METHODS FOR IMPROVING INFLAMMATORY BOWEL DISEASE DIAGNOSIS

• US 20130225439A1
• Filed: 03/15/2013
• Published: 08/29/2013
• Est. Priority Date: 10/21/2011
• Status: Active Grant

• Current Assignee: Prometheus Laboratories Incorporated (Merck & Co., Inc.)
• Original Assignee: Nestec SA (Nestle SA)
• Inventors: Princen, Fred, Croner, Lisa J., Singh, Sharat, Lockton, Steven, Fletcher, Frederick A., Stockfisch, Thomas
• Granted Patent: US 8,715,943 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/4737 C-reactive protein
• CPC Class Codes: G01N 2333/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: G01N 2333/70525 ICAM molecules, e.g. CD50, CD54, CD102
• CPC Class Codes: G01N 2333/70542 CD106
• CPC Class Codes: G01N 2333/775 Apolipopeptides
• CPC Class Codes: G01N 2800/065 Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
• CPC Class Codes: G01N 2800/60 Complex ways of combining multiple protein biomarkers for diagnosis
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/40 Population genetics; Linkage disequilibrium
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16H 50/30 for calculating health indices; for individual health risk assessment
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The present invention provides methods and systems to predict and diagnose inflammatory bowel disease (IBD) and subtypes such as ulcerative colitis (UC) and Crohn'"'"'s disease (CD) by detecting the presence, absence, level, and/or genotype of one or more sero-genetic-inflammation markers. Advantageously, with the present invention, it is possible to provide a diagnosis of IBD versus non-IBD, to rule out IBD that is inconclusive for CD and UC, and to differentiate between CD and UC with increased accuracy.

Claims

1. A method for diagnosing inflammatory bowel disease (IBD) and/or a clinical subtype thereof in an individual, said method comprising:

(a) analyzing a sample obtained from said individual to determine the presence or level or genotype of at least each the following markers to obtain a marker profile;

(i) the presence or level of each of the serological markers ASCA-A, ASCA-G, ANCA, pANCA, anti-OmpC antibody, anti-CBir1 antibody, anti-FlaX antibody, and anti-A4-Fla2 antibody;

(ii) the presence or level of each of the inflammation markers VEGF, ICAM, VCAM, SAA, and CRP; and

(iii) the genotype of each of the genetic markers ATG16L1, ECM1, NKX2-3, and STAT3;

(b) applying a first random forest statistical analysis to said marker profile to obtain a decision whether said sample is an IBD sample or a non-IBD sample;

(c) if said sample is an IBD sample, then applying a decision tree to said IBD sample to determine if said IBD sample is or is not an inconclusive sample; and

(d) if said IBD sample is not an inconclusive sample, then applying a second random forest statistical analysis to said IBD sample to determine a clinical subtype of IBD.

2. The method of claim 1, wherein the presence or level of each of said serological markers or inflammation markers is independently detected with a hybridization assay, amplification-based assay, immunoassay, or immunohistochemical assay.

3. The method of claim 1, wherein the genotype of each of said genetic markers is independently detected by genotyping for the presence or absence of a single nucleotide polymorphism (SNP) in each of said genetic markers.

4. The method of claim 3, wherein said SNP is selected from the group consisting of ATG16L1 SNP rs2241880, ECM1 SNP rs3737240, NKX2-3 SNP rs10883365, STAT3 SNP rs744166, and a combination thereof.

5. The method of claim 1, wherein the presence or level of pANCA is used to determine the value of pANCA2.

6. The method of claim 5, wherein said IBD sample is an inconclusive sample when said IBD sample has an ANCA level≧

Q3, is pANCA2 positive, and has anti-CBir1 antibody or anti-Fla2 antibody or anti-FlaX antibody level≧

Q3.

7. The method of claim 5, wherein said IBD sample is an inconclusive sample when said IBD sample is pANCA2 positive and expresses two out of three markers selected from anti-CBir1 antibody, anti-Fla2 antibody, and anti-FlaX antibody≧

Q3.

8. The method of claim 1, wherein the clinical subtype of IBD is Crohn'"'"'s disease (CD) or ulcerative colitis (UC).

9. The method of claim 1, wherein said second random forest statistical analysis is based upon (i) the presence or level of ASCA-A, ASCA-G, ANCA, pANCA, anti-OmpC antibody, anti-CBir1 antibody, anti-FlaX antibody, anti-A4-Fla2 antibody, and VEGF in said sample and (ii) the genotype of ECM1 and STAT3 in said sample.

10. The method of claim 1, wherein said sample is selected from the group consisting of serum, plasma, whole blood, and stool.

11. The method of claim 5, wherein when pANCA values are 0 or +1, the pANCA2 value is negative.

12. The method of claim 5, wherein when pANCA values are +2, +3 or +4, the pANCA2 value is positive.

13. The method of claim 11, wherein in said pANCA2 negative sample, further comprising:

(a) determining whether the pANCA2 negative sample is directly predictive of Crohn'"'"'s disease by measuring a serum marker panel, wherein the serum marker panel is a member of the group consisting of ASCA-IgA, ASCA-IgG, and OmpC-IgA and comparing each of the serum markers of the serum marker panel to a cutoff value to determine if the sample is consistent with Crohn'"'"'s disease;

or

(b) determining whether the pANCA2 negative sample is consistent with Crohn'"'"'s disease by measuring a CD count panel, wherein the CD count panel is a member of the group consisting of ASCA-IgA, ASCA-IgG, OmpC-IgA, CBir1-IgG, A4-Fla2-IgG and FlaX-IgG to form a CD count value, wherein when the CD count value is greater than or equal to 2, the sample is consistent with Crohn'"'"'s disease;

or

(c) determining whether the pANCA2 negative sample having a pANCA value of zero is consistent or inconsistent with IBD by measuring ANCA ELISA and comparing the ANCA ELISA value to a reference value and designating the sample as being consistent or inconsistent with IBD based on the ANCA ELISA value;

or

(d) determining whether the pANCA2 negative sample having a pANCA value of zero is consistent with IBD or is inconclusive of IBD by measuring ANCA ELISA and comparing the ANCA ELISA value and considering the CD count value to determine whether the sample is consistent or inconsistent with IBD or is consistent with IBD and inconclusive for Crohn'"'"'s disease or ulcerative colitis;

or

(e) determining whether the pANCA2 negative sample having a pANCA value of one is consistent or inconsistent with IBD by measuring ANCA ELISA and comparing the ANCA ELISA value to a cut-off value to determine whether the sample is consistent with IBD or is inconsistent with IBD.

14. The method of claim 13, wherein in part (a) above, if the value for ASCA-IgA is greater than or equal to 69 EU/mL, then designating the sample as being consistent with Crohn'"'"'s disease.

15. The method of claim 13, wherein in part (a) above, if the value of ASCA-IgG is greater than or equal to 40 EU/mL, then designating the sample as being consistent with Crohn'"'"'s disease.

16. The method of claim 13, wherein in part (a) above, if the value for OmpC-IgA is greater than or equal to 60 EU/mL, then designating the sample as being consistent with Crohn'"'"'s disease.

17. The method of claim 13, wherein in part (b) above, further comprising assigning the CD count value by determining if:

i. ASCA-IgA is greater than or equal to 8.5 EU/mL, then adding +1 to the CD count value;

ii. ASCA-IgG is greater than or equal to 17.8 EU/mL, then adding +1 to the CD count value;

iii. OmpC-IgA is greater than or equal to 10.9 EU/mL, then adding +1 to the CD count value;

iv. 2 or more flagellin markers are above their respective reference ranges, then adding +1 to the CD count value;

v. any flagellin is greater than or equal to 100 EU/mL, then adding +1 to the CD count value;

and if the total CD count value is greater than or equal to 2, then designating that the sample is consistent with Crohn'"'"'s disease.

18. The method of claim 17, wherein the reference value for CBir1-IgG is greater than or equal to 78.4 EU/mL.

19. The method of claim 17, wherein the reference value for A4-Fla2-IgG is greater than or equal to 44.8 EU/mL.

20. The method of claim 17, wherein the reference value for FlaX-IgG is greater than or equal to 33.4 EU/mL.

21. The method of claim 13, wherein in part (c) above, if pANCA is zero (not detected), further comprising:

i. designating the sample as being inconsistent with IBD if ANCA ELISA is less than 20 EU/mL;

or

ii. designating the sample as being consistent with ulcerative colitis if ANCA ELISA is greater than 27.4 EU/mL;

or

iii. designating the sample as being inconsistent with IBD if ANCA ELISA is greater than or equal to 20 and less than or equal to 27.4 EU/mL and the CD count value is zero;

or

iv. designating the sample as being consistent with IBD but inconclusive for Crohn'"'"'s disease or ulcerative colitis if ANCA ELISA is greater than or equal to 20 and less than or equal to 27.4 EU/mL and the CD count value is one.

22. The method of claim 13, wherein when pANCA is one, further comprising:

i. designating the sample as being inconsistent with IBD if ANCA ELISA is less than 13.7 EU/mL;

or

ii. designating that the sample as being consistent with ulcerative colitis if ANCA ELISA is greater than or equal to 13.7 EU/mL.