first polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
  • a second polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
  • a third polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 617;
  • a fourth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 894;
  • a fifth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 903;
  • a sixth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1011;
  • a seventh polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1513;
  • a eight polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1920;
  • a ninth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1987;
  • a tenth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 2412.
  • the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis.
  • the biological sample is selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and biopsy, such as colon biopsy.
  • the biological sample is a colon biopsy.
  • the biological sample is blood plasma or serum, even more preferably serum.
  • sample preparation comprises the following steps:
  • step ii) subjecting the product of step i) to homogenization.
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate, cetyltrimethylammonium, N- methyldioctylammine or any combination thereof.
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate.
  • the lysis buffer comprises sodium deoxycholate, urea and tri ethyl ammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM triethylammonium bicarbonate buffer.
  • the product of step i) and/or product of step ii) is subjected to enzymatic digestion.
  • the enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
  • the levels of the at least one or at least ten polypeptides may be detected and quantified by detecting and quantifying the levels of at least one fragment, such as at least two fragments, at least three fragments, at least four fragments, at least five fragments, at least six fragments, at least seven fragments, at least eight fragments, at least nine fragments or at least ten fragments for each of said polypeptides.
  • the length of each fragment is in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
  • the product of step i) and/or product of step ii) is subjected to treatment suitable for reducing and alkylating cysteines.
  • the levels of the at least one polypeptide or the at least ten polypeptides is measured by mass spectroscopy, preferably liquid chromatography mass spectroscopy (LC- MS), especially high performance liquid chromatography mass spectroscopy
  • HPLC-MS reverse phase liquid chromatography mass spectroscopy
  • ESI-MS electro-spray ionization mass spectroscopy
  • GC-MS gas chromatography
  • APCI-MS atmospheric pressure chemical ionization mass spectroscopy
  • CE-MS capillary electrophoresis mass spectroscopy
  • MS-MS tandem mass spectroscopy
  • the at least one polypeptide is at least 5 polypeptides, preferably at least 10 polypeptides, more preferably at least 100 polypeptides, even more preferably at least 200 polypeptides and most preferably at least 500 polypeptides.
  • the levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
  • the length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
  • the comparison i.e. step b
  • the comparison is performed using software-based statistical and
  • the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo.
  • Figure 1 Algorithm of treatment step up of UC obtain clinical remission according to severiety of disease (Danese S et al. Aliment Pharmacol Ther 2014, 39, 1095- 1 103).
  • Figure 2 Grey Results of a PLS predictive model based on proteomic data (2896 proteins analyzed in total, i.e. SEQ ID NOs: 1 to 2896) from 19 untreated patients at debut of disease. Black: Observed outcome of disease 1 year after debut of disease.
  • Figure 3 Grey Results of a PLS predictive model based on proteomic data (10 proteins analyzed in total, i.e. SEQ ID NOs: 27, 213, 617, 894, 903, 101 1, 1513, 1920, 1987 or 2412) from 19 untreated patients at debut of disease.
  • Black Observed outcome of disease 1 year after debut of disease.
  • Figure 4a is a principal component analysis (PCA) of samples with all identified proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
  • PCA principal component analysis
  • Figure 4b is a partial least squares regression analysis (PLS) of samples with all identified proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
  • PLS partial least squares regression analysis
  • Figure 5a is a partial least squares regression analysis (PLS) of samples with the 50 most important proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
  • PLS partial least squares regression analysis
  • Figure 5b is a principal component analysis (PCA) of samples with the 50 most important proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
  • PCA principal component analysis
  • Figure 6 represents a network analysis of the 50 most important proteins in the PLS model.
  • UC and CD are the major forms of IBD and affect, with increasing prevalence - 0,5% to 1.0% of the population in Europe.
  • the affected persons colon and rectum are in a state of chronic inflammation. This chronic inflammation is accompanied by ulcers, diarrhea with blood and mucus and further gastrointestinal and
  • UC may be classified into distinct sub-groups based on proteomics data and that it may be possible to predict the clinical course of the disease based on proteomics.
  • proteomic pattern/ fingerprint which is linked to the severity of the disease. If such a proteomic pattern/ fingerprint exists, such data may be used to predict clinical course of UC at an early stage.
  • Such a diagnostic test would create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
  • colon biopsy samples from patients newly diagnosed with UC were collected in order to investigate whether there is a pattern/ fingerprint based on proteomic data at debut of disease which is correlated with the clinical picture 12 months after debut of the disease (example la).
  • sample preparation including tissue lysis, reduction & alkylation, enzymatic digestion, TMT labelling and fractionation (example lb).
  • sample preparation including tissue lysis, reduction & alkylation, enzymatic digestion, TMT labelling and fractionation (example lb).
  • the peptides, oligopeptides and polypeptides thus obtained were quantified by LC-MS/MS (example lc) and the data analyzed using software-based statistical data analysis (example ld).
  • SEQ ID Nos: l to 2896 represents the most promising polypeptides to study when predicting the course of UC.
  • Figure 2 is a visual illustration of the correlation between proteomic data at debut of disease (grey) and the degree of illness 12 months after debut of the disease (black). As it appears from figure 2, there is almost a complete overlap between the black and the grey curves; clearly indicating high correlation.
  • the proteomic data presented in figure 2 is based on proteomic data of the polypeptides as set forth in SEQ ID Nos: l to 2896.
  • a first aspect of the present invention relates to a method, such as an in-vitro method, of predicting clinical course of ulcerative colitis in a subject, such as a human, the method comprising the following step(s):
  • each of said at least ten polypeptides comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896;
  • step b) comparing the results obtained in step a) with
  • step a) the results obtained in step a) are compared with:
  • second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months, such as at least 12 months or at least 24 months, after the second control sample was taken; and/or
  • step a) the results obtained in step a) are compared with:
  • control sample from a second control subject wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after onset of disease and the third control sample was taken at onset of disease.
  • step a) the results obtained in step a) are compared with:
  • control sample from a second control subject wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months after onset of disease and the third control sample was taken at onset of disease.
  • step a) the results obtained in step a) are compared with:
  • control sample from a second control subject wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after onset of disease and the third control sample was taken at onset of disease.
  • the subject to be diagnosed has not previously been diagnosed with UC.
  • the clinical course of ulcerative colitis is the clinical picture, such as degree of illness or severity of disease, at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months or 24 months after debut of disease, i.e. after debut of UC.
  • onset of disease refers to the point in time when a subject experiences symptoms of the disease, e.g. when a subject for the first time experiences symptoms of the disease. At that time, the subject typically contacts a physician to further investigate what is causing the symptoms.
  • polypeptide refers to a long, continuous peptide chain, preferably of at least 100 amino acids in length, such as at least 80 amino acids in length, at least 60 amino acids in length or at least 40 amino acids in length.
  • polypeptide is intended to include both polypeptides which has not been subjected to post -translational modification and polypeptide which has been subjected to post-translational modification.
  • Glycosylation and phosphorylation are examples of very common post-translational modifications.
  • Post-translational modification refers to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modification.
  • lipidation often targets a protein or part of a protein attached to the cell membrane.
  • Other forms of post- translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue.
  • the formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds.
  • the term“detecting and quantifying” in the context of detecting and quantifying the levels of at least one or at least ten polypeptide(s), as referred to herein, may be conducted directly on the polypeptide(s) as such (e.g. by western blotting), indirectly on fragments of the polypeptide(s) (e.g. by enzymatic digestion followed by LC-MS/MS as disclosed in example 1 of the present application) or by other means suitable for detecting and quantifying the levels of the polypeptide(s).
  • a number of other techniques suitable for detecting and quantifying the levels of one or more polypeptides are well known to the skilled person.
  • fragment refers to a subsequence of a polypeptide.
  • Fragments can vary in size from as few as 7 amino acid residues to the full length of the intact polypeptide, but are preferably 7 to 70 amino acids long and even more preferably 7-55 amino acids long.
  • sequence identity is generally expressed as a percentage and refers to the percent of amino acid residues or nucleotides, as appropriate, that are identical as between two sequences when optimally aligned.
  • sequence identity means the sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute/National Institutes of Health
  • the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
  • the three classes may be differentiated based on a clinical evaluation of symptoms, calprotectin levels in faces and/or by endoscopy to examine the degree of colon inflammation.
  • Mild ulcerative colitis is typically treated with topical steroids and/or amino salicylates.
  • Moderate ulcerative colitis is typically treated with prednisolone, budesonide and/or immunosuppressants.
  • Severe ulcerative colitis is typically in need of surgical treatment and/or treatment with anti TNF.
  • the present invention represents a new diagnostic tool which makes it possible to predict the clinical course of UC at an early stage.
  • the severity of UC at onset of disease is different from the predicted course of UC. Said in other words, the severity of UC at onset of disease is different from the severity of disease 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months or 24 months after onset of disease.
  • the clinical outcomes of UC are based on the required treatment level to obtain disease remission, using the step-up algorithm guidelines ECCO and the three levels proposed by Danese et al. (Danese S, Siegel CA, Peyrin-Biroulet L. Review article: integrating budesonide-MMX into treatment algorithms for mild-to-moderate ulcerative colitis. Aliment Pharmacol Ther; Dignass A, Lindsay JO, Sturm A, et al. Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 2: current management. J Crohns Colitis 2012;6:991-1030). The following three disease outcome levels after 1 year were used; mild, moderate and severe.
  • the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896; preferably with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others.
  • the proviso clarifying that the 10 polypeptides referred to are 10 different polypeptides, i.e. they do not have the same amino acid sequence.
  • each of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896; preferably with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides.
  • a first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: l
  • none of the remaining at least 9 polypeptides comprises or consists of the amino acid sequence set forth in SEQ ID NO: l .
  • the top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least one of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least two of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least three of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least four of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least five of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least six of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least seven of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least eight of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • At least nine of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • each and all of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011 , SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
  • a first polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
  • a second polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
  • a third polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 617;
  • a fourth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 894;
  • a fifth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 903;
  • a sixth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 101 1;
  • a seventh polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1513;
  • a eight polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1920;
  • a ninth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1987;
  • a tenth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 2412.
  • the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis.
  • Protein abundances, and in particular posttranslational modifications, on which the above method is based are constantly changing with the state of a tissue. Since the state of one tissue may affect other tissues in the body, it may be that the observed differences in protein levels that are associated with different degrees of illness also may be detected in other parts of the body.
  • the biological sample, the first control sample, the second control sample and the third control sample are selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and a biopsy sample.
  • the biological sample, the first control sample, the second control sample and the third control sample are a colon biopsy.
  • the sample is preferably subjected to sample preparation to release intracellular proteins. Adding a lysis buffer to the biopsy sample is a common way of releasing intracellular proteins, but the skilled person will also be aware of a number of other means for releasing the intracellular proteins.
  • such a sample preparation comprises the following steps:
  • step ii) subjecting the product of step i) to homogenization.
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate,
  • the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate. Even more preferably, the lysis buffer comprises sodium deoxycholate, urea and triethylammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM
  • the samples may be subjected to treatment with an alkylating agent.
  • Iodoacetamide is one example of an alkylating agent which is an irreversible inhibitor of all cysteine peptidases.
  • the product of step i) and/or product of step ii) is subjected to treatment suitable for reducing and alkylating cysteines.
  • the proteins are preferably subjected to some kind of enzymatic digestion.
  • the person skilled in the art will easily know how the proteins should be digested in order for the samples to be analysed using e.g LC-MS/MS.
  • Two non-limiting examples of enzymes that may be used for such enzymatic digestion is Lys-C and trypsin.
  • the product of step i) and/or product of step ii) are subjected to enzymatic digestion.
  • the enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
  • the polypeptides in the sample are subjected to pre-enzymatic digestion using Lys-C, preferably for a time period in the range 4-12 hours, such as 5-10 hours, or 6-9 hours or 7-9 hours such as 8 hours.
  • the sample is preferably subjected to enzymatic digestion using trypsin.
  • trypsin : protein weight ratio is in the range 1 : 10 to 1 :30, such as 1 :20.
  • the levels of the at least ten polypeptides may be detected and quantified by detecting and quantifying the levels of at least one fragment, such as at least two fragments, at least three fragments, at least four fragments, at least five fragments, at least six fragments, at least seven fragments, at least eight fragments, at least nine fragments or at least ten fragments for each of said polypeptides.
  • the length of each fragment is in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
  • the samples may be subjected to treatment with an alkylating agent after having been subjected to enzymatic digestion.
  • an alkylating agent which is an irreversible inhibitor of all cysteine peptidases.
  • the level of each protein may be measured e.g. by mass spectroscopy.
  • the levels of the at least ten polypeptides is measured by mass spectroscopy, preferably liquid chromatography mass
  • LC-MS liquid chromatography mass spectroscopy
  • HPLC-MS high performance liquid chromatography mass spectroscopy
  • reverse phase liquid chromatography mass LC-MS
  • RPLC-MS electro-spray ionization mass spectroscopy
  • ESI-MS electro-spray ionization mass spectroscopy
  • GC-MS gas chromatography
  • APCI-MS atmospheric pressure chemical ionization mass spectroscopy
  • CE-MS capillary electrophoresis mass spectroscopy
  • MS-MS tandem mass spectroscopy
  • the at least 10 polypeptides are at least 100 polypeptides, even more preferably at least 200 polypeptides and most preferably at least 500 polypeptides.
  • the levels of the at least ten polypeptides are detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
  • the length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10- 55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
  • the comparison i.e. step b
  • the comparison is performed using software-based statistical and bioinformatics data analysis.
  • the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo.
  • ACN acetonitrile
  • BCA bicinchoninic acid
  • DTT dithiothreitol
  • FA formic acid
  • IAA iodoacetamide
  • IBD inflammatory bowel disease
  • SDC sodium deoxycholate
  • TEAB Triethylammonium bicarbonate buffer
  • TFA trifluoroacetic acid
  • TMT Tandem Mass Tag
  • the recruited subjects were 19 patients admitted at the University Hospital of North Norway who newly have been diagnosed with UC, i.e. at the debut of the disease. All participants were informed and signed a written consent.
  • the Regional Network of North Norway The recruited subjects were 19 patients admitted at the University Hospital of North Norway who newly have been diagnosed with UC, i.e. at the debut of the disease. All participants were informed and signed a written consent.
  • ulcerative colitis disease activity index (UCDAI) which is based on clinical signs (score 0-12) and on endoscopic evaluation of the colon during colonoscopy (grade 0-3) ( Gastroenterology 1987, 92, 1894-1898). 1 1 patients were found to suffer from mild UC, 3 patients were found to suffer from moderate UC and the last five patients were found to suffer from severe UC.
  • UDAI ulcerative colitis disease activity index
  • Lysis buffer 5% Sodium deoxycholate (SDC) and 8 M urea in 100 mM
  • lysis buffer 250 m ⁇ cooled lysis buffer is filled into 38 MagNA Lyser Green Bead tubes (Roche Diagnostics AG, Rotnch, Switzerland). 19 debut samples were placed between the beads in said tubes. The tubes are kept on ice until lysis is completed and thereafter the tubes are homogenized in a MagNA Lyzer instrument (Roche Diagnostics AG, Rotnch, Switzerland) at 6500 rpm for 35 sec. The homogenized samples hereinafter being referred to as cell lysates.
  • a CaCl 2 solution, 0.6 pg Lys-C (enzyme-to -protein ratio 1 : 100, w/w), water, and 1M TEAB is added to the reduced samples to a final concentration of 1 mM calcium chloride, 6 M urea, and 100 mM TEAB.
  • Protein predigest is performed under gentle agitation for 8 hours at 37°C.
  • CaCh solution, 3 pg trypsin (enzyme-to-protein ratio 1 :20, w/w), water, and 1M TEAB is added to a final concentration of 1 mM calcium chloride, 1 M urea, and 100 mM TEAB. Tryptic protein digest is performed under gentle agitation for 16 hours at 37°C.
  • the reduced samples that has been subjected to enzymatic treatment hereinafter hereinafter being referred to as fragment samples.
  • TMT labelling is performed according to the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). 0.4 mg TMT reagent is used for 25 pg peptides from samples with starting protein concentrations > 2200 pg/ml. The amount of TMT reagent is increased for lower starting concentrations. A total of 100 pg of the differently labelled peptides is mixed in equal amounts in a Protein LoBind tube (Eppendorf AG, Hamburg, Germany). The TMP-labelled samples are hereinafter referred to as labelled samples.
  • the labelled samples are acidified with 50% formic acid to a final concentration of 2.5 % formic acid and pH ⁇ 2.
  • the precipitated sodium deoxycholate (SDC) is removed by centrifugation at 16,000 g for 15 min.
  • the supernatants are carefully transferred to fresh Protein LoBind tubes (Eppendorf AG, Hamburg, Germany).
  • Acetonitrile is removed by evaporation in a vacuum concentrator.
  • the samples which have been subjected to SDC and acetonitrile removal are hereinafter referred to as free samples.
  • TFA is added to each of the free samples to a concentration of 0.1%.
  • the samples are fractionated into 8 fractions using PierceTM High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Samples exceeding 300 m ⁇ are loaded stepwise by repeating the following: Add 300 m ⁇ to the column, centrifuge 2 min at 3000 x g, discard flow-through. The fractions are subjected to evaporation until dryness in a vacuum concentrator and re-dissolved in 10 m ⁇ 0.1% TFA for subsequent LC- MS/MS analyses.
  • the fractions were analyzed with a nano-LC-MS/MS system (EASY-nLC 1000 coupled to a Q Exactive mass spectrometer, both Thermo Fisher Scientific, Bremen, Germany).
  • the Q Exactive mass spectrometer was run in positive mode, with the following global settings:
  • Chromatographic peak width 15 s 15 s, default charge state 2, full MS survey scans from 400 to 2000 m/z, resolution 70,000, AGC target value 3e6, maximum injection time 100 ms for MS scans. Subject the 10 most intense peaks to MS/MS with the following settings: resolution 17,500, dynamic exclusion 10 s, underfill ratio 1%, charge states +2, +3 and +4, exclude isotopes, normalized collision energy 31, isolation window 2 m/z, AGC target value le5 and maximum injection time 50 ms, fixed first mass 120.
  • the raw data are processed with a quantitative proteomics software package, e.g. MaxQuant (Cox, J., Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein
  • MS2/TMTsixplex is set as quantification method.
  • the raw files are loaded and their experiment name and fraction numbers specified.
  • the configured fasta file is searched with the following parameters: Enzyme:
  • peptides 1, Use only unmodified peptides and: TRUE, Modifications included in protein quantification: Oxidation (M) and Acetyl (Protein N-term), Peptides used for protein quantification: Razor, Discard unmodified counterpart peptides: TRUE, Fabel min. ratio count: 2, Use delta score: FAFSE, iBAQ: FAFSE, iBAQ log fit: FAFSE, Match between runs: TRUE, Matching time window [min]: 0.7, Alignment time window [min]: 20, Find dependent peptides: FAFSE, Decoy mode: revert, Include contaminants: TRUE, Advanced ratios:
  • TRUE Second peptides: FAFSE, Calculate peak properties: FAFSE, Main search max. combinations: 200, Advanced site intensities: TRUE, Max. peptide mass [Da]: 4600, Min. peptide length for unspecific search: 8, Max. peptide length for unspecific search: 25, Razor protein FDR: TRUE, Disable MD5: FAFSE, Max mods in site table: 3, Match unidentified features: FAFSE, MS/MS tol. (FTMS): 20 ppm, Top MS/MS peaks per Da interval. (FTMS): 12, Da interval.
  • FTMS MS/MS deisotoping
  • FTMS MS/MS deisotoping tolerance
  • FTMS MS/MS deisotoping tolerance unit
  • MS/MS higher charges MS/MS: TRUE
  • MS/MS water loss TRUE
  • MS/MS ammonia loss TRUE
  • MS/MS dependent losses TRUE
  • MS/MS recalibration FAFSE.
  • the quantification data are loaded into Perseus (Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M. Y., Geiger, T., Mann, M., Cox, J., The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Meth 2016, 13, 731- 740). Identifications labelled as only identified by site are deleted, hits in reverse database and potential contaminants are removed. A log2 transformation is performed. The intensities are normalized. If a standard channel was used, this can be done by subtracting its reporter ion intensities from the other channels of the same experiment and subsequent Z-score transformation (matrix access: columns).
  • Example le Verification of the in-vitro method of predicting clinical course of ulcerative colitis
  • a predictive model was build utilizing a multivariate model in SIMCA 14.1 (Umetrics) where a OPLS model was utilized to build a model for separating the patients with a severe outcome from the patients with moderate or mild outcome. Relative protein ratios were used as input in the model, and the model was filtered to obtain no missing values in quantified proteins.
  • Example 2 Prognostic model for predicting the 1-year outcome for patients with ulcerative colitis based on proteomics data.
  • Colon biopsies were lysed in 250 m ⁇ cold buffer (5% sodium deoxycholate (SDC), 8 M urea, 100 mM triethylammonium bicarbonate buffer pH 8.5 (TEAB)) in MagNA Lyser Green Bead tubes (Roche Diagnostics AG, Rotnch, Switzerland) with a MagNA Lyser Instrument (Roche Diagnostics AG, Rotnch, Switzerland) for 35 seconds at 6500 rpm. The samples were frozen and stored at -70 °C until further preparation.
  • SDC sodium deoxycholate
  • TEAB triethylammonium bicarbonate buffer pH 8.5
  • the protein concentration was determine with a bicinchoninic acid (BCA) assay (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific) according to the manufacturer’s protocol.
  • BCA bicinchoninic acid
  • the TEAB concentration for the BCA assay in standards and samples was 5 mM TEAB.
  • DTT dithiothreitol
  • IAA iodoacetamide
  • Lys-C 0.6 pg endoproteinase Lys-C (Wako Chemicals GmbH, Neuss, Germany) were added for a 1 : 100 enzyme-to -protein ratio (w/w).
  • the sample compositions were adjusted to 1 mM calcium chloride, 6M urea and 100 mM TEAB with calcium chloride solution, water, and 1 M TEAB.
  • the Lys-C predigestion was facilitated under gentle agitation for 8 hours at 37°C.
  • trypsin Sequencing Grade Modified Trypsin, Promega Corporation, Madison, USA
  • the sample compositions were adjusted to 1 mM calcium chloride, 1 M urea, and 100 mM TEAB with calcium chloride solution, water, and 1 M TEAB. Tryptic digestion was facilitated for 16 hours at 37°C under gentle agitation.
  • TMT tandem mass tag
  • TMTsixplex reagents For each sample, 25 pg peptides were labeled with the remaining TMTsixplex reagents as described in the manufacturer’s protocol. 0.4 mg TMT reagent were used to label 25 pg peptides, if the starting protein concentration was > 2200 pg/ml in the respective sample. Samples with lower starting concentrations were labeled with more TMT reagent to achieve complete labeling.
  • the detergent SDC was precipitated by adding 50% formic acid (FA) solution to a final concentration of 2.5% FA. A pH ⁇ 2 was confirmed with pH indicator paper.
  • FA formic acid
  • the samples were adjusted to a concentration of 0.1% TFA by adding 10% trifluoroacetic acid (TFA) solution.
  • TFA trifluoroacetic acid
  • the fractionation into 8 fractions was performed with PierceTM High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, Rockford, USA) as described in the manufacturer’s protocol.
  • Proteins were identified and quantified from the mass spectrometry data with MaxQuant. l3
  • Protein intensities were log2-transformed in Perseus.14 The identifications with labels“potential contaminant”,“reverse” or“only identified by site” were removed. The standard intensities were subtracted from the sample intensities of the same experiment and the resulting intensities were Z- score normalized (matrix access: columns).
  • the core of the network labelled A in figure 6, is dominated by the fibrinogen proteins Fibrinogen Alpha, Beta and Gamma chain (FGA, FGB and FGG).
  • Fibrinogen is most commonly recognized for the involvement in blood clotting and wound healing as an acute phase protein.
  • fibrinogen has been linked to inflammation as well and has been reported to have pro-inflammatory properties in several diseases, e.g. multiple sclerosis, Alzheimer’s disease, rheumatoid arthritis, kidney fibrosis and several types of cancer.
  • the fibrins are also linked to other well-known parts of the immune system, like complement component C6 (C6), a part of the adaptive immune response, which forms pores in the cell membrane of target cells.
  • C6 complement component
  • fibrinogens are interlinked with the glycoprotein Ceruplasmin (CP), known for oxidizing cupper to Cu 2+ , as well as platelet basic protein (PPBP), which among many other functions is known to stimulate the formation and secretion of plasminogen activator.
  • CP glycoprotein Ceruplasmin
  • PPBP platelet basic protein
  • Network A is linked through a metalloproteinase (MMP2) to the network labelled B, which consists of proteins involved in splicing (YBX1 , CD2BP2 and SRSF9) and other proteins involved in translation of mRNA to proteins (NACA and BTF3).
  • MMP2 metalloproteinase
  • YBX1 proteins involved in splicing
  • CD2BP2 proteins involved in splicing
  • SRSF9 proteins involved in translation of mRNA to proteins
  • NACA and BTF3 proteins involved in translation of mRNA to proteins
  • Network C consists of 4 proteins, all located in the mitochondria of the cells.
  • P-Arrestin2 (ARRB2) which is a G-protein coupled receptor signaling for various cytoplasmic signaling within inflammation 17
  • HIP1 Huntington interacting protein
  • MRPL44 39S ribosomal proteins that interact with P-Arrestin2
  • MRPL51 39S ribosomal proteins that interact with P-Arrestin2
  • MRPL44 has previously been shown to be involved in tumor development.
  • MRPL51 on the other hand has previously been reported in a dextran sodium sulphate (DSS)-induced colitis model, where MRPL51 was shown to be regulated at transcript level but without being able to explain the role of the protein in IBD.
  • DSS dextran sodium sulphate
  • the independent network labelled D has two Rab proteins at its core, Rabl3 and Rab22A, which have their main importance in membrane trafficking. Both Rab 13 and Rab22A have been reported to be involved in inflammation as well as inflammation-related diseases like cancer. Mistargeting of Rab 13 to basolateral sites has been observed in patients with Crohn's Disease. Network D seems to influence permeability of the intestine, which is known to be dysregulated in patients with IBD. These data indicate that intestinal permeability is also of importance for predicting the outcome of the patients.
  • proteins described above as members of the networks there are other proteins that are of importance for the clustering of patients. The majority of these proteins seems to be involved in immune system or energy metabolism pathways. In general, the biological functions of the proteins seem to fit to the severity of IBD, which is strengthening the background of the statistical model for predicting the outcome of IBD patients.

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Abstract

The present invention relates generally to the field of inflammatory bowel disease (IBD), and in particular ulcerative colitis (UC). The present invention provides methods for predicting clinical course of UC in a patient. By being able to predict the clinical course of UC, it is now possible to conclude on a therapeutic approach adapted to reflect the actual clinical picture of the patient at an early stage. Such a diagnostic test will create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.

Description

METHOD OF PREDICTING STAGE OF ULCERATIVE COLITIS
Field of the invention
The present invention relates generally to the field of inflammatory bowel disease (IBD), and in particular ulcerative colitis (UC). The present invention provides methods for predicting clinical course of UC in a patient. By being able to predict the clinical course of UC, it is now possible to conclude on a therapeutic approach adapted to reflect the actual clinical picture of the patient at an early stage. Such a diagnostic test will create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
Background of the invention
The global incidence of IBD, including Crohn’s disease (CD) and UC, is increasing. In Europe, 2.2 million predominantly younger people are suffering from the disease. The immune response is dysregulated in IBD such as in other autoimmune diseases (Mucosal immunology 2014, 7, 1139-1150). The cause of the disease is unknown, but it is generally accepted that IBD represents a dysregulation of the intestinal immune tolerance to microbiological and/or nutritional antigens. This dysregulation results from a combination of environmental and genetic factors, in which dysfunctional gene products may initiate inflammation via signaling pathways that have been defined to varying degrees, including innate, adaptive and T-regulatory immunity systems.
In UC, treatment decision is typically dependent on the stage of the disease, which may vary considerably among patients. Patients with mild manifestations are usually treated with aminosalicylates, whereas corticosteroids are typically prescribed for those with moderate disease and cyclosporine is typically given to patients with severe disease. In CD, decisions about drug therapy are dependent on both location and behavior of the disease. Despite that, the medication in CD usually includes aminosalicylates and antibiotics to treat mild mucosal disease, corticosteroids to moderate disease, and biological molecules to treat fistulizing disease. Also, aminosalicylates, azathioprine, mercaptopurine, methotrexate, metronidazole, and associations can be used as maintenance therapies.
Notwithstanding their reduced cost, these drugs can generate several side effects. Moreover, these therapies do not achieve clinical remission and they can lead to the onset of other conditions such as renal impairment. At the same time the classical treatments are widely used, new therapeutic strategies are under development in the attempt of improving the patient’s life quality. The new therapies aim to reduce the side effects and to treat patients who do not respond satisfactorily to conventional therapies. These involve the manipulation of the microbiome using antibiotics, probiotics, prebiotics, diet, and fecal microbiota transplantation.
The goal of medical treatment of IBD, and UC in particular, is to rapidly induce remission while at the same time preventing complications of the disease itself and its treatment. The choice of treatment depends on a number of factors such as severity, localization and the course of the disease.
WO2016/049772 discloses protein biomarkers and methods for their use in diagnosing IBD, UC and CD. There is also a disclosure of methods for diagnosing the severity of UC disease, but there is no mentioning of a method suitable to predict the clinical course of UC.
At the onset of disease the symptoms of UC are usually mild, and it is difficult to predict how the disease will progress for the next 12 months. The lack of reliable tests to predict the clinical course of UC has made it difficult to conclude on a therapeutic approach which reflects the actual clinical picture at an early stage. To reduce side effects as much as possible, the golden mean (figure 1) has been to start treatment assuming that the clinical course is mild. If the patient does not respond to the treatment, the next therapeutic approach has been to assume that the clinical course is moderate. If this treatment approach also fails, it is concluded that the clinical course is severe ulcerative colitis and the therapeutic approach is adapted accordingly.
However, there are also several groups currently investigating a top-down treatment, where the patients are treated with expensive biological drugs early in the disease development in order to remove the inflammation as much as possible. Thereafter the patients are kept on a minimum anti-inflammatory treatment with steroids and 5 -ASA. However, this approach might cause severe side effects caused by the biological for patients that could have been helped with local steroids and/or 5-ASA.
Predictors of clinical outcome will create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
To the best of our knowledge, there are no general agreement of clinical marker nor biomarkers at debut of disease indicating the need of future treatment. Moreover, there are no knowledge of any pathophysiological profile at debut of disease that can predict future clinical outcomes.
Thus, there is a continuous need for reliable tests to predict the clinical course of UC at an early stage.
Summary of the invention
The present invention has solved the above need by providing a method for predicting clinical course of UC in a patient. By being able to predict the clinical course of UC, it is now possible to conclude on a therapeutic approach which at an early stage is adapted to reflect the actual clinical picture.
Thus, a first aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
a) detecting and quantifying in a biological sample from said subject the levels of at least ten polypeptides or fragments thereof; each of said at least ten polypeptides comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896;
b) comparing the results obtained in step a) with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 1 month after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after the third control sample was taken.
In one embodiment according to the present invention, the results obtained in step a) are compared with i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 4 months after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 4 months after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 4 months after the third control sample was taken.
In one embodiment according to the present invention, the results obtained in step a) are compared with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 6 months after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months after the third control sample was taken.
In one embodiment according to the present invention, the results obtained in step a) are compared with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 12 months after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after the second control sample was taken; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after the third control sample was taken.
In one embodiment according to the present invention, the results obtained in step a) are compared with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 24 months after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 24 months after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 24 months after the third control sample was taken.
In one embodiment according to the present invention, the results obtained in step a) are compared with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 1 month after onset of disease and the first control sample was taken at onset of disease; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after onset of disease and the second control sample was taken at onset of disease; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after onset of disease and the third control sample was taken at onset of disease.
In one embodiment according to the present invention, the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 6 months after onset of disease and the first control sample was taken at onset of disease; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months after onset of disease and the second control sample was taken at onset of disease; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months after onset of disease and the third control sample was taken at onset of disease.
In another embodiment according to the present invention, the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 12 months after onset of disease and the first control sample was taken at onset of disease; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after onset of disease and the second control sample was taken at onset of disease; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after onset of disease and the third control sample was taken at onset of disease. In another embodiment according to the present invention, the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 24 months after onset of disease and the first control sample was taken at onset of disease; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 24 months after onset of disease and the second control sample was taken at onset of disease; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 24 months after onset of disease and the third control sample was taken at onset of disease.
In one embodiment according to the present invention,
the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the first control sample;
the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the second control sample; and
the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) are similar, such as closest, to the levels of said at least ten polypeptides or fragments thereof in the third control sample.
In one embodiment, onset of disease is the first time the subject experiences symptoms of ulcerative colitis.
In another embodiment, onset of disease is when the subject experiences symptoms of ulcerative colitis.
In another embodiment according to the first aspect of the present invention, onset of disease is at an early stage of ulcerative colitis, such as the first time the subject experiences symptoms of ulcerative colitis. In one embodiment according to the present invention, the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis.
In an embodiment according to the first aspect of the present invention, the subject to be diagnosed:
has not previously been diagnosed with ulcerative colitis; and/or
experiences symptoms of ulcerative colitis for the first-time.
In yet another embodiment according to the invention,
- the first control subject has been diagnosed with moderate ulcerative colitis within 12 months, such as within 24 months, from the onset of disease;
- the second control subject has been diagnosed with severe ulcerative colitis within 12 months, such as within 24 months, from the onset of disease; and
- the third control subject has been diagnosed with mild ulcerative colitis within 12 months, such as within 24 months, from the onset of disease.
In yet another embodiment according to the first aspect of the present invention,
- the first control subject has been diagnosed with moderate ulcerative colitis within 5 years from the onset of disease;
- the second control subject has been diagnosed with severe ulcerative colitis within 5 years from the onset of disease; and
- the third control subject has been diagnosed with mild ulcerative colitis within 5 years from the onset of disease.
In one embodiment according to the present invention, the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
In one embodiment according to the present invention, the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896 with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others.
In one embodiment according to the present invention, the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity, with any one of SEQ ID NOs: 1 to 2896 with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides.
The top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least one of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least two of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least three of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least four of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least five of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least six of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least seven of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least eight of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least nine of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, each and all of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In one embodiment according to the first aspect of the present invention, one of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27, 213, 617, 894, 903, 1011, 1513, 1920, 1987 or 2412 respectively. In another embodiment according to the first aspect of the present invention,
- a first polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
- a second polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
- a third polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 617;
- a fourth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 894;
- a fifth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 903;
- a sixth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 101 1;
- a seventh polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1513;
- a eight polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1920;
- a ninth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 1987; and
- a tenth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity sequence identity with SEQ ID NOs: 2412.
In yet another embodiment according to the first aspect of the present invention, each of said at least ten polypeptides comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 2896. In yet another embodiment according to the first aspect of the present invention, each of said at least ten polypeptides consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1 to 2896.
In another embodiment according to the first aspect of the present invention, step b) is performed using software-based statistical and bioinformatics data analysis.
In another embodiment according to the present invention, the biological sample, first control sample, second control sample and third control sample are selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and biopsy, such as colon biopsy.
If the biological sample, first control sample, second control sample and third control sample are colon biopsy, it is preferred that the colon biopsy is subjected to sample preparation prior to step a).
In one embodiment, such a sample preparation comprises the following steps:
i) adding lysis buffer, such as the one defined in example lb, to the colon
biopsy; and
ii) subjecting the product of step i) to homogenization.
In another embodiment according to the first aspect of the present invention, the biological sample, first control sample, second control sample and third control sample are a colon biopsy; and the colon biopsy is subjected to sample preparation prior to step a); the sample preparation comprising the following steps:
i) adding a liquid, such as water, to the colon biopsy;
ii) subjecting the product of step i) to homogenization; and
iii) adding lysis buffer to the product of step ii).
According to one embodiment, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate,
cetyltrimethyl ammonium, N-methyldioctylammine or any combination thereof.
Preferably, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate. Even more preferably, the lysis buffer comprises sodium deoxycholate, urea and triethylammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM
triethylammonium bicarbonate buffer. In another embodiment according to the present invention, the product of step i) and/or product of step ii) is subjected to enzymatic digestion. The enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
The product of step i), product of step ii) and/or the product of step iii) is preferably subjected to treatment suitable for reducing and alkylating cysteines.
In one embodiment according to the first aspect of the present invention, the levels of the at least ten polypeptides or fragments thereof are detected and quantified by mass spectroscopy, preferably liquid chromatography mass spectroscopy (LC-MS), especially high performance liquid chromatography mass spectroscopy (HPLC-MS) or reverse phase liquid chromatography mass spectroscopy (RPLC-MS); electro- spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC-MS), atmospheric pressure chemical ionization mass spectroscopy (APCI-MS), capillary electrophoresis mass spectroscopy (CE-MS), tandem mass spectroscopy (MS-MS); or any combination thereof.
In one embodiment according to the first aspect of the present invention, levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
In one embodiment according to the present invention, the levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides. The length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10- 55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
In another embodiment, the length of each of said fragments is in the range 5-70 amino acids long, such as in the range 10-70.
In one embodiment according to the present invention, the comparison (i.e. step b) is performed using software-based statistical and bioinformatics data analysis. In one preferred embodiment according to the present invention, the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo. An alternative aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
a) detecting and quantifying in a biological sample from said subject the levels of at least one, such as at least ten, polypeptide(s) or a fragment thereof; the at least one polypeptide comprising an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896;
b) comparing the results obtained in step a) with reference values of levels of said at least one polypeptide or a fragment thereof from subjects suffering from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis respectively;
wherein
- the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from mild ulcerative colitis;
- the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from moderate ulcerative colitis; and
- the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from severe ulcerative colitis.
A preferred embodiment according to the alternative aspect of the present invention relates to an in-vitro method of predicting clinical course of ulcerative colitis in a subject, the method comprising the following step(s):
a) detecting and quantifying in a biological sample from said subject the levels of at least ten polypeptides or fragments thereof; each of said at least ten
polypeptides comprising an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID
NOs: 1 to 2896;
b) comparing the results obtained in step a) with reference values of levels of said at least ten polypeptides or fragments thereof from subjects suffering from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis respectively; wherein
- the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from mild ulcerative colitis;
- the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from moderate ulcerative colitis; and
- the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) is closest to the reference values from subjects suffering from severe ulcerative colitis.
In one embodiment according to the alternative aspect of the present invention, the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
In one embodiment according to the alternative aspect of the present invention, the at least one polypeptide or each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896; with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others.
In another embodiment according to the alternative aspect of the present invention, the at least one polypeptide or each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896.
In a preferred embodiment according to the alternative aspect of the present invention, each of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896; with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides.
The top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894. In one embodiment according to the alternative aspect of the present invention, the at least one polypeptide comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least one of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least two of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least three of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least four of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least five of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least six of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least seven of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least eight of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, at least nine of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the alternative aspect of the present invention, each and all of the at least ten polypeptides comprise an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894. In a preferred embodiment according to the alternative aspect of the present invention,
- a first polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
- a second polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
- a third polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 617;
- a fourth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 894;
- a fifth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 903;
- a sixth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1011;
- a seventh polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1513;
- a eight polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1920;
- a ninth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1987; and
- a tenth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 2412.
In one embodiment according to the alternative aspect of the present invention, the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis.
In another embodiment according to the alternative aspect of the present invention, the biological sample is selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and biopsy, such as colon biopsy. In a preferred embodiment, the biological sample is a colon biopsy. In another preferred embodiment the biological sample is blood plasma or serum, even more preferably serum.
If the biological sample is colon biopsy, it is preferred that the colon biopsy is subjected to sample preparation prior to step a). In one embodiment, such a sample preparation comprises the following steps:
i) adding lysis buffer, such as the one defined in example lb, to the colon
biopsy; and
ii) subjecting the product of step i) to homogenization.
According to one embodiment according to the alternative aspect of the present invention, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate, cetyltrimethylammonium, N- methyldioctylammine or any combination thereof. Preferably, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate. Even more preferably, the lysis buffer comprises sodium deoxycholate, urea and tri ethyl ammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM triethylammonium bicarbonate buffer.
In another embodiment according to the alternative aspect of the present invention, the product of step i) and/or product of step ii) is subjected to enzymatic digestion. The enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
If the product of step i) and/or product of step ii) has been subjected to enzymatic digestion, the levels of the at least one or at least ten polypeptides may be detected and quantified by detecting and quantifying the levels of at least one fragment, such as at least two fragments, at least three fragments, at least four fragments, at least five fragments, at least six fragments, at least seven fragments, at least eight fragments, at least nine fragments or at least ten fragments for each of said polypeptides. Preferably, the length of each fragment is in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long. In another embodiment according to the alternative aspect of the present invention, the product of step i) and/or product of step ii) is subjected to treatment suitable for reducing and alkylating cysteines.
In another embodiment according to the alternative aspect of the present invention, the levels of the at least one polypeptide or the at least ten polypeptides is measured by mass spectroscopy, preferably liquid chromatography mass spectroscopy (LC- MS), especially high performance liquid chromatography mass spectroscopy
(HPLC-MS) or reverse phase liquid chromatography mass spectroscopy (RPLC- MS); electro-spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC-MS), atmospheric pressure chemical ionization mass spectroscopy (APCI-MS), capillary electrophoresis mass spectroscopy (CE-MS), tandem mass spectroscopy (MS-MS); or any combination thereof.
According to another embodiment according to the alternative aspect of the present invention, the at least one polypeptide is at least 5 polypeptides, preferably at least 10 polypeptides, more preferably at least 100 polypeptides, even more preferably at least 200 polypeptides and most preferably at least 500 polypeptides.
In one embodiment according to the alternative aspect of the present invention, the levels of the at least ten polypeptides is detected and quantified by detecting and quantifying fragments of said at least ten polypeptides. The length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
In one embodiment according to the alternative aspect of the present invention, the comparison (i.e. step b) is performed using software-based statistical and
bioinformatics data analysis. In one preferred embodiment according to the alternative aspect of the present invention, the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo.
Brief description of drawings
Figure 1 Algorithm of treatment step up of UC obtain clinical remission according to severiety of disease (Danese S et al. Aliment Pharmacol Ther 2014, 39, 1095- 1 103). Figure 2 Grey: Results of a PLS predictive model based on proteomic data (2896 proteins analyzed in total, i.e. SEQ ID NOs: 1 to 2896) from 19 untreated patients at debut of disease. Black: Observed outcome of disease 1 year after debut of disease.
Figure 3 Grey: Results of a PLS predictive model based on proteomic data (10 proteins analyzed in total, i.e. SEQ ID NOs: 27, 213, 617, 894, 903, 101 1, 1513, 1920, 1987 or 2412) from 19 untreated patients at debut of disease. Black: Observed outcome of disease 1 year after debut of disease.
Figure 4a is a principal component analysis (PCA) of samples with all identified proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
Figure 4b is a partial least squares regression analysis (PLS) of samples with all identified proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
Figure 5a is a partial least squares regression analysis (PLS) of samples with the 50 most important proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
Figure 5b is a principal component analysis (PCA) of samples with the 50 most important proteins taken into account, circles indicate mild or moderate outcome after one year and diamonds indicate severe outcome after one year.
Figure 6 represents a network analysis of the 50 most important proteins in the PLS model.
Detailed description of the invention
Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of medicine, biochemistry, bioinformatics and biology.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out. UC and CD are the major forms of IBD and affect, with increasing prevalence - 0,5% to 1.0% of the population in Europe. The affected persons colon and rectum are in a state of chronic inflammation. This chronic inflammation is accompanied by ulcers, diarrhea with blood and mucus and further gastrointestinal and
extraintestinal manifestations.
To the best of our knowledge, the major studies have been designed to show the differences between different types of IBD and diagnosing the severity of the disease, but not been able to predict the clinical course of UC. To personalize the treatment and thereby increase the quality of life for the patients, it is necessary to conclude on a therapeutic approach adapted to reflect the actual clinical picture of the patient at an early stage, preferably at the onset of the disease.
Based on the idea that protein abundances, and in particular posttranslational modifications, are constantly changing with the state of a tissue, it was
hypothesized that UC may be classified into distinct sub-groups based on proteomics data and that it may be possible to predict the clinical course of the disease based on proteomics.
In order to investigate this further, it was decided to launch a project where a group of patients were followed from debut of disease, i.e. onset of disease, until l2months after debut of disease. By evaluating the degree of illness l2months after debut of disease and studying proteomic data at debut of disease, it was
hypothesized that it would be possible to establish the existence of a proteomic pattern/ fingerprint which is linked to the severity of the disease. If such a proteomic pattern/ fingerprint exists, such data may be used to predict clinical course of UC at an early stage. Such a diagnostic test would create new knowledge of high clinical utility for the clinician, create a personalized therapy for the patient, improve the prognosis for the patient and reduce health costs for the society.
Thus, colon biopsy samples from patients newly diagnosed with UC were collected in order to investigate whether there is a pattern/ fingerprint based on proteomic data at debut of disease which is correlated with the clinical picture 12 months after debut of the disease (example la).
The collected samples were subjected to sample preparation, including tissue lysis, reduction & alkylation, enzymatic digestion, TMT labelling and fractionation (example lb). The peptides, oligopeptides and polypeptides thus obtained were quantified by LC-MS/MS (example lc) and the data analyzed using software-based statistical data analysis (example ld).
The obtained data were then compared with the degree of illness 12 months after debut of disease in search of a pattem/fingerprint which may be used to predict the course of UC. Surprisingly, it was discovered that such patterns/ fingerprints exist. Among all the polypeptides that were quantified (example lc), SEQ ID Nos: l to 2896 represents the most promising polypeptides to study when predicting the course of UC.
Figure 2 is a visual illustration of the correlation between proteomic data at debut of disease (grey) and the degree of illness 12 months after debut of the disease (black). As it appears from figure 2, there is almost a complete overlap between the black and the grey curves; clearly indicating high correlation. The proteomic data presented in figure 2 is based on proteomic data of the polypeptides as set forth in SEQ ID Nos: l to 2896.
Even though SEQ ID Nos: l to 2896 were found to represent the most promising polypeptides to study when predicting the course of UC, the question arises whether it would be possible to predict the course of UC based on a much smaller data set.
To investigate this further, it was decided to study the correlation when the number of polypeptides in the proteomic data set was reduced to 5, 10 and 25 polypeptides respectively.
As expected, the correlation between proteomic data at debut of disease and the degree of illness 12 months after debut of the disease is decreasing with decreasing number of polypeptides in the proteomic data set, but it was surprising that quantification of only 10 polypeptides is sufficient to predict the clinical course of UC (figure 3). Among all the polypeptides set forth in SEQ ID Nos: l to 2896, SEQ ID Nos: 27, 213, 617, 894, 903, 1011, 1513, 1920, 1987 and 2412 were found to represent the 10 most promising polypeptides to study when predicting the course of UC on basis of a small data set.
Thus, a first aspect of the present invention relates to a method, such as an in-vitro method, of predicting clinical course of ulcerative colitis in a subject, such as a human, the method comprising the following step(s):
a) detecting and quantifying in a biological sample from said subject the levels of at least ten polypeptides; each of said at least ten polypeptides comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896;
b) comparing the results obtained in step a) with
- the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 1 month after the first control sample was taken; and/or
- the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after the second control sample was taken; and/or
- the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after the third control sample was taken.
In one embodiment the results obtained in step a) are compared with:
- the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 6 months, such as at least 12 months or at least 24 months, after the first control sample was taken; and/or
- the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months, such as at least 12 months or at least 24 months, after the second control sample was taken; and/or
- the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months, such as at least 12 months or at least 24 months, after the third control sample was taken.
In one embodiment the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 1 month after onset of disease and the first control sample was taken at onset of disease; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a second
control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after onset of disease and the third control sample was taken at onset of disease.
In one embodiment the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 6 months after onset of disease and the first control sample was taken at onset of disease; and/or ii) the levels of said at least ten polypeptides or fragments thereof in a second
control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months after onset of disease and the third control sample was taken at onset of disease.
In one embodiment the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 12 months after onset of disease and the first control sample was taken at onset of disease; and/or ii) the levels of said at least ten polypeptides or fragments thereof in a second
control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after onset of disease and the second control sample was taken at onset of disease; and/or iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after onset of disease and the third control sample was taken at onset of disease.
In one embodiment the subject to be diagnosed has not previously been diagnosed with UC.
In one embodiment according to the present invention, the clinical course of ulcerative colitis is the clinical picture, such as degree of illness or severity of disease, at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months or 24 months after debut of disease, i.e. after debut of UC.
The term“onset of disease” refers to the point in time when a subject experiences symptoms of the disease, e.g. when a subject for the first time experiences symptoms of the disease. At that time, the subject typically contacts a physician to further investigate what is causing the symptoms.
The term "polypeptide", as used herein, refers to a long, continuous peptide chain, preferably of at least 100 amino acids in length, such as at least 80 amino acids in length, at least 60 amino acids in length or at least 40 amino acids in length.
In the context of the present invention, the term“polypeptide” is intended to include both polypeptides which has not been subjected to post -translational modification and polypeptide which has been subjected to post-translational modification.
Glycosylation and phosphorylation are examples of very common post-translational modifications.
Post-translational modification (PTM) refers to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation, which can promote protein folding and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation, often targets a protein or part of a protein attached to the cell membrane. Other forms of post- translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds.
It is to be understood that the term“detecting and quantifying” in the context of detecting and quantifying the levels of at least one or at least ten polypeptide(s), as referred to herein, may be conducted directly on the polypeptide(s) as such (e.g. by western blotting), indirectly on fragments of the polypeptide(s) (e.g. by enzymatic digestion followed by LC-MS/MS as disclosed in example 1 of the present application) or by other means suitable for detecting and quantifying the levels of the polypeptide(s). A number of other techniques suitable for detecting and quantifying the levels of one or more polypeptides are well known to the skilled person.
The term "fragment" as used herein, refers to a subsequence of a polypeptide.
Fragments can vary in size from as few as 7 amino acid residues to the full length of the intact polypeptide, but are preferably 7 to 70 amino acids long and even more preferably 7-55 amino acids long.
The term "sequence identity," as used herein, is generally expressed as a percentage and refers to the percent of amino acid residues or nucleotides, as appropriate, that are identical as between two sequences when optimally aligned. For the purposes of this invention, sequence identity means the sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute/National Institutes of Health
(Bethesda, Maryland) and has been described in printed publications (see, e.g., Altschul et ah, J. Mol. Biol, 215(3), 403-10 (1990)).
In one embodiment according to the present invention, the clinical course of ulcerative colitis is selected from mild ulcerative colitis, moderate ulcerative colitis and severe ulcerative colitis.
The skilled person will know that degree of illness in respect of UC typically is classified as mild, moderate or severe. These three classes are typically
differentiated by the therapeutic approach applied to treat said conditions, c.f. figure 1. Optionally, the three classes may be differentiated based on a clinical evaluation of symptoms, calprotectin levels in faces and/or by endoscopy to examine the degree of colon inflammation.
As stated above, mild, moderate and severe UC may be differentiated by the therapeutic approach typically applied to treat said conditions, c.f. figure 1. Mild ulcerative colitis is typically treated with topical steroids and/or amino salicylates. Moderate ulcerative colitis is typically treated with prednisolone, budesonide and/or immunosuppressants. Severe ulcerative colitis is typically in need of surgical treatment and/or treatment with anti TNF.
When a patient experiences symptoms of UC for the first time, it is highly unlikely that the patient suffers from severe UC. It is far more common that the patient suffers from mild or moderate UC at debut of disease. A patient suffering from mild UC at debut of disease may suffer from mild, moderate or severe UC 12 months after debut of disease. The present invention represents a new diagnostic tool which makes it possible to predict the clinical course of UC at an early stage.
In one preferred embodiment according to the present invention, the severity of UC at onset of disease is different from the predicted course of UC. Said in other words, the severity of UC at onset of disease is different from the severity of disease 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months or 24 months after onset of disease.
The clinical outcomes of UC are based on the required treatment level to obtain disease remission, using the step-up algorithm guidelines ECCO and the three levels proposed by Danese et al. (Danese S, Siegel CA, Peyrin-Biroulet L. Review article: integrating budesonide-MMX into treatment algorithms for mild-to-moderate ulcerative colitis. Aliment Pharmacol Ther; Dignass A, Lindsay JO, Sturm A, et al. Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 2: current management. J Crohns Colitis 2012;6:991-1030). The following three disease outcome levels after 1 year were used; mild, moderate and severe. These outcomes were defined by the treatment level needed for clinical remission; 5-ASA per oral or per rectal (mild), need of oral steroids and/or thioprines (moderate) and need of anti-TNF and/or surgery (severe) (see figure 1). Clinical remission was defined by ulcerative colitis clinical score (UCCS) <2 according to Feagan et al. (Feagan BG, Greenberg GR, Wild G, et al. Treatment of Ulcerative Colitis with a Humanized Antibody to the a4b7 Integrin. New England Journal of Medicine 2005; 352:2499-2507).
In one embodiment according to the present invention, the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with any one of SEQ ID NOs: 1 to 2896; preferably with the proviso that each polypeptide represents a unique amino acid sequence as compared to the others. The proviso clarifying that the 10 polypeptides referred to are 10 different polypeptides, i.e. they do not have the same amino acid sequence.
In a preferred embodiment, each of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896; preferably with the proviso that the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2896 is unique for each of said at least ten polypeptides. Said in other words, if a first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: l, none of the remaining at least 9 polypeptides comprises or consists of the amino acid sequence set forth in SEQ ID NO: l . The top 10 polypeptides which differentiate most between mild, moderate and severe UC are SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
Thus, in another embodiment according to the present invention, at least one of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least two of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least three of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least four of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least five of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least six of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 101 1, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least seven of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least eight of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, at least nine of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011, SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In another embodiment according to the present invention, each and all of the at least ten polypeptides comprise or consist of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 617, SEQ ID NOs: 1920, SEQ ID NOs: 1513, SEQ ID NOs: 27, SEQ ID NOs: 903, SEQ ID NOs: 1011 , SEQ ID NOs: 2412, SEQ ID NOs: 1987, SEQ ID NOs: 213 and SEQ ID NOs: 894.
In a preferred embodiment according to the present invention, - a first polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 27;
- a second polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 213;
- a third polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 617;
- a fourth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 894;
- a fifth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 903;
- a sixth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 101 1;
- a seventh polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1513;
- a eight polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1920;
- a ninth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 1987; and
- a tenth polypeptide of the at least ten polypeptides comprises or consists of an amino acid sequence having at least 80%, such as at least 90%, at least 95% or 100% sequence identity with SEQ ID NOs: 2412.
In one embodiment according to the present invention, the in-vitro method of predicting clinical course of ulcerative colitis in a subject is an in-vitro method of predicting clinical course of ulcerative colitis in a subject that previously has been diagnosed with ulcerative colitis. Protein abundances, and in particular posttranslational modifications, on which the above method is based are constantly changing with the state of a tissue. Since the state of one tissue may affect other tissues in the body, it may be that the observed differences in protein levels that are associated with different degrees of illness also may be detected in other parts of the body. Thus, in one embodiment according to the present invention, the biological sample, the first control sample, the second control sample and the third control sample are selected from the group consisting of plasma, urine, cerebrospinal fluid, sputum, mucous secretions, synovial fluid, lymph fluid, bursa fluid, feces and a biopsy sample. Preferably, the biological sample, the first control sample, the second control sample and the third control sample are a colon biopsy.
In the embodiment where the biological sample, the first control sample, the second control sample and the third control sample are a biopsy, such as a colon biopsy, the sample is preferably subjected to sample preparation to release intracellular proteins. Adding a lysis buffer to the biopsy sample is a common way of releasing intracellular proteins, but the skilled person will also be aware of a number of other means for releasing the intracellular proteins.
In one embodiment, such a sample preparation comprises the following steps:
i) adding lysis buffer, such as the one defined in example lb, to the colon
biopsy; and
ii) subjecting the product of step i) to homogenization.
According to one embodiment, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, dodecyl sulfate, laurate,
cetyltrimethylammonium, N-methyldioctylammine or any combination thereof. Preferably, the lysis buffer comprises a detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate. Even more preferably, the lysis buffer comprises sodium deoxycholate, urea and triethylammonium bicarbonate buffer, such as 5% by weight sodium deoxycholate, 8M urea and lOOmM
triethylammonium bicarbonate buffer.
Now having released the intracellular proteins, the samples may be subjected to treatment with an alkylating agent. Iodoacetamide is one example of an alkylating agent which is an irreversible inhibitor of all cysteine peptidases. Thus, in one embodiment the product of step i) and/or product of step ii) is subjected to treatment suitable for reducing and alkylating cysteines.
If the samples are to be analysed using e.g LC-MS/MS, the proteins are preferably subjected to some kind of enzymatic digestion. The person skilled in the art will easily know how the proteins should be digested in order for the samples to be analysed using e.g LC-MS/MS. Two non-limiting examples of enzymes that may be used for such enzymatic digestion is Lys-C and trypsin.
In another embodiment according to the present invention, the product of step i) and/or product of step ii) are subjected to enzymatic digestion. The enzymatic digestion preferably comprising subjecting the product of step i) and/or the product of step ii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
In a preferred embodiment according to the present invention the polypeptides in the sample are subjected to pre-enzymatic digestion using Lys-C, preferably for a time period in the range 4-12 hours, such as 5-10 hours, or 6-9 hours or 7-9 hours such as 8 hours. After the pre-enzymatic digestion, the sample is preferably subjected to enzymatic digestion using trypsin. In one embodiment according to the present invention, the trypsin : protein weight ratio is in the range 1 : 10 to 1 :30, such as 1 :20.
If the product of step i) and/or product of step ii) has been subjected to enzymatic digestion, the levels of the at least ten polypeptides may be detected and quantified by detecting and quantifying the levels of at least one fragment, such as at least two fragments, at least three fragments, at least four fragments, at least five fragments, at least six fragments, at least seven fragments, at least eight fragments, at least nine fragments or at least ten fragments for each of said polypeptides. Preferably, the length of each fragment is in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10-55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
In another embodiment according to the present invention, the samples may be subjected to treatment with an alkylating agent after having been subjected to enzymatic digestion. Iodoacetamide is one example of an alkylating agent which is an irreversible inhibitor of all cysteine peptidases.
Now having released and digested the intracellular proteins, the level of each protein may be measured e.g. by mass spectroscopy. In another embodiment according to the present invention, the levels of the at least ten polypeptides is measured by mass spectroscopy, preferably liquid chromatography mass
spectroscopy (LC-MS), especially high performance liquid chromatography mass spectroscopy (HPLC-MS) or reverse phase liquid chromatography mass
spectroscopy (RPLC-MS); electro-spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC-MS), atmospheric pressure chemical ionization mass spectroscopy (APCI-MS), capillary electrophoresis mass spectroscopy (CE-MS), tandem mass spectroscopy (MS-MS); or any combination thereof. A huge number of polypeptides formed basis for the plot shown in figure 2.
However as shown in figure 3, ten of these polypeptides were found to be significantly correlated with the degree of illness 12 months after debut of disease.
According to one embodiment, the at least 10 polypeptides are at least 100 polypeptides, even more preferably at least 200 polypeptides and most preferably at least 500 polypeptides.
In one embodiment according to the present invention, the levels of the at least ten polypeptides are detected and quantified by detecting and quantifying fragments of said at least ten polypeptides. The length of each fragment may be in the range 5-70 amino acids long, more preferably 7-55 amino acids long, even more preferably 10- 55 amino acids long, such as e.g. 15-55 or 20-55 amino acids long.
In one embodiment according to the present invention, the comparison (i.e. step b) is performed using software-based statistical and bioinformatics data analysis. In one preferred embodiment according to the present invention, the comparison is performed by batch correcting the obtained data using the“EB method for adjusting batch effect” (disclosed in Biostatistics. 2007 Jan;8(l): l 18-27. Epub 2006 Apr 21. which is hereby incorporated by reference in its entirety) and then preferably analyzing the batch-corrected data using principal component analysis, limma package, Cluster Profiler and revigo.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
Examples
ACN, acetonitrile; BCA, bicinchoninic acid; DTT, dithiothreitol; FA, formic acid; IAA, iodoacetamide; IBD, inflammatory bowel disease; SDC, sodium deoxycholate; TEAB, Triethylammonium bicarbonate buffer; TFA, trifluoroacetic acid; TMT, Tandem Mass Tag
Example la: Biopsy collection
The recruited subjects were 19 patients admitted at the University Hospital of North Norway who newly have been diagnosed with UC, i.e. at the debut of the disease. All participants were informed and signed a written consent. The Regional
Committee of Medical Ethics of North Norway and the Norwegian Social Science Data Services approved the study and the storage of biological material. Debut of disease
Colonic mucosal biopsies were sampled from the most severe inflammation in the UC patients at the debut of the disease. Biopsies were (10-15 mg wet weight) kept frozen at - 70 °C for further processing as described below, the samples hereinafter being referred to as debut samples.
12 months after debut of disease
Further, the patients were also subjected to evaluation of the degree of illness using the scoring system ulcerative colitis disease activity index (UCDAI) which is based on clinical signs (score 0-12) and on endoscopic evaluation of the colon during colonoscopy (grade 0-3) ( Gastroenterology 1987, 92, 1894-1898). 1 1 patients were found to suffer from mild UC, 3 patients were found to suffer from moderate UC and the last five patients were found to suffer from severe UC.
Example lb: Sampling preparation
Tissue lysis
Lysis buffer: 5% Sodium deoxycholate (SDC) and 8 M urea in 100 mM
triethylammonium bicarbonate buffer (TEAB)
250 mΐ cooled lysis buffer is filled into 38 MagNA Lyser Green Bead tubes (Roche Diagnostics AG, Rotkreuz, Switzerland). 19 debut samples were placed between the beads in said tubes. The tubes are kept on ice until lysis is completed and thereafter the tubes are homogenized in a MagNA Lyzer instrument (Roche Diagnostics AG, Rotkreuz, Switzerland) at 6500 rpm for 35 sec. The homogenized samples hereinafter being referred to as cell lysates.
Measurement of protein concentration by BCA assay
5 mΐ of cell lysates were diluted with 95 mΐ water to a final concentration of 5 mM TEAB. Further solutions for the measurement were prepared by dilution with 5 mM TEAB. These solutions were measured against bovine serum albumin (BSA) standards in 5 mM TEAB. BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific) was performed according to the manufacturer’s protocol in order to estimate the protein concentration in the cell lysates.
Reduction and alkylation
An aliquot of 60 pg protein was taken from the cell lysates and transferred to protein LoBind tubes (Eppendorf AG, Hamburg, Germany). Cysteines were reduced by adding DTT to a concentration of 5 mM and incubating for 30 min at 54 °C, followed by alkylation with 10 mM iodoacetamide for 30 min at room temperature in the dark. Excess iodoacetamide was quenched by adding DTT corresponding to 5 mM and incubation for 5 minutes at room temperature (Analytical Chemistry 2011, 83, 8484-8491). The reduced and alkylated samples are hereinafter referred to as reduced samples.
Enzymatic cleavage
A CaCl2 solution, 0.6 pg Lys-C (enzyme-to -protein ratio 1 : 100, w/w), water, and 1M TEAB is added to the reduced samples to a final concentration of 1 mM calcium chloride, 6 M urea, and 100 mM TEAB. Protein predigest is performed under gentle agitation for 8 hours at 37°C. CaCh solution, 3 pg trypsin (enzyme-to-protein ratio 1 :20, w/w), water, and 1M TEAB is added to a final concentration of 1 mM calcium chloride, 1 M urea, and 100 mM TEAB. Tryptic protein digest is performed under gentle agitation for 16 hours at 37°C. The reduced samples that has been subjected to enzymatic treatment hereinafter being referred to as fragment samples.
TMT labeling
Aliquots of the fragment samples corresponding to 25 pg peptides is transferred to new Protein LoBind tubes (Eppendorf AG, Hamburg, Germany). TMT labelling is performed according to the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). 0.4 mg TMT reagent is used for 25 pg peptides from samples with starting protein concentrations > 2200 pg/ml. The amount of TMT reagent is increased for lower starting concentrations. A total of 100 pg of the differently labelled peptides is mixed in equal amounts in a Protein LoBind tube (Eppendorf AG, Hamburg, Germany). The TMP-labelled samples are hereinafter referred to as labelled samples.
SDC and acetonitrile removal
The labelled samples are acidified with 50% formic acid to a final concentration of 2.5 % formic acid and pH < 2. The precipitated sodium deoxycholate (SDC) is removed by centrifugation at 16,000 g for 15 min. The supernatants are carefully transferred to fresh Protein LoBind tubes (Eppendorf AG, Hamburg, Germany). Acetonitrile is removed by evaporation in a vacuum concentrator. The samples which have been subjected to SDC and acetonitrile removal are hereinafter referred to as free samples.
Fractionation
TFA is added to each of the free samples to a concentration of 0.1%. The samples are fractionated into 8 fractions using Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Samples exceeding 300 mΐ are loaded stepwise by repeating the following: Add 300 mΐ to the column, centrifuge 2 min at 3000 x g, discard flow-through. The fractions are subjected to evaporation until dryness in a vacuum concentrator and re-dissolved in 10 mΐ 0.1% TFA for subsequent LC- MS/MS analyses.
Example lc: LC-MS/MS
The fractions were analyzed with a nano-LC-MS/MS system (EASY-nLC 1000 coupled to a Q Exactive mass spectrometer, both Thermo Fisher Scientific, Bremen, Germany).
2.0 pg peptides per sample were injected. The peptides were concentrated on a reversed-phase trap column (Acclaim PepMap® 100, Cl 8, particle size 3 pm, pore size 100 A, inner diameter 75 pm, length 2 cm, nanoViper, Thermo Fisher
Scientific) with 0.1% formic acid (FA) at a flow rate of 20 pl/min. The peptides were separated on a reversed-phase main column (EASY-Spray, PepMAP® RSFC, Cl 8, particle size 2 pm, pore size 100 A, inner diameter 75 pm, length 50 cm, Thermo Fisher Scientific). A binary solvent gradient (solvent A: 0.1% FA, solvent B: acetonitrile, 0.1% FA) was used at 60 °C column temperature and 200 nl/min flow rate. The ACN proportion was increased from 2% to 5% over 19 min, further to 30% at 180 min and to 100% at 200 min. The column is regenerated with 100% ACN for additional 10 min.
The Q Exactive mass spectrometer was run in positive mode, with the following global settings:
Chromatographic peak width 15 s, default charge state 2, full MS survey scans from 400 to 2000 m/z, resolution 70,000, AGC target value 3e6, maximum injection time 100 ms for MS scans. Subject the 10 most intense peaks to MS/MS with the following settings: resolution 17,500, dynamic exclusion 10 s, underfill ratio 1%, charge states +2, +3 and +4, exclude isotopes, normalized collision energy 31, isolation window 2 m/z, AGC target value le5 and maximum injection time 50 ms, fixed first mass 120.
Example Id: Data evaluation
The raw data are processed with a quantitative proteomics software package, e.g. MaxQuant (Cox, J., Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein
quantification. Nat Biotech 2008, 26, 1367-1372). The newest version at the filing date of the present application of the respective fasta file is downloaded from https://www.uniprot.org/, and configured in the MaxQuant sequence database configurations. The TMT reporter ion distribution is changed in the MaxQuant PTM configuration according to the manufacturer’s Certificate of Analysis and MaxQuant is re-started. The reporter ion
MS2/TMTsixplex is set as quantification method. The raw files are loaded and their experiment name and fraction numbers specified.
The configured fasta file is searched with the following parameters: Enzyme:
Trypsin/P (specific), fixed modifications: Carbamidomethyl (C), variable
modifications: Oxidation (M) and Acetyl (Protein N-term) (Note 16), max. 2 missed cleavages, PSM FDR: 0.01, Protein FDR: 0.01 Site FDR: 0.01, Use Normalized Ratios For Occupancy: TRUE, Min. peptide Fength: 7, Min. score for unmodified peptides: 0, Min. score for modified peptides: 40, Min. delta score for unmodified peptides: 0, Min. delta score for modified peptides: 6, Min. unique peptides: 0, Min. razor peptides: 1, Min. peptides: 1, Use only unmodified peptides and: TRUE, Modifications included in protein quantification: Oxidation (M) and Acetyl (Protein N-term), Peptides used for protein quantification: Razor, Discard unmodified counterpart peptides: TRUE, Fabel min. ratio count: 2, Use delta score: FAFSE, iBAQ: FAFSE, iBAQ log fit: FAFSE, Match between runs: TRUE, Matching time window [min]: 0.7, Alignment time window [min]: 20, Find dependent peptides: FAFSE, Decoy mode: revert, Include contaminants: TRUE, Advanced ratios:
TRUE, Second peptides: FAFSE, Calculate peak properties: FAFSE, Main search max. combinations: 200, Advanced site intensities: TRUE, Max. peptide mass [Da]: 4600, Min. peptide length for unspecific search: 8, Max. peptide length for unspecific search: 25, Razor protein FDR: TRUE, Disable MD5: FAFSE, Max mods in site table: 3, Match unidentified features: FAFSE, MS/MS tol. (FTMS): 20 ppm, Top MS/MS peaks per Da interval. (FTMS): 12, Da interval. (FTMS): 100, MS/MS deisotoping (FTMS): TRUE, MS/MS deisotoping tolerance (FTMS): 7, MS/MS deisotoping tolerance unit (FTMS): ppm, MS/MS higher charges (FTMS): TRUE, MS/MS water loss (FTMS): TRUE, MS/MS ammonia loss (FTMS): TRUE, MS/MS dependent losses (FTMS): TRUE, MS/MS recalibration (FTMS): FAFSE.
The quantification data are loaded into Perseus (Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M. Y., Geiger, T., Mann, M., Cox, J., The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Meth 2016, 13, 731- 740). Identifications labelled as only identified by site are deleted, hits in reverse database and potential contaminants are removed. A log2 transformation is performed. The intensities are normalized. If a standard channel was used, this can be done by subtracting its reporter ion intensities from the other channels of the same experiment and subsequent Z-score transformation (matrix access: columns). The results are visualized as described in Tyanova, S., Cox, J., Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research. Methods in molecular biology (Clifton, N.J.) 2018, 1711, 133-148.
Example le: Verification of the in-vitro method of predicting clinical course of ulcerative colitis
A predictive model was build utilizing a multivariate model in SIMCA 14.1 (Umetrics) where a OPLS model was utilized to build a model for separating the patients with a severe outcome from the patients with moderate or mild outcome. Relative protein ratios were used as input in the model, and the model was filtered to obtain no missing values in quantified proteins.
Results of the predictive model based on proteomic data (2896 proteins analyzed in total, i.e. SEQ ID NOs: 1 to 2896) from 19 untreated patients at debut of disease is shown in figure 2.
Results of the predictive model based on i) proteomic data (10 proteins analyzed in total, i.e. SEQ ID NOs: 27, 213, 617, 894, 903, 1011 , 1513, 1920, 1987 or 2412) from 19 untreated patients at debut of disease is shown in figure 3.
Example 2: Prognostic model for predicting the 1-year outcome for patients with ulcerative colitis based on proteomics data.
Patient data:
Figure imgf000040_0001
Biopsy lysis:
Colon biopsies were lysed in 250 mΐ cold buffer (5% sodium deoxycholate (SDC), 8 M urea, 100 mM triethylammonium bicarbonate buffer pH 8.5 (TEAB)) in MagNA Lyser Green Bead tubes (Roche Diagnostics AG, Rotkreuz, Switzerland) with a MagNA Lyser Instrument (Roche Diagnostics AG, Rotkreuz, Switzerland) for 35 seconds at 6500 rpm. The samples were frozen and stored at -70 °C until further preparation.
Protein concentration measurement:
The protein concentration was determine with a bicinchoninic acid (BCA) assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific) according to the manufacturer’s protocol. The TEAB concentration for the BCA assay in standards and samples was 5 mM TEAB.
Reduction, alkylation, and quenching of alkylating agent:
l ,4-dithiothreitol (DTT) was added to a concentration of 5 mM to 60 pg protein aliquots in ProteinLoBind tubes (Eppendorf AG, Hamburg, Germany). Cysteines were reduced under incubation at 54°C for 30 min. The alkylating agent
iodoacetamide (IAA) was added to a concentration of 15 mM and cysteines were alkylated during incubation for 30 min at room temperature in the dark. DTT was added to a concentration of 5 mM to quench remaining IAA.
Lys-C/trypsin tandem digestion:
0.6 pg endoproteinase Lys-C (Wako Chemicals GmbH, Neuss, Germany) were added for a 1 : 100 enzyme-to -protein ratio (w/w). The sample compositions were adjusted to 1 mM calcium chloride, 6M urea and 100 mM TEAB with calcium chloride solution, water, and 1 M TEAB. The Lys-C predigestion was facilitated under gentle agitation for 8 hours at 37°C.
3 pg trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, USA) were added to the predigested samples for a 1 :20 enzyme-to-protein ratio (w/w). The sample compositions were adjusted to 1 mM calcium chloride, 1 M urea, and 100 mM TEAB with calcium chloride solution, water, and 1 M TEAB. Tryptic digestion was facilitated for 16 hours at 37°C under gentle agitation.
TMT labelling:
Equal peptide amounts from each sample were mixed to produce a standard. This standard mixture was labeled with the tandem mass tag (TMT) reagent TMT6-126 as described in the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, USA).
For each sample, 25 pg peptides were labeled with the remaining TMTsixplex reagents as described in the manufacturer’s protocol. 0.4 mg TMT reagent were used to label 25 pg peptides, if the starting protein concentration was > 2200 pg/ml in the respective sample. Samples with lower starting concentrations were labeled with more TMT reagent to achieve complete labeling.
The labeled standard and 5 differently labeled samples were mixed in equal amounts to in total lOOpg peptides for each fractionation mixture. Detergent removal and high pH reversed-phase fractionation:
The detergent SDC was precipitated by adding 50% formic acid (FA) solution to a final concentration of 2.5% FA. A pH < 2 was confirmed with pH indicator paper.
The samples were centrifuged at 16,000 g for 15 min and the supernatant was transferred to new Protein LoBind tubes to separate it from the pelleted detergent. Acetonitrile, which was added to the samples during TMT labeling, was removed in a vacuum concentrator because it would interfere with the fractionation procedure.
The samples were adjusted to a concentration of 0.1% TFA by adding 10% trifluoroacetic acid (TFA) solution. The fractionation into 8 fractions was performed with Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, Rockford, USA) as described in the manufacturer’s protocol.
Samples exceeding 300 mΐ were loaded stepwise onto the fractionation columns by centrifugation for 2 minutes after each loading. The collected fractions were dried in a vacuum concentrator and redissolved in 10 mΐ 0.1% TFA for subsequent LC- MS/MS analyses.
The absorption of these solutions at 205 nm was measured with a Nanodrop 2000 (Thermo Fisher Scientific, Bremen, Germany). The corresponding peptide concentration was calculated based on an extinction coefficient of 31 mg/ml.12
LC-MS/MS:
The fractions were analyzed with an EASY-nLC 1000 coupled to a Q Exactive mass spectrometer (both
Thermo Fisher Scientific, Bremen, Germany).
2.0 pg peptides of each fraction were injected and concentrated on a reversed-phase trap column. Separation was performed with a gradient of 0.1% FA in water and 0.1% FA in acetonitrile on a reversed-phase main column. The proportion of the acetonitrile solvent was increased over 19 min from 0% to 5%, to 30% at 180 min, and up to 100% at 200 min. More details on the FC-MS/MS settings are provided in the supporting information.
Processing of mass spectrometry data:
Proteins were identified and quantified from the mass spectrometry data with MaxQuant. l3
Protein intensities were log2-transformed in Perseus.14 The identifications with labels“potential contaminant”,“reverse” or“only identified by site” were removed. The standard intensities were subtracted from the sample intensities of the same experiment and the resulting intensities were Z- score normalized (matrix access: columns).
Multivariate analysis of data:
All statistical multivariate handling of data was conducted in SIMCA P+ 15.0 (Umetrics, Sweden). Normalized protein levels were imported and all proteins with missing data were removed from the dataset before further data handling.
Results and discussion
Pinpointing the important features in a prospective proteomics analysis is challenging and requires strict evaluation for the clinical usefulness of the biomarkers. The process from the first promising data to the ready-to-use is long with many obstacles. However, it is presented herein the first promising prospective results from a proteomics study from colon biopsies of UC patients.
In an unsupervised PCA model (figure 4a) the data is quite spread, however there is a trend that the patients with severe l-year outcome seems to cluster slightly more towards the lower left side than the rest. This indicates that there are generally differences expressed in patients that will have a more severe course of the disease already at time of diagnosis. In a supervised PLS model, where we want to separate the group with the severe l-year outcome from those with mild to moderate outcome, the separation between the groups becomes very clear (figure 4b). The model also shows excellent separation of the patient’s outcome severity (Y- variable) with a R2Y of 0.995, and an acceptable predictive Q2 value of 0.673. However, as the model is based on a total of almost 8.500 proteins, there is a necessity to reduce the number of proteins before the data can be utilized in a clinical environment. In addition, most of the identified proteins are probably not important for the statistical model; hence, these proteins contribute only noise, but no additional predictive strength, to the model.
To circumvent the problems with noise and to reduce the number of proteins, several PLS models were built with different numbers of the most important proteins, choosing the most important proteins in the PLS model in figure 4b.
Reducing the number of variables in a predictive model is a trade-off between reducing noise and keeping important information. In this dataset, it seems that the 50 most important proteins are sufficient for building the best predictive model. With the resulting PLS model (Figure 5a), we see an excellent separation of the 1- year outcome status of the patients. As seen in the graphical representation of the model, the patients are clearly separated by the tl compartment. Upon reduction of the number of proteins from approximately 8.500 to 50, the predictive Q2 value increased from 0.673 to 0.869. The relatively high Q2 value testifies that the model has good predictive power for the l-year outcome.
In order to investigate the model further, we also created unsupervised PCA plots of the included patients, with only the 50 most important proteins taken into account (figure 5b). The resulting unsupervised PCA shows that the groups open up a bit more, especially the group with mild to moderate outcome, however, the separation is still excellent which strengthens the postulation that this model can predict the 1 - year outcome of UC patients.
Biological relevance of proteins:
The 50 most important proteins from the model shown in figure 4b, and further used in the models presented in figure 5a and 5b, show distinct networks when processed through functional processing association network searches. In general there are two separate networks (figure 6), where the major network consists of 3 interlinked networks (Figure 6A, 6B and 6C).
The core of the network, labelled A in figure 6, is dominated by the fibrinogen proteins Fibrinogen Alpha, Beta and Gamma chain (FGA, FGB and FGG).
Fibrinogen is most commonly recognized for the involvement in blood clotting and wound healing as an acute phase protein. However, fibrinogen has been linked to inflammation as well and has been reported to have pro-inflammatory properties in several diseases, e.g. multiple sclerosis, Alzheimer’s disease, rheumatoid arthritis, kidney fibrosis and several types of cancer. The fibrins are also linked to other well-known parts of the immune system, like complement component C6 (C6), a part of the adaptive immune response, which forms pores in the cell membrane of target cells. In addition, the fibrinogens are interlinked with the glycoprotein Ceruplasmin (CP), known for oxidizing cupper to Cu2+, as well as platelet basic protein (PPBP), which among many other functions is known to stimulate the formation and secretion of plasminogen activator.
Network A is linked through a metalloproteinase (MMP2) to the network labelled B, which consists of proteins involved in splicing (YBX1 , CD2BP2 and SRSF9) and other proteins involved in translation of mRNA to proteins (NACA and BTF3). The cold-shock protein YBX1 has been shown to be a key protein in inflammation through regulation of interleukin release and has been linked to resistance against Janus kinase (JAK) inhibitors.
Network C consists of 4 proteins, all located in the mitochondria of the cells. In network C there are two upregulated proteins in patients with severe compared to mild or moderate outcome, P-Arrestin2 (ARRB2) which is a G-protein coupled receptor signaling for various cytoplasmic signaling within inflammation 17 and Huntington interacting protein (HIP1) which has been shown to regulate arthritis severity. In addition, two 39S ribosomal proteins that interact with P-Arrestin2 are downregulated, MRPL44 and MRPL51. MRPL44 has previously been shown to be involved in tumor development. MRPL51 on the other hand has previously been reported in a dextran sodium sulphate (DSS)-induced colitis model, where MRPL51 was shown to be regulated at transcript level but without being able to explain the role of the protein in IBD.
The independent network labelled D has two Rab proteins at its core, Rabl3 and Rab22A, which have their main importance in membrane trafficking. Both Rab 13 and Rab22A have been reported to be involved in inflammation as well as inflammation-related diseases like cancer. Mistargeting of Rab 13 to basolateral sites has been observed in patients with Crohn's Disease. Network D seems to influence permeability of the intestine, which is known to be dysregulated in patients with IBD. These data indicate that intestinal permeability is also of importance for predicting the outcome of the patients.
In addition to the proteins described above as members of the networks, there are other proteins that are of importance for the clustering of patients. The majority of these proteins seems to be involved in immune system or energy metabolism pathways. In general, the biological functions of the proteins seem to fit to the severity of IBD, which is strengthening the background of the statistical model for predicting the outcome of IBD patients.

Claims

1. An in-vitro method of predicting clinical course of ulcerative colitis in a
subject, the method comprising the following step(s):
a) detecting and quantifying in a biological sample from said subject the levels of at least ten polypeptides or fragments thereof; each of said at least ten polypeptides comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1 to 2896;
b) comparing the results obtained in step a) with
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 1 month after the first control sample was taken; and/or
ii) the levels of said at least ten polypeptides or fragments thereof in a
second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 1 month after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 1 month after the third control sample was taken.
2. The in-vitro method according to claim 1, wherein the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 6 months after the first control sample was taken; and/or ii) the levels of said at least ten polypeptides or fragments thereof in a second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 6 months after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 6 months after the third control sample was taken.
3. The in-vitro method according to claim 1, wherein the results obtained in step a) are compared with:
i) the levels of said at least ten polypeptides or fragments thereof in a first control sample from a first control subject; wherein the first control subject has been diagnosed with moderate ulcerative colitis at least 12 months after the first control sample was taken; and/or ii) the levels of said at least ten polypeptides or fragments thereof in a second control sample from a second control subject; wherein the second control subject has been diagnosed with severe ulcerative colitis at least 12 months after the second control sample was taken; and/or
iii) the levels of said at least ten polypeptides or fragments thereof in a third control sample from a third control subject; wherein the third control subject has been diagnosed with mild ulcerative colitis at least 12 months after the third control sample was taken.
4. The in-vitro method according to claim 1, wherein
the clinical course of ulcerative colitis is moderate ulcerative colitis if the results obtained in step a) are similar to the levels of said at least ten polypeptides or fragments thereof in the first control sample;
the clinical course of ulcerative colitis is severe ulcerative colitis if the results obtained in step a) are similar to the levels of said at least ten polypeptides or fragments thereof in the second control sample; and
the clinical course of ulcerative colitis is mild ulcerative colitis if the results obtained in step a) are similar to the levels of said at least ten polypeptides or fragments thereof in the third control sample.
5. The in-vitro method according to claim 1, wherein onset of disease is when the subject experiences symptoms of ulcerative colitis.
6. The in-vitro method according to claim 1, wherein
- the first control subject has been diagnosed with moderate ulcerative colitis within 12 months, such as within 24 months, from the onset of disease; - the second control subject has been diagnosed with severe ulcerative colitis within 12 months, such as within 24 months, from the onset of disease; and
- the third control subject has been diagnosed with mild ulcerative colitis within 12 months, such as within 24 months, from the onset of disease.
7. The in-vitro method according to claim 1, wherein the biological sample, first control sample, second control sample and third control sample are selected from the group consisting of a colon biopsy, urine and blood; most preferably a colon biopsy.
8. The in-vitro method according to claim 1, wherein
- the subject has not previously been diagnosed with ulcerative colitis; and/or the subject experiences for the first-time symptoms of ulcerative colitis.
9. The in-vitro method according to claim 1, wherein one of the at least ten
polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 27, 213, 617, 894, 903, 101 1, 1513, 1920, 1987 or 2412 respectively.
10. The in-vitro method according to claim 1, wherein
- a first polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 27;
- a second polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 213;
- a third polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 617;
- a fourth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 894;
- a fifth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 903;
- a sixth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 1011;
- a seventh polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs:
1513; - a eight polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 1920;
- a ninth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 1987; and
- a tenth polypeptide of the at least ten polypeptides comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 2412.
11. The in-vitro method according to claim 1 , wherein each of said at least ten
polypeptides comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 2896.
12. The in-vitro method according to claim 1, wherein each of said at least ten
polypeptides consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1 to 2896.
13. The method according to claim 1, wherein step b) is performed using software- based statistical and bioinformatics data analysis.
14. The method according to claim 7, wherein the biological sample, first control sample, second control sample and third control sample are a colon biopsy; and the colon biopsy is subjected to sample preparation prior to step a); the sample preparation comprising the following steps:
i) adding a liquid, such as water, to the colon biopsy;
ii) subjecting the product of step i) to homogenization; and
iii) adding lysis buffer to the product of step ii).
15. The method according to claim 14, wherein the lysis buffer comprises a
detergent selected from the group consisting of deoxycholate, such as sodium deoxycholate, dodecyl sulfate, laurate, cetyltrimethylammonium, N- methyldioctylammine or any combination thereof.
16. The method according to claim 14, wherein the product of step i), product of step ii) and/or the product of step iii) is subjected to enzymatic digestion. The method according to claim 16, wherein enzymatic digestion comprises subjecting the product of step i), the product of step ii) and/or the product of step iii) to enzymatic pre-treatment using Lys-C, optionally followed by enzymatic treatment using trypsin.
17. The method according to claim 14, wherein the product of step i), product of step ii) and/or the product of step iii) is subjected to treatment suitable for reducing and alkylating cysteines.
18. The method according to claim 1, wherein the levels of the at least ten
polypeptides or fragments thereof are detected and quantified by mass spectroscopy, preferably liquid chromatography mass spectroscopy (LC-MS), especially high performance liquid chromatography mass spectroscopy (HPLC- MS) or reverse phase liquid chromatography mass spectroscopy (RPLC-MS); electro-spray ionization mass spectroscopy (ESI-MS), gas chromatography (GC- MS), atmospheric pressure chemical ionization mass spectroscopy (APCI-MS), capillary electrophoresis mass spectroscopy (CE-MS), tandem mass
spectroscopy (MS-MS); or any combination thereof.
19. The method according to claim 1, wherein levels of the at least ten polypeptides are detected and quantified by detecting and quantifying fragments of said at least ten polypeptides.
20. The method according to claim 1 or 20, wherein the length of each of said
fragments is in the range 5-70 amino acids long.
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