WO2023175177A1

WO2023175177A1 - Differential diagnosis of crohn's disease and ulcerative colitis

Info

Publication number: WO2023175177A1
Application number: PCT/EP2023/056958
Authority: WO
Inventors: Emmanuel FARGE; Didier Meseure
Original assignee: Institut Curie; Centre National De La Recherche Scientifique; Sorbonne Universite
Priority date: 2022-03-18
Filing date: 2023-03-17
Publication date: 2023-09-21

Abstract

Inflammatory Bowel Diseases (IBDs) represent 3 million cases a year in a continent like the US. Differentially diagnosing Crohn's Disease (60% of IBDs) from Ulcerative Recto-Colitis (URC, 40% of IBDs) is required for differential treatments, but involves time-consuming and expensive batteries of more than 10 analyses, including repeated biopsies, with a 10-20% probability of miss- diagnosis. Here we find that the phosphorylation of GSK3β on Ser9 (pSer9GSK3β) is a single strong discriminant marker of URC patient biopsies compared to Crohn patient biopsies, with a 2-7% probability of miss-diagnosis. pSer9GSK3β is thus an innovative unique and simple sharp test to diagnose IBD type (URC and Crohn's).

Description

Differential diagnosis of Crohn’s disease and Ulcerative colitis

Technical field of the invention

The invention relates to methods for the differential diagnosis of Crohn’s disease and Ulcerative colitis. The invention is in particular related to methods suitable for the early diagnosis of Crohn’s disease and Ulcerative colitis. The invention also relates to kits suitable for performing a diagnosis of Crohn’s disease or Ulcerative recto colitis for a patient having or developing an inflammatory bowel disease.

Background of the invention

Inflammatory bowel diseases (IBDs) are chronic and systemic inflammatory conditions that primarily affect the gastrointestinal tract and result in significant morbidity and health issues. IBD are often associated with extra-intestinal manifestations and autoimmune disorders. IBDs’ origin remains unclear, but a complex interplay of genetic predisposition, microbiota composition and dysregulation of the immune system is believed to be key factor in the development of this kind of diseases within a patient. IBDs represent approximately 200 000 cases each year in France, and 3 million cases in USA.

Ulcerative colitis and Crohn's disease are the two main forms of inflammatory bowel diseases. Both conditions are characterized by chronic inflammation of the digestive tract. Although they share many similarities, there are key differences between the two diseases. These diseases are recognized as important causes of gastrointestinal disease both in children and in adults. Both diseases often develop in teenagers and young adults although the disease can occur at any age.

Ulcerative colitis is a condition in which the inflammatory response and morphologic changes remain confined to the colon, with variable degrees of proximal extension. Inflammation is limited primarily to the mucosa and consists of continuous involvement of variable severity with ulceration, oedema, and haemorrhage along the length of the colon. The characteristic histologic findings are acute and chronic inflammation of the mucosa by polymorphonuclear leukocytes and mononuclear cells, crypt abscesses, distortion of the mucosal glands, and goblet cell depletion.

Crohn’s disease can involve any part of the gastrointestinal tract from the oropharynx (i.e. the mouth) to the perianal area (i.e. the colon). Diseased segments frequently are separated by intervening normal bowel. Inflammation can be transmural, often extending through to the serosa, resulting in sinus tracts or fistula formation. Histologic findings include small superficial ulcerations over a Peyer’s patch (aphthoid ulcer) and focal chronic inflammation extending to the submucosa.

UC and CD are associated with both intestinal and extra-intestinal manifestations. While Ulcerative Colitis and Crohn’s disease have a number of similarities in their clinical presentations, since their associated symptoms are very similar, it is of the utmost importance to diagnose if a patient has or develop an Ulcerative colitis or a Crohn’s disease. The specific causes of both UC and Crohn's disease are not known and both diseases have similar types of contributing factors such as environmental, genetic and an inappropriate response by the body's immune system. Therefore, it is difficult to determine if a person has Crohn’s disease or Ulcerative Colitis. Nonetheless, the correct diagnosis is imperative, as treatment is different for the two diseases.

Differential diagnosis of Crohn’s disease (approximately 60% of IBDs cases) from Ulcerative Colitis (approximately 40 % of IBDs cases) requires around 15 (Crohn) to 20 (UC) chemical, morphological, biological and disease evolutionarily criteria than can take from 1 to 2 months to establish a diagnosis, which suffers from a lack of confidence due to a high miss-diagnostic ratio.

Besides, the medication used recently in the treatment of either Crohn’s disease or Ulcerative colitis differs. The surgical treatment of either Crohn’s disease or ulcerative colitis also differs. Thus, it is important to determine as early as possible if a subject having an IBD is suffering from Crohn’s disease or from Ulcerative colitis. Nowadays, diagnosis of either Crohn’s disease or Ulcerative colitis includes evaluation of clinical images, requiring radiologic imaging techniques, such as X- rays, CT scans and Magnetic Resonance Imaging, and/or gastrointestinal endoscopy, possibly with tissue sampling, the samples being later used in tissue imaging analysis. All these tools are expansive as well as time consuming. Furthermore, the sampling method through gastrointestinal endoscopy is most of the time uncomfortably perceived by the patients, noting that the diagnosis method currently available requires several repeated biopsies.

The main issues with the current diagnosis methods is that it is still difficult to clearly distinguish between Crohn’s disease and Ulcerative colitis, which may lead to wrong diagnostic, with potential deleterious effects if a wrong treatment is prescribed to the patient. Hence, there is a need for new diagnostic tools which overcome at least one of the above issues, in particular which facilitate the sampling (e.g. a faster sampling that does not require the use of complex imagery devices) and enhance the confidence in the diagnosis (e.g. by providing a more robust assessment in the differential diagnosis of either Crohn’s disease or Ulcerative colitis).

By being able to distinguish between CD and UC in a reliable manner, the means of the invention can be used to afford administer, in good time, the drug treatments which are currently available to attempt to combat or at least alleviate IBDs, in particular Crohn’s disease or Ulcerative Colitis, in particular Ulcerative Recto-Colitis. Since these drug treatments usually give rise to major side effects for the patient, the means of the invention provide very clear advantages as regards the general health of the patient.

Crohn’s disease is sequentially treated through the administration of budesonide, glucocorticoids, steroids, azathioprine, and inhibitors of TNFa as the severity and the chronicity of the disease increases.

Ulcerative colitis is sequentially treated with mesalazine (also known as mesalamine, 5-aminosalicylic acid, or 5-ASA), azathioprine, 5-ASA and budesonide, topic 5-ASA and systemic steroids, and alternatively cyclosporine infliximab, 6-mercaptopurine, as the seventy and chronicity of the disease increases.

Cell therapies, based on the introduction of new regenerative stem cells, or immune system regulatory cells, are currently tested at pre-clinic and clinic trials stages.

The inventors of the present invention have identified that distinct levels of phosphorylation of Glycogen synthase kinase-3 (GSK3) are representative of either Crohn’s disease or Ulcerative colitis, and that the phosphorylation state of this protein can accordingly be used as a biomarker of Ulcerative Colitis, in particular Ulcerative Recto-Colitis, and Crohn’s disease. It should be understood that the invention relies on the measurement of levels of phosphorylation of Glycogen synthase kinase-3 (GSK3), and not on the presence or absence (i.e. level of protein expression) of Glycogen synthase kinase-3 (GSK3) in tissue or cells of patients. The detection of Glycogen synthase kinase-3 (GSK3) in samples obtained from patients with CD or UC does not allow discrimination between these two diseases, unlike the measurement of phosphorylated Glycogen synthase kinase-3 (GSK3) as disclosed herein. Indeed, the inventors identified that when the majority of Glycogen synthase kinase-3 is not phosphorylated on Ser9, a patient having an inflammatory bowel disease is more likely to have Crohn’s disease than Ulcerative Colitis. On the contrary, when the majority of Glycogen synthase kinase-3 is phosphorylated on Ser9, a patient having an inflammatory bowel disease is more likely to have an Ulcerative Colitis, in particular an Ulcerative Recto-Colitis, than Crohn’s disease. More particularly, the inventors propose establishing the phosphorylation profile of GSK3 protein and using this profile as a signature of a differential diagnostic between Ulcerative Colitis, in particular Ulcerative Recto-Colitis, and Crohn’s disease.

The application provides means which are specifically adapted for this purpose.

The means of the invention in particular use the measurement or assay of the phosphorylation level of Glycogen synthase kinase-3-beta (GSK3|3), and the means of the inventions comprise:

- methods which comprise the measurement or assay of the levels of GSK3[3 with phosphorylated Ser9 of;

- methods for treating either CD or UC, after the diagnosis performed according to the diagnostic method of the invention;

- products or reagents which are specifically adapted to the measurement or assay of this levels of phosphorylation; also including in particular means for measuring level expression of GSK3[3, the level of GSK3[3 with either phosphorylated or not phosphorylated Ser9, the level of GSK3[3 with phosphorylated Ser9, the level of GSK3[3 with not phosphorylated Ser9;

- manufactured articles, compositions, pharmaceutical compositions, kits, tubes or solid supports comprising such products or reagents;

- computer systems (in particular a computer program product and computer device) which are specifically adapted to implementing the means of the invention.

An objective of the present invention is to provide a method for diagnosing IBDs. More particularly, an objective of the present invention is to provide a method to diagnose Crohn’s disease and Ulcerative Colitis, in particular Ulcerative Recto- Colitis, in a patient having an IBD. Among other benefits, the invention reduces the time and the expanses required for the specific diagnosis of either Crohn’s disease or Ulcerative Colitis in a differential diagnosis. The invention provides a more comfortable method for the patients, minimizing the need for endoscopic procedures for diagnosing CD or UC.

Summary of the invention

The application pertains to means for detecting or for diagnosing inflammatory Bowel Diseases (IBDs), in particular Crohn’s Disease and Ulcerative Colitis, in particular Ulcerative Recto-Colitis. In particular, the invention provides a method suitable to establish a differential diagnostic in a patient having an IBD between a patient who has a Crohn’s disease or a patient who has an Ulcerative Colitis, more particularly Ulcerative recto-Colitis. The method comprises the determination of a phosphorylated Ser9 on Glycogen synthase kinase-3 (GSK3) protein.

Glycogen synthase kinase-3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. GSK-3 is encoded by two paralogous genes, GSK-3 alpha (GSK3A) and GSK-3 beta (GSK3B) leading to the expression of two GSK3 isoforms: GSK3-alpha (GSK3a or GSK3a) and GSK3-beta (GSK3[3 or GSK3b). Glycogen synthase kinase 3|3 is a multifunctional protein kinase involved in many cellular activities including development, differentiation and diseases. GSK3[3 is thought to be constitutively activated by autophosphorylation of the specific tyrosine residue Tyr216 and inactivated by phosphorylation of the specific serine residue Ser9 induced by insulin. GSK3[3 is ubiquitously expressed in all types of cells and tissues and is particularly abundant in the brain. GSK3[3 regulates many cellular functions, including cell proliferation, cell survival, gene expression, cellular aURCitecture, neural development and plasticity. Tyr216-phosphorylated GSK3[3 is constitutively active and is inactivated by phosphorylation at the N-terminal Ser9 by several protein kinases, such as PKA, PKB, p90RSK and Akt. Phosphorylated Ser9 binds to a pocket for a priming phosphorylation in the substrate-binding region and reduces the binding affinity for substrates.

In the present invention, the method for determining if a patient has Crohn’s disease or Ulcerative Colitis, in particular Ulcerative Recto-Colitis, requires the measurement of the level of Human GSK3[3 exhibiting phosphorylated Ser9 (or the level of Human GSK3[3 exhibiting non-phosphorylated Ser9) only. Of course, measurements of other parameters can be performed to assess more closely the health status of the patient. References for GSK3[3 amino acid sequence and for the gene encoding the protein may be found in online databases. The skilled artisan may refer in particular to Entrez reference No. 2932 and UniProt reference P49841 for more details about human GSK3[3. A reference sequence for GSK3[3 may correspond to the sequence of SEQ ID No. 1 . In the present invention, Ser9-GSK3-[3 refers to all forms of GSK3-[3, including GSK3-[3 with phosphorylated Ser9 amino acid or non-phosphorylated Ser9 amino acid residue.

In accordance with one aspect of the invention, a method of the invention is a method for detecting or diagnosing if a subject suffering from an inflammatory bowel disease suffers from Crohn’s disease or ulcerative colitis, in particular, it is provided a method for differentially determining if a patient has or is likely to develop Crohn’s diseases, or if a patient has or is likely to develop ulcerative colitis.

Based on the level (or grade) of GSK3-[3 with phosphorylated Ser9 in a biological sample previously obtained from a subject, a differential diagnosis between Crohn’s disease and ulcerative colitis in a patient having an inflammatory bowel disease can be provided.

The determination in said subject of the grade of phosphorylation of Ser9-GSK3- [3 can simply be quantified by analysis of the phosphorylated and nonphosphorylated Serine on position 9 of GSK3-[3, and hence quantifying the proportion of GSK3-[3 with phosphorylated Ser9 within the overall quantity of GSK3-[3 within a biological sample issued from a patient.

Short description of the drawings

These and further aspects of the invention will be explained in greater detail by way of examples and with reference to the accompanying drawings in which:

Fig.1 : Labelling of slides from Crohn’s patient (1 A), Ulcerative recto-colitis patient (1 B, 1 C) biopsies with an anti-phospho-Ser9-GSK3[3 antibody.

Fig. 2 : p-GSK3b scores in Crohn v/s URC. Illustrates the score values attributed to the level GSK3[3 with phosphorylated Ser9 in patients having Crohn’s disease and in a patients having an Ulcerative colitis (one point is one patient).

Fig. 3 : Pictures of a tissue slide labelled with an anti-phospho-Ser9-GSK3[3 antibody illustrating an imagery treatment before counting labelled cells. Fig. 4: Illustration of the diagnostic of patients having either UC or CD based on the analysis of the percentage of stained cells visible at magnification x100 (grade 3 cells).

Detailed description of specific embodiments of the invention

In a first aspect, the invention concerns a method for determining if a subject suffering from an inflammatory bowel disease (IBD) suffers from Crohn’s disease or ulcerative colitis (UC), wherein the method comprises the steps of:

- providing a biological sample previously obtained from the subject;

- determining a level of phosphorylation of Ser9-GSK3-[3 in the sample;

- attributing a score to the level of phosphorylation of Ser9-GSK3-[3, wherein a score of phosphorylation of Ser9-GSK3-[3 lower than a threshold value is indicative that the subject suffers from Crohn’s disease, and/or wherein a score of phosphorylation of Ser9-GSK3-[3 higher or equal to a threshold value is indicative that the subject suffers from an ulcerative colitis, in particular ulcerative recto-colitis.

A biological sample obtained from the subject can be any biological sample, such as tissue, blood, urine, whole cell lysate. Methods of obtaining a biological sample are well known in the art and include obtaining samples from surgically excised tissue. Tissue, blood, urine and cellular samples can also be obtained without the need for invasive surgery, for example by puncturing the subject with a fine needle and withdrawing cellular material, including by biopsy. In certain embodiments, samples taken from a patient can be treated or processed to obtain processed biological samples such as supernatant, whole cell lysate, or fractions or extract from cells obtained directly from the patient. In other embodiments, biological samples issued from a patient can also be used with no further treatment or processing. In a preferred embodiment, the biological sample obtained from the subject is a tissue sample, in particular issued from intestinal tract, like colon, rectum, ileum or duodenum, preferably obtained by biopsy. A biological sample issued from a subject may, for example, be a sample removed or collected or susceptible of being removed or collected from an internal organ or tissue of said subject, in particular from intestinal tract, like colon, rectum, ileum or duodenum, or a biological fluid from said subject such as the blood, serum, plasma or urine, in particular an intracorporal fluid such as blood. The method of the invention is in particular carried out on a sample that comprises epithelial cells.

The measurement or assay may be carried out in a biological sample which has been collected or removed from said subject and which has been transformed, for example by extraction and/or purification of proteins and/or polypeptides and/or peptides, or by extraction and/or purification of a protein fraction such as serum or plasma extracted from blood. In an embodiment the measurement or assay is performed on epithelial cells obtained from the sample obtained from the patient.

The subject may be any human who has, develops, or is suspected to have or develop an Inflammatory Bowel Disease. In particular the subject has been diagnosed with IBD. The subject may be a child, an adolescent, an adult. The subject may or may not have been treated for symptoms associated with IBD.

The term “measure” or “assay” or equivalent term is to be construed as being in accordance with its general use in the field, and refers to the quantification of a measured compound. The level of phosphorylation of Ser9-GSK3[3 in the biological sample may correspond to the proportion or the concentration of GSK3[3 with phosphorylated Ser9 within the whole quantity of GSK3[3 within the sample (/.e. comprising all forms of GSK3[3, with phosphorylated Ser9 and not phosphorylated Ser9). The proportion may be expressed as a percentage (%) of GSK3[3 with phosphorylated Ser9 of the total quantity of GSK3[3 within the sample. Alternatively, instead of determining a percentage, a proportion or a concentration of GSK3[3 with phosphorylated Ser9 within the whole quantity of GSK3[3 within the sample, the level of GSK3[3 with phosphorylated Ser9 in the sample may be determined and a score may be associated to the level of GSK3[3 with phosphorylated Ser9. The score is an arbitrary value attributed according to semi-quantitative or fully quantitative analysis of the proportion of GSK3[3 with phosphorylated Ser9 within the biological sample, as compared to known reference values.

The term “score” reflects the degree of GSK3[3 with phosphorylated Ser9 within the biological sample. A score associated to the level of GSK3[3 with phosphorylated Ser9 may be defined as an arbitrary value issued from the semi- quantitative analysis by the labelling of histological slides labelled with an anti- phospho-Ser9GSK3[3 antibody, wherein a value of “0” corresponds to the absence of labelling of the anti-phospho-Ser9GSK3[3 antibody, a value of “3” corresponds to the strongest labelling observed. Histological slides or histological slides used for creating the base reference of the grade may be issued from patients whose health situation is known. Accordingly, a reference database may be created from the labelling results observed on slides issued from patients with Crohn’s disease or from slides issued from patients with Ulcerative Colitis.

In an alternative embodiment, in particular when the quantity of GSK3[3 with phosphorylated Ser9 is indirectly quantified by measuring the quantity GSK3[3 with non-phosphorylated Ser9 and deduced from the overall quantity of GSK3[3 (ie. GSK3[3 with phosphorylated Ser9 and GSK3[3 with non-phosphorylated Ser9, the term “score” reflects the degree of GSK3[3 with non-phosphorylated Ser9 within the biological sample. A score associated to the level of GSK3[3 with non- phosphorylated Ser9 may be defined as an arbitrary value issued from the semi- quantitative analysis by the labelling of histological slides labelled with an anti- phospho-Ser9GSK3[3 antibody, wherein a value of “0” corresponds to the absence of labelling of the anti-Ser9GSK3[3 antibody, a value of “3” corresponds to the strongest labelling observed. Histological slides used for creating the base reference of the grade may be issued from patients whose health situation is known. Accordingly, a reference database may be created from the labelling results observed on slides issued from patients with Crohn’s disease or from slides issued from patients with Ulcerative Colitis.

The score associated to the level GSK3[3 with phosphorylated Ser9 may be deduced or determined from measurement or assay values obtained from the subject, in particular by statistical inference and/or statistical classification, for example with respect to (pre)-established reference cohorts in accordance with their known clinical situation regarding CD or UC, correlated to the level of GSK3[3 with phosphorylated Ser9 within biological samples issued from patients of the reference cohorts.

In addition to measuring the level of GSK3[3 with phosphorylated Ser9 in the biological sample obtained from said subject, a method of the invention may thus further comprise a step for deducing or determining the score corresponding to the level of GSK3[3 with phosphorylated Ser9 from the value(s) associated to the level of phosphorylation of Ser9-GSK3-[3. This step for deduction or determination is a step in which the values for the measurements or assays obtained for said subject are analysed in order to infer therefrom the score of GSK3[3 with phosphorylated Ser9.

The score may be deduced or determined by comparing the values for measurements obtained from said subject with their values, or the distribution of their values, in reference cohorts which have already been set up as a function of their health situation (i.e. patients with CD or patients with UC), in order to classify said subject into that of those reference cohorts to which it has the highest probability of belonging (i.e. to attribute the likeliness of having either CD or UC to said subject).

Accordingly, a reference grade value score may be created wherein; “0” corresponds to the absence of labelling by the anti-phospho-Ser9GSK3[3 antibody contacted with the sample issued from the patient; “1” corresponds to a light staining of the anti-phospho-Ser9GSK3[3 antibody on the sample issued from the patient; “2” corresponds to a moderate staining of the anti-phospho- Ser9GSK3[3 antibody on the sample issued from the patient; “3” corresponds to the strongest staining of the anti-phospho-Ser9GSK3[3 antibody on the sample issued from the patient. The analysis of the level of GSK3[3 with phosphorylated Ser9 may be performed by direct observation of the sample. In a preferred embodiment of the method, a reference grade is created by fully quantitative analysis of reference samples issued from patients whose health condition is known (i.e. patients having an UC, patients having CD). The quantitative analysis may be performed by optical density analysis of the labelling of an anti-phospho- Ser9GSK3[3 antibody on a the reference samples, wherein “0” corresponds to the absence of labelling of the anti-phospho-Ser9GSK3[3 antibody on the sample issued from the patient; “1” corresponds to a light staining of the anti-phospho- Ser9GSK3[3 antibody on the sample issued from the patient; “2” corresponds to a moderate staining of the anti-phospho-Ser9GSK3[3 antibody on the sample issued from the patient; “3” corresponds to the strongest staining of the anti- phospho-Ser9GSK3[3 antibody on the sample issued from the patient.

The determination of the absence of labelling, the light staining, the moderate staining and the highest staining may be issued from tests performed in reference cohorts which have already been set up as a function of their health situation. If desired or required, the distribution of the measurement values obtained for the individuals of a cohort may be smoothed so that it approaches a Gaussian law.

The score quantifies the level of GSK3[3 with phosphorylated Ser9 within the biological sample, and is compared to known values, issued from previous experiments for which a score of the level of GSK3[3 with phosphorylated Ser9 has been determined and the clinical situation (i.e. either a patient having CD or a patient having a UC) has been determined with known methods. From these experiments, the inventors were able to classify the patients depending on the score attributed to the level of expression of GSK3[3 with phosphorylated Ser9. Accordingly, the method permits to allow a threshold value to distinguish patients having Crohn’s disease from patients having Ulcerative colitis.

In a preferred embodiment of the invention, the score value ranges from 0 (no labelling) to 3 (maximum labelling)

In a preferred embodiment of the invention, when the score is lower than 1 , the patient is likely to have or develop Crohn’s disease. In a preferred embodiment of the invention, when the score is lower than or equal to 1 , the patient is likely to have or develop Crohn’s disease.

In a preferred embodiment of the invention, when the score is higher than 2, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto- colitis.

In a preferred embodiment of the invention, when the score is higher than or equal to 2, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto-colitis.

In a preferred embodiment of the invention, when the score is higher than 1 ,5, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto-colitis.

In a preferred embodiment of the invention, when the score is higher than 1 , the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto- colitis.

In a particular embodiment of the invention, when the score is lower than or equal to 1 , the patient is likely to have or develop Crohn’s disease, when the score is higher than or equal to 2, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto-colitis, and when the score is comprised between 1 and 2, the health status of the patient needs further analysis.

In a particular embodiment of the invention, when the score is lower than or equal to 1 , the patient is likely to have or develop Crohn’s disease, when the score is higher than or equal to 1 ,5, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto-colitis, and when the score is comprised between 1 and 1 ,5, the health status of the patient needs further analysis.

In a particular embodiment of the invention, when the score is lower than or equal to 1 ,5, the patient is likely to have or develop Crohn’s disease, when the score is higher than or equal to 2 the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto-colitis, and when the score is comprised between 1 ,5 and 2, the health status of the patient needs further analysis.

In a preferred embodiment of the invention, the threshold value is determined according to the scores attributed within reference cohorts to patients having CD and to patients having UC.

In a preferred embodiment of the invention, when the score is lower than 1 , the patient is likely to have Crohn’s disease, and when the score is higher than 2, the patient is likely to have or develop Ulcerative colitis, in particular Ulcerative Recto- colitis.

In a particular embodiment of the invention, the method is performed with an anti- GSK3[3 selected from the following list: from Cell Signaling Technology:

Phospho-GSK-3[3 (Ser9) (5B3) Rabbit mAb #9323;

Phospho-GSK-3[3 (Ser9) (D85E12) XP® Rabbit mAb #5558;

Phospho-GSK-3[3 (Ser9) Rabbit Antibody #9336. from Sigma Aldrich:

Anti-nonphospho-GSK3 (Ser9) Antibody beta specific Antibody, clone 12B2, from mouse

In a particular embodiment of the invention, the method is performed with an anti- GSK3[3 selected from the following list:

GSK-3[3 (27C10) Rabbit mAb #9315

GSK-3[3 (D5C5Z) XP® Rabbit mAb #12456

GSK-3[3 (3D10) Mouse mAb #9832

A list of relevant antibodies that can be used in the method of the invention may be found in Front. Mol. Neurosci., 17 November 2016 (https://doi.orq/10.3389/fnmol.2016.00123) Novel Non-phosphorylated Serine 9/21 GSK3[3/a Antibodies: Expanding the Tools for Studying GSK3 Regulation In an embodiment, the method may comprise the measurement of the level of phosphorylation of Ser9-GSK3-[3 in several, different, samples issued from the same patient. As an example, several tissue slides issued from a biopsy obtained from a single patient may be provided for performing the method of the invention.

In an embodiment of the invention, the level of phosphorylation of Ser9-GSK3-[3 in the sample is determined on a tissue biopsy, in particular a tissue slice, the level of phosphorylated-Ser9-GSK3-[3 being measured by immunohistochemistry with an anti-phosphorylated-Ser9-GSK3-[3 antibody (for example an antibody listed here above), and the score is attributed by visual analysis of the tissue biopsy after the immunochemistry reaction, especially as performed by an anatomo-cytopathologist, by counting visible cells (i.e. cells that are stained following the immunohistochemistry reaction). According to the method:

- if a majority (absolute or relative) of cells are not visibly stained at magnification x400, a score of “0” is attributed;

- if a majority (absolute or relative) of cells are visibly stained at magnification x400, a score of “1” is attributed;

- if a majority (absolute or relative) of cells are visibly stained at magnification x200, a score of “2” is attributed;

- if a majority (absolute or relative) of cells are visibly stained at magnification x100, a score of “3” is attributed.

The score may thus be dependent from the stained cells observed throughout the whole tissue biopsy. Absolute majority means that more than half of cells present within the biopsy belong to the same category (e.g. either not visibly stained at magnification x400, or visibly stained at magnification x400, or visibly stained at magnification x200, or visibly stained at magnification x100). Relative majority means that a group of cells is the most present as compared to the other group of cells.

The score attributed to the sample corresponds to the level of phosphorylation of Ser9-GSK3-[3, and this score has to be compared to a threshold value that is indicative that the subject suffers from Crohn’s disease when the attributed score is lower than the threshold, or to an ulcerative colitis, in particular ulcerative recto- colitis, when the attributed score is higher or equal than the threshold value. In a particular embodiment, the threshold value that discriminates a patient having Crohn’s disease from a patient having an ulcerative colitis is 1 ,5, as illustrated on figure 2.

In a particular embodiment, a score of “0.5” is attributed when a staining is slightly visible when magnification x400 is reached but does not have the same intensity as compared to the staining viewed at magnification x200.

In an embodiment of the invention, it is provided several, different, biological sample issued from a single patient, and a mean value of the level of phosphorylation of Ser9-GSK3-[3 is calculated from the score attributed to each biological sample.

In an embodiment of the invention, it is provided a method for determining if a subject suffering from an inflammatory bowel disease (IBD) suffers from a Crohn’s disease (CD) or an ulcerative colitis (UC), wherein the method comprises the steps of:

- providing a biological sample, in particular a tissue biopsy, more particularly a tissue slice, previously obtained from the subject;

- determining a level of phosphorylation of Ser9-GSK3-[3 in the sample;

- attributing a score to the level of phosphorylation of Ser9-GSK3-[3, the score being representative of a percentage of stained cells, wherein the level of phosphorylation of Ser9-GSK3-[3 in the sample is determined according to the following protocol:

- in the biological sample, labelling, for example by immunohistochemistry, cells that have phosphorylated-Ser9-GSK3-[3,

- counting cells that have visible staining at magnification x100 (i.e. grade 3 cells), when labelled cells that are visible at magnification x100 represent more than 14,5% of the overall cells within the biological sample, classifying the patient from whom the biological tissue is issued as likely to have ulcerative colitis, and when labelled cells that are visible at magnification x100 represent less than 14,5% of the overall cells within the biological sample, classifying the patient from whom the biological tissue is issued as likely to have Crohn’s disease.

The visual analysis of the tissue biopsy may be performed by an anatomo- cytopathologist, and the score of each tissue biopsy is given by the pathologist after visual inspection of the tissue biopsy according to the method disclosed here above. Alternatively, analysis may be performed through the use a software that has been programmed by data learning of scores previously attributed by a pathologist according to the method disclosed here above. Tissue samples with scores of 0, 1 , 2 and 3 (and 0.5 if used) may be issued from prior tests, issued from samples which have already been set up as a function of their health situation.

A detailed example of the method is present in the example section of the present disclosure.

In an embodiment, the level of phosphorylation of Ser9-GSK3-[3 in the sample is determined on a tissue biopsy, in particular a tissue slice, the level of phosphorylated-Ser9-GSK3-[3 being measured by immunohistochemistry with an anti-phosphorylated-Ser9-GSK3-[3 antibody, and a score is attributed by counting cells that have visible staining at magnification x100 (i.e. grade 3 cells),

- when labelled cells that are visible at magnification x100 represent more than 14,5% of the overall cells within the tissue biopsy, classifying the patient from whom the biological tissue is issued as likely to have ulcerative colitis,

- when labelled cells that are visible at magnification x100 represent less than 14,5% of the overall cells within the tissue biopsy, classifying the patient from whom the tissue biopsy is issued as likely to have Crohn’s disease.

In an alternative embodiment, the level of non-phosphorylated Ser9-GSK3[3 is measured and compared to the overall amount of GSK3[3 to indirectly quantify the proportion of GSK3-[3 with phosphorylated Ser9 within the overall quantity of GSK3-[3 within a biological sample issued from a patient.

The measurement is generally carried out by an immunometric method using specific antibodies, and by expression of the measurements made thereby in quantities by weight or international units using a standard curve. Examples of specific antibodies includes antibodies which specifically binds to GSK3[3 with phosphorylated Ser9, and/or GSK3[3 with non-phosphorylated Ser9, and/or GSK3[3. A value for the measurement of the level of phosphorylation of GSK3[3 may, for example, be expressed as the optical density of labelling of this protein per surface of a biological slice).

The method of the invention may optionally comprise determining one or more clinical factors of said subject, such as selected from sex, age, body mass index.

The method of the invention may optionally comprise measuring one or more biological factor(s) selected from the concentration of the downstream of pSer9GSK3[3 nuclear pcatenin, the level of expression of pcatenin, the presence or absence of pcatenin. Other discriminative downstream molecules related to pSer9GSK3[3 targets, like but not limited to Crebs, Jun-B or RTLR, may be relevant and enhance sensitivity of the method.

As indicated above, it is preferable to administer a different treatment to patients having Crohn’s disease as compared to patients having Ulcerative colitis.

Consequently, the method of the invention may be encompassed within a treatment method, more particularly a method for determining which treatment should be administered to a subject. Said treatment may in particular be a treatment aimed at blocking or slowing down the progress of the disease.

In an aspect of the invention, it is provided a method wherein GSK3[3 with phosphorylated Ser9 is used as a marker in the diagnosis of Crohn’s disease. More particularly, when GSK3[3 with phosphorylated Ser9 is lower than a threshold value, or lower than a particular concentration, within a biological sample previously obtained from a patient, the patient having an IBD is likely to have or develop Crohn’s disease.

In an aspect of the invention, it is provided a method wherein GSK3[3 with nonphosphorylated Ser9 is used as a marker in the diagnosis of Crohn’s disease. More particularly, when GSK3[3 with non-phosphorylated Ser9 is higher than a threshold value, or higher than a particular concentration, within a biological sample previously obtained from a patient, the patient having an IBD is likely to have or develop Crohn’s disease.

It is also provided a method wherein GSK3[3 with phosphorylated Ser9 is used as a marker in the diagnosis of Ulcerative colitis. More particularly, when GSK3[3 with phosphorylated Ser9 is higher than a threshold value, or higher than a particular concentration, within a biological sample previously obtained from a patient, the patient having an IBD is likely to have or develop Ulcerative colitis.

It is also provided a method wherein GSK3[3 with non-phosphorylated Ser9 is used as a marker in the diagnosis of Ulcerative colitis. More particularly, when GSK3[3 with non-phosphorylated Ser9 is lower than a threshold value, or lower than a particular concentration, within a biological sample previously obtained from a patient, the patient having an IBD is likely to have or develop Ulcerative colitis.

The present invention also concerns a method for treating an Inflammatory Bowel Disease, in particular Crohn’s disease, in a patient and comprising:

- providing a biological sample previously obtained from the subject;

- determining a level of phosphorylation of Ser9-GSK3-[3 in the sample; the determination of a level of phosphorylation of Ser9-GSK3-[3 being performed according to any method disclosed herein, in particular the level of phosphorylation of Ser9-GSK3-[3 may be determined by direct quantification of phosphorylated-Ser9-GSK3-[3, or indirectly by quantification of non- phosphorylated-Ser9-GSK3-[3; - attributing to the level of phosphorylation of Ser9-GSK3-[3 a score, and wherein a score of phosphorylation of Ser9-GSK3-[3 is lower than a threshold value indicating that the subject suffers from Crohn’s disease;

- administering to the patient budesonide, glucocorticoids, steroids, azathioprine, and/or inhibitors of TNFa for treating Crohn’s disease.

The present invention also concerns a method for treating an Inflammatory Bowel Disease in a patient identified as having Ulcerative Colitis, in particular Ulcerative Recto-Colitis, comprising:

- providing a biological sample previously obtained from the subject;

- determining a level of phosphorylation of Ser9-GSK3-[3 in the sample; the determination of a level of phosphorylation of Ser9-GSK3-[3 being performed according to any method disclosed herein, in particular the level of phosphorylation of Ser9-GSK3-[3 may be determined by direct quantification of phosphorylated-Ser9-GSK3-[3, or indirectly by quantification of non- phosphorylated-Ser9-GSK3-[3;

- attributing to the level of phosphorylation of Ser9-GSK3-[3 a score, and wherein if a score of phosphorylation of Ser9-GSK3-[3 is higher than a threshold value indicating that the subject suffers from Ulcerative colitis, in particular Ulcerative Recto-colitis,

- administering to the patient mesalazine (5-ASA), azathioprine, 5-ASA and budesonide, 5-ASA and steroids, cyclosporine, and infliximab, and/or 6- mercaptopurine, for treating Ulcerative Colitis.

The present invention also concerns a method for treating an Inflammatory Bowel Disease in a patient and comprising:

- providing a biological sample previously obtained from the subject;

- administering to the patient budesonide, glucocorticoids, steroids, or azathioprine, and/or inhibitors of TNFa for treating Crohn’s disease. and wherein if a score of phosphorylation of Ser9-GSK3-[3 is higher than a threshold value indicating that the subject suffers from Ulcerative colitis, in particular Ulcerative Recto-colitis,

- administering to the patient mesalazine (5-ASA), azathioprine, 5-ASA and budesonide, 5-ASA and steroids, cyclosporine and infliximab, and/or 6- mercaptopurine, for treating Ulcerative Colitis.

Examples illustrating the invention Example 1.

Material and method

Immunohistochemistry (IHC) assay was performed using anti-phospho-GSK3 antibody. Paraffin-embedded tissue blocks, obtained at the time of the initial diagnosis, were retrieved from the archives of the Department of Diagnostic and Theranostic Medicine, Institut Curie. Sections of 3 pm in thickness were cut with a microtome from the paraffin-embedded tissue blocks of normal breast tissue, pre-invasive lesions and IBCs. Tissue sections were deparaffinized and rehydrated through a series of xylene and ethanol washes. Briefly, key figures included: (i) antigen retrieval in 0.1 mol/L citrate buffer, pH 6 (BioCare, Pacheco, CA, USA) in a pressure cooker (4 min); (ii) blocking of endogenous peroxidase activity by immersing sections in 3% hydrogen peroxide in methanol for 15 min a nd subsequently rinsing them in water and PBS; (iii) incubation with primary antibodies against the targeted antigen; (iv) immunodetection with a biotin- conjugated secondary antibody formulation that recognizes rabbit and mouse immunoglobulins, followed by peroxidase-labeled streptavidin and linking with a rabbit biotinylated antibody against mouse immunoglobulin G (DAKO SA) and (v) chromogenic revelation with DAB and counterstaining with Mayer’s hematoxylin. All immunostainings were processed and analyzed using a Leica BOND RX research automated immunostaining device, leading to a semi quantitative histological score (HScore = intensity x frequency) for interpretation (0 = negative staining, 1 = weak staining, 2 = moderate staining and 3 = strong staining). Antibodies specificity was confirmed by a panel of human tissues containing lymphocytes.

As illustrated on figure 1 , a tissue slide issued from a sample obtained from a patient having a Crohn disease has been labelled with an antibody binding to GSK3-[3 with phosphorylated Ser9. A score of 0,5 is obtained (see fig. 1A). A tissue slide issued from a sample obtained from a patient having Ulcerative colitis has been labelled with an antibody binding to GSK3-[3 with phosphorylated Ser9. A score of 3 is obtained (see fig. 1 B).

As illustrated on figure 2, a score of pGSK3 [3 labelling higher or equal to 1 .5 diagnoses a URC with an error of 2.5% on all patients screened that are in fact Crohn (2), and a score strictly smaller than 1 .5 diagnoses a Crohn with an error of 7% on all patients screened that are in fact URC. These results clearly illustrate that the method of the invention leads to an improved diagnosis of the disease in the patient having an inflammatory bowel disease.

Example 2.

Material and method

The biological sample was stained by immunohistochemistry with the anti- Phospho-GSK-3[3 (Ser9) antibody from CellSignal ing #9323, clone 5B3 obtained in rabbit.

Immunohistochemistry (IHC) was performed with a Leica BOND RX machine and all tissue slides obtained from the biological sample were scanned with a x40 objective by an Ultra Fast IntelliSite slide scanner (Philips Healthcare, Philips, The Netherlands). Virtual slide creation was performed using the Image Management System (IMS) (Philips Healthcare, Philips, The Netherlands) with 40-fold magnification (0.25 pm/pixel) in the iSyntax format. Digital image analysis was performed by HALO-AI v3.5 software (Indica Labs, Albuquerque, NM). Tissue annotations were performed manually under the supervision of an experienced anatomo-cytopathologist. This study included 26 cases, 11 of which were known to be Crohn's Disease (CD) and 15 of which were known to be Hemorrhagic Rectocolitis (UC). Immunohistochemistry (IHC) protocol for FFPE samples

The slides were labelled according to the following protocol:

1 - the slides were let to dry at Room Temperature (RT) overnight

2- the slides were then melted in paraffin (1 H, 58°C)

3- Paraffin was then removed using

- 3 Xylene baths (3x5min)

- 2 ethanol bath 100% (2x3 min)

- 1 ethanol bath 90% (3 min)

- 1 ethanol bath 70% (3 min)

4- the resulting was put in distilled water

5- Decrosslinking slides was performed with EDTA (pH9) (90°C, 20min), and the obtained sample was let cool in buffer for 20m.

6- Endogenous peroxidase was used to block the sample (peroxidase block or H2O2 3%, RT, 5m in)

7- a washing step was then performed in TBST 1X (1x3min)

8- a specific protein block (protein block, RT, 10min) (remove by gently flicking the slides, no wash) was recovered

9- a anti-Phospho-GSK-3[3 (Ser9) antibody (anti-P-GSK3) is added on the slides (1/50, 4°C, Overnight)

10- Wash in TBST 1X (2x5min)

11 - Secondary Antibody conjugated with HRP (RT, 30min) is added on the slides

12- Wash in TBST 1X (2x5min)

13- Chromogenic revelation, DAB (3,3 -Diaminobenzidine) (RT, 5min)

14- Wash in TBST 1X (2x5min)

15- Chromogenic revelation, DAB (3,3 -Diaminobenzidine) (RT, 5min)

16- Wash in TBST 1X (2x5min)

17- Dehydrate and Mount under coverslip.

The 5B3 antibody detects endogenous levels of GSK-3[3 only when its serine 9 is phosphorylated. The antibody may show weak cross-reactivity with the phosphorylated form of GSK-3a due to high sequence homology. First, a segmentation network adapted to nuclei segmentation (Nuclei Seg (HALO Al)) was used. Then the Multiplex IHC v3.2 algorithm was used to measure the labelling and attribute a score in the previously detected cells.

As illustrated on Figure 3, a digital image at magnification x20 is provided (Fig. 3A). Then, contrast has been enhanced by changing the display parameters (Fig. 3B). The segmentation network adapted to nuclei segmentation is then performed (Fig. 3C). Labelling intensity is thereafter measured in nuclei and cytoplasm of cells (Fig. 3D). If cell with phosphorylated- Ser9-GSK3-[3 is visible at magnification x100, this cell is considered to be labelled.

Results obtained from the method disclosed above are illustrated on Figure 4. From the results, a threshold allowing discrimination between UC patients and CD patients has been observed when 14,5% of grade 3 cells (i.e. stained cells visible at magnification x100) are present within the sample. When patients have less than 14.5% of grade 3 cells, they are diagnosed as having Crohn disease. When patients have more than 14.5% of grade 3 cells, they are diagnosed as having ulcerative colitis.

The present invention has been described in terms of specific embodiments, which are illustrative of the invention and not to be construed as limiting. More generally, it will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and/or described hereinabove.

Reference numerals in the claims do not limit their protective scope.

Use of the verbs "to comprise", "to include", "to be composed of", or any other variant, as well as their respective conjugations, does not exclude the presence of elements other than those stated.

Use of the article "a", "an" or "the" preceding an element does not exclude the presence of a plurality of such elements.

Claims

1 . An in vitro or ex vivo method for determining if a subject suffering from an inflammatory bowel disease (IBD) suffers from a Crohn’s disease or an ulcerative colitis (UC), wherein the method comprises the steps of:

- providing a biological sample previously obtained from the subject;

- determining a level of phosphorylation of Ser9-GSK3-[3 in the sample;

2. The method according to claim 1 , wherein the score is dependent from the optical density of labelled GSK3-[3 with phosphorylated Ser9 or non- phosphorylated-Ser9 in the sample as compared to reference optical densities of labelled GSK3-[3 with phosphorylated Ser9 or non- phosphorylated-Ser9 issued from a reference.

3. The method according to claim 1 , wherein the level of phosphorylation of- Ser9-GSK3-[3 is determined by quantifying the amount of phosphorylated- Ser9-GSK3-[3 in the sample, the expression level of GSK3-[3, and determining the proportion of phosphorylated-Ser9-GSK3-[3 within expressed GSK3-[3, or wherein the level of phosphorylation of-Ser9-GSK3-[3 is determined by quantifying the amount of non-phosphorylated-Ser9- GSK3-[3 in the sample, the expression level of GSK3-[3, and determining the proportion of phosphorylated-Ser9-GSK3-[3 within expressed GSK3-[3.

4. The method of any one of claims 1 to 3, wherein the threshold value is issued from a reference database comprising the scores corresponding to the level of phosphorylation of Ser9-GSK3-[3 of patients having Crohn’s disease and/or patients having Ulcerative colitis. The method of any one of claims 1 to 4, wherein a value comprised between 0 and 3 is attributed to the level of phosphorylation of Ser9-GSK3- P, and a value of phosphorylation of Ser9-GSK3-p lower than or equal to 1 .5, more particularly lower than or equal to 1 , is indicative that the subject suffers from Crohn’s disease. The method of any one of claims 1 to 5, wherein a value comprised between 0 and 3 is attributed to the level of phosphorylation of Ser9-GSK3- P, and a value of phosphorylation of Ser9-GSK3-p higher than or equal to 1 .5, in particular higher than or equal to 2, is indicative that the subject suffers from Ulcerative Colitis, in particular Ulcerative Recto-colitis. The method of any one of claims 1 to 6, wherein the level of phosphorylation of Ser9-GSK3-p in the sample is determined on a tissue biopsy, in particular a tissue slice, the level of phosphorylated-Ser9-GSK3-p being measured by immunohistochemistry with an anti-phosphorylated- Ser9-GSK3-p antibody, and the score is attributed by visual analysis of the tissue biopsy after the immunochemistry reaction, and wherein:

- if a majority (absolute or relative) of cells are visibly stained magnification x400, a score of “1” is attributed;

- if a majority (absolute or relative) of cells are visibly stained at magnification x100, a score of “3” is attributed. The method according to any one of claims 1 to 6, wherein the level of phosphorylation of Ser9-GSK3-p in the sample is determined on a tissue biopsy, in particular a tissue slice, the level of phosphorylated-Ser9-GSK3-p being measured by immunohistochemistry with an anti-phosphorylated- Ser9-GSK3-p antibody, and a score is attributed by counting cells that have visible staining at magnification x100 (i.e. grade 3 cells), - when labelled cells that are visible at magnification x100 represent more than 14,5% of the overall cells within the tissue biopsy, classifying the patient from whom the biological tissue is issued as likely to have ulcerative colitis,

9. The method according to any one of claims 1 to 8, wherein the biological sample is a tissue, blood, urine, or whole cell lysate, in particular is a tissue sample from intestinal tract, more particularly is a tissue sample from colon, rectum, ileum or duodenum, preferably obtained by biopsy.

10. The method according to any one of claims 1 to 9, wherein the determination of the level of phosphorylation of Ser9-GSK3-[3 is performed by Western Blot, ELISA, protein microarray, or immunohistochemistry, in particular with an antibody specifically binding to GSK3-[3 with phosphorylated Ser9.

11 . The method according to claim 10, wherein the immunohistology is followed by optical density analysis of phosphorylated-Ser9-GSK3-[3 within the sample.

12. A kit for performing the method of any one of claims 1 to 11 , which comprises reagents for determining the level of phosphorylation of Ser9- GSK3-[3 in a sample previously obtained from a subject.