Serological Markers in IBD: Are They Ready for Prime Time

1. Serological Markers in IBD: Are They Ready for Prime Time? Raymond Cross, M.D. Assistant Professor of Medicine Division of Gastroenterology and Hepatology Director, IBD Program Acting Chief, VA GI Service

2. What are the Serological Markers in IBD? pANCA (perinuclear staining pattern) Loss of perinuclear pattern after DNAase Differentiate from the �other pANCAs� Antibody against myeloperoxidase Antibody against cathepsin G, elastase, lysozyme, and lactoferrin ASCA (anti-Saccharomyces cerevisiae) Both IgG and IgA Recognize mannose in the cell wall mannan of Saccharomyces cerevisiae Loss of perinuclear pattern after DNAase digestionLoss of perinuclear pattern after DNAase digestion

3. What are the Serological Markers in IBD-2? Omp C IgG only Recognize outer membrane porin C protein in E. coli I2 IgA only Recognizes novel homologue of bacterial transcription-factor families from a Pseudomonas fluorescens-associated sequence Cbir 1 flagellin IgG BACKGROUND & AIMS: Antibody responses to certain microbial antigens define heterogeneous groups of Crohn's patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by investigations in the C3H/HeJBir mouse model, has been identified as a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn's disease (CD). The aim of this study was to evaluate serum response to CBir1 flagellin in CD patients and to compare this response to responses defined previously to oligomannan (anti-Saccharomyces cerevisiae antibody), I2, OmpC, and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1-associated phenotypes. METHODS: A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were assessed for clinical phenotype associations. RESULTS: The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. CONCLUSIONS: Serum responses to CBir1 independently identify a unique subset of patients with complicated CD. This bacterial antigen was identified in a murine model and has a similar pattern of aberrant reactivity in a subset of CD patientBACKGROUND & AIMS: Antibody responses to certain microbial antigens define heterogeneous groups of Crohn's patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by investigations in the C3H/HeJBir mouse model, has been identified as a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn's disease (CD). The aim of this study was to evaluate serum response to CBir1 flagellin in CD patients and to compare this response to responses defined previously to oligomannan (anti-Saccharomyces cerevisiae antibody), I2, OmpC, and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1-associated phenotypes. METHODS: A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were assessed for clinical phenotype associations. RESULTS: The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. CONCLUSIONS: Serum responses to CBir1 independently identify a unique subset of patients with complicated CD. This bacterial antigen was identified in a murine model and has a similar pattern of aberrant reactivity in a subset of CD patient

4. Why Use Serological Markers in Clinical Practice? Differentiate IBD from functional bowel disorders Accurately diagnose Crohn�s or UC in a patient with: Severe colitis Indeterminate colitis Predict disease course or complications in IBD CD phenotype Severity of disease Risk of pouchitis

5. Frequency of pANCA in UC Patients and Controls in a Referral Center Previous studies from this laboratory showed that serum anti-neutrophil cytoplasmic antibodies, distinct from those associated with active Wegener's granulomatosis, are present in the majority of patients with ulcerative colitis. In this study, the specificity of anti-neutrophil cytoplasmic antibodies for ulcerative colitis as compared with other colitides and diarrheal illness was determined. In a blinded study, test samples of serum were screened for anti-neutrophil immunoglobulin G in a fixed neutrophil enzyme-linked immunosorbent assay. Levels of neutrophil binding by immunoglobulin G and titers of anti-neutrophil immunoglobulin G in sera from patients with ulcerative colitis and patients with ulcerative colitis post colectomy were each significantly greater than the levels and titers for normal controls and patients with a variety of other colitides and diarrheal illnesses. Levels of neutrophil binding for colonic Crohn's disease, bacterial/amoebic colitis, the irritable bowel syndrome with diarrhea, and other miscellaneous diarrheal illnesses were not significantly different than the levels for normal controls. Although the levels of binding for collagenous colitis were significantly less than the levels for ulcerative colitis, they were significantly greater than the levels for normal controls. Patterns of neutrophil binding by serum samples that were positive in the enzyme-linked immunosorbent assay were determined by indirect immunofluorescence. Perinuclear staining was the predominant pattern shown by sera from patients with ulcerative colitis. The combination of a positive value in the enzyme-linked immunosorbent assay and a perinuclear immunofluorescence pattern was 60% sensitive and 94% specific for ulcerative colitis. It is concluded that anti-neutrophil cytoplasmic antibodies in ulcerative colitis are not simply an epiphenomenon related to inflammation of the colon. Identification of the antigen(s) to which these autoantibodies are directed may facilitate understanding of the underlying immune response and may allow development of an assay that is more sensitive and specific for ulcerative colitis.Previous studies from this laboratory showed that serum anti-neutrophil cytoplasmic antibodies, distinct from those associated with active Wegener's granulomatosis, are present in the majority of patients with ulcerative colitis. In this study, the specificity of anti-neutrophil cytoplasmic antibodies for ulcerative colitis as compared with other colitides and diarrheal illness was determined. In a blinded study, test samples of serum were screened for anti-neutrophil immunoglobulin G in a fixed neutrophil enzyme-linked immunosorbent assay. Levels of neutrophil binding by immunoglobulin G and titers of anti-neutrophil immunoglobulin G in sera from patients with ulcerative colitis and patients with ulcerative colitis post colectomy were each significantly greater than the levels and titers for normal controls and patients with a variety of other colitides and diarrheal illnesses. Levels of neutrophil binding for colonic Crohn's disease, bacterial/amoebic colitis, the irritable bowel syndrome with diarrhea, and other miscellaneous diarrheal illnesses were not significantly different than the levels for normal controls. Although the levels of binding for collagenous colitis were significantly less than the levels for ulcerative colitis, they were significantly greater than the levels for normal controls. Patterns of neutrophil binding by serum samples that were positive in the enzyme-linked immunosorbent assay were determined by indirect immunofluorescence. Perinuclear staining was the predominant pattern shown by sera from patients with ulcerative colitis. The combination of a positive value in the enzyme-linked immunosorbent assay and a perinuclear immunofluorescence pattern was 60% sensitive and 94% specific for ulcerative colitis. It is concluded that anti-neutrophil cytoplasmic antibodies in ulcerative colitis are not simply an epiphenomenon related to inflammation of the colon. Identification of the antigen(s) to which these autoantibodies are directed may facilitate understanding of the underlying immune response and may allow development of an assay that is more sensitive and specific for ulcerative colitis.

6. Prevalence of ASCA in Patients with CD and UC and Controls in the Different Assays BACKGROUND & AIMS: Anti-Saccharomyces cerevisiae antibody (ASCA) is a serologic marker associated with Crohn's disease (CD). Although there is still discussion on its clinical value, several companies each promote their own ASCA assay to be used in the gastroenterologist's practice at considerable expense. The aim of this study was to determine whether different ASCA assays agree sufficiently well for the results to be used interchangeably. METHODS: Blood obtained from a large cohort of IBD patients with inflammatory bowel disease (IBD; 100 with CD, 100 with ulcerative colitis [UC]) and 178 controls (100 healthy blood donors and 78 patients with non-IBD diarrheal illnesses) was studied with 4 different ASCA assays. Sensitivity, specificity, and positive predictive value were compared. Agreement between assays was evaluated. RESULTS: Sensitivity of ASCA for CD ranged between 41% and 76%. Sensitivity was inversely related to specificity and positive predictive value. Results correlated well overall (range = 0.54-0.90) and the different ROC curves showed good agreement. When recalculated cutoff points were used, interchangeability increased. However, large differences were seen when absolute values were compared. CONCLUSIONS: A large range in sensitivities and specificities of ASCA for CD is seen with different ASCA assays, mainly as a consequence of the cutoff value chosen for each individual assay. Although agreement between and within assays is good, caution is important when absolute values are used. Standardization of ASCA measurements is greatly needed.BACKGROUND & AIMS: Anti-Saccharomyces cerevisiae antibody (ASCA) is a serologic marker associated with Crohn's disease (CD). Although there is still discussion on its clinical value, several companies each promote their own ASCA assay to be used in the gastroenterologist's practice at considerable expense. The aim of this study was to determine whether different ASCA assays agree sufficiently well for the results to be used interchangeably. METHODS: Blood obtained from a large cohort of IBD patients with inflammatory bowel disease (IBD; 100 with CD, 100 with ulcerative colitis [UC]) and 178 controls (100 healthy blood donors and 78 patients with non-IBD diarrheal illnesses) was studied with 4 different ASCA assays. Sensitivity, specificity, and positive predictive value were compared. Agreement between assays was evaluated. RESULTS: Sensitivity of ASCA for CD ranged between 41% and 76%. Sensitivity was inversely related to specificity and positive predictive value. Results correlated well overall (range = 0.54-0.90) and the different ROC curves showed good agreement. When recalculated cutoff points were used, interchangeability increased. However, large differences were seen when absolute values were compared. CONCLUSIONS: A large range in sensitivities and specificities of ASCA for CD is seen with different ASCA assays, mainly as a consequence of the cutoff value chosen for each individual assay. Although agreement between and within assays is good, caution is important when absolute values are used. Standardization of ASCA measurements is greatly needed.

7. Sensitivity, Specificity, and Positive and Negative Predictive Value of ASCA BACKGROUND & AIMS: Anti-Saccharomyces cerevisiae antibody (ASCA) is a serologic marker associated with Crohn's disease (CD). Although there is still discussion on its clinical value, several companies each promote their own ASCA assay to be used in the gastroenterologist's practice at considerable expense. The aim of this study was to determine whether different ASCA assays agree sufficiently well for the results to be used interchangeably. METHODS: Blood obtained from a large cohort of IBD patients with inflammatory bowel disease (IBD; 100 with CD, 100 with ulcerative colitis [UC]) and 178 controls (100 healthy blood donors and 78 patients with non-IBD diarrheal illnesses) was studied with 4 different ASCA assays. Sensitivity, specificity, and positive predictive value were compared. Agreement between assays was evaluated. RESULTS: Sensitivity of ASCA for CD ranged between 41% and 76%. Sensitivity was inversely related to specificity and positive predictive value. Results correlated well overall (range = 0.54-0.90) and the different ROC curves showed good agreement. When recalculated cutoff points were used, interchangeability increased. However, large differences were seen when absolute values were compared. CONCLUSIONS: A large range in sensitivities and specificities of ASCA for CD is seen with different ASCA assays, mainly as a consequence of the cutoff value chosen for each individual assay. Although agreement between and within assays is good, caution is important when absolute values are used. Standardization of ASCA measurements is greatly neededBACKGROUND & AIMS: Anti-Saccharomyces cerevisiae antibody (ASCA) is a serologic marker associated with Crohn's disease (CD). Although there is still discussion on its clinical value, several companies each promote their own ASCA assay to be used in the gastroenterologist's practice at considerable expense. The aim of this study was to determine whether different ASCA assays agree sufficiently well for the results to be used interchangeably. METHODS: Blood obtained from a large cohort of IBD patients with inflammatory bowel disease (IBD; 100 with CD, 100 with ulcerative colitis [UC]) and 178 controls (100 healthy blood donors and 78 patients with non-IBD diarrheal illnesses) was studied with 4 different ASCA assays. Sensitivity, specificity, and positive predictive value were compared. Agreement between assays was evaluated. RESULTS: Sensitivity of ASCA for CD ranged between 41% and 76%. Sensitivity was inversely related to specificity and positive predictive value. Results correlated well overall (range = 0.54-0.90) and the different ROC curves showed good agreement. When recalculated cutoff points were used, interchangeability increased. However, large differences were seen when absolute values were compared. CONCLUSIONS: A large range in sensitivities and specificities of ASCA for CD is seen with different ASCA assays, mainly as a consequence of the cutoff value chosen for each individual assay. Although agreement between and within assays is good, caution is important when absolute values are used. Standardization of ASCA measurements is greatly needed

8. Accuracy of Serological Markers in Differentiating IBD from Controls OBJECTIVES: Correct diagnosis of inflammatory bowel disease (IBD), especially the differentiation between Crohn's disease (CD) and ulcerative colitis (UC), is highly important toward treatment and prognosis. Serological markers are noninvasive diagnostic tools that could be of value in differentiating CD from UC, in cases of indeterminate colitis, and in the identification of subgroups in IBD. The aim of this study was to evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) for IBD. METHODS: ASCA and pANCA were studied in a large cohort of consecutive IBD patients (n = 582: 407 CD, 147 UC, and 28 indeterminate colitis), patients with non-IBD diarrheal illnesses (n = 74), and healthy controls (n = 157). An indirect immunofluorescence technique and a standardized ELISA were performed for detection of pANCA and ASCA, respectively. RESULTS: Prevalence of ASCA and pANCA was high in CD patients (59.7%) and UC (49.7%) patients, respectively. Positivity for both markers was significantly lower in healthy and non-IBD controls. Accuracy data (sensitivity, specificity, PPV, and NPV, respectively) for differentiating IBD from controls are as follows: ASCA+: 60% (243/407), 91% (345/378), 88% (243/276), and 68% (345/509); pANCA+: 50% (73/147), 95% (605/638), 69% (73/106), and 89% (605/679); ASCA+/pANCA-: 56% (229/407), 94% (355/378), 91% (229/252), and 67% (355/533); and pANCA+/ASCA-: 44% (65/147), 97% (620/638), 78% (65/83), and 88% (620/702). CONCLUSIONS: Specificity of serological markers for IBD is high, but low sensitivity makes them less useful as diagnostic tests. The combination of tests is probably more powerful, although, clinical subgroups still need to be defined. The usefulness of these markers in indeterminate colitis needs to be studied prospectively.OBJECTIVES: Correct diagnosis of inflammatory bowel disease (IBD), especially the differentiation between Crohn's disease (CD) and ulcerative colitis (UC), is highly important toward treatment and prognosis. Serological markers are noninvasive diagnostic tools that could be of value in differentiating CD from UC, in cases of indeterminate colitis, and in the identification of subgroups in IBD. The aim of this study was to evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) for IBD. METHODS: ASCA and pANCA were studied in a large cohort of consecutive IBD patients (n = 582: 407 CD, 147 UC, and 28 indeterminate colitis), patients with non-IBD diarrheal illnesses (n = 74), and healthy controls (n = 157). An indirect immunofluorescence technique and a standardized ELISA were performed for detection of pANCA and ASCA, respectively. RESULTS: Prevalence of ASCA and pANCA was high in CD patients (59.7%) and UC (49.7%) patients, respectively. Positivity for both markers was significantly lower in healthy and non-IBD controls. Accuracy data (sensitivity, specificity, PPV, and NPV, respectively) for differentiating IBD from controls are as follows: ASCA+: 60% (243/407), 91% (345/378), 88% (243/276), and 68% (345/509); pANCA+: 50% (73/147), 95% (605/638), 69% (73/106), and 89% (605/679); ASCA+/pANCA-: 56% (229/407), 94% (355/378), 91% (229/252), and 67% (355/533); and pANCA+/ASCA-: 44% (65/147), 97% (620/638), 78% (65/83), and 88% (620/702). CONCLUSIONS: Specificity of serological markers for IBD is high, but low sensitivity makes them less useful as diagnostic tests. The combination of tests is probably more powerful, although, clinical subgroups still need to be defined. The usefulness of these markers in indeterminate colitis needs to be studied prospectively.

9. Utility of Serodiagnostics in Pediatric IBD: Use of a Two-Step Assay

10. IgA Antibody to I2 in Patients with CD or UC and Controls BACKGROUND & AIMS: Enteric microorganisms are implicated in the pathogenesis of Crohn's disease (CD), but no clear bacterial or viral species has been identified. In this study, representational difference analysis (RDA) was used to isolate DNA segments preferentially abundant in lamina propria mononuclear cells of lesional mucosa vs. adjacent uninvolved mucosa. METHODS: Two RDA-derived microbial sequences were isolated (I1 and I2) and identified as novel homologues of the ptxR and tetR bacterial transcription-factor families. RESULTS: Quantitative competitive polymerase chain reaction of paraffin-embedded intestinal specimens from 212 patients showed that I2 DNA was present in many CD colonic lesions (43%), but was infrequent in other colonic specimens (9% of ulcerative colitis lesions and 5% of non-inflammatory bowel disease diseases; P<0.0001). I2 was prevalent in ileal specimens, regardless of disease status (43%-54%). Enzyme-linked immunosorbent assay analysis of 150 individuals with an I2 glutathione-S-transferase fusion protein showed frequent immunoglobulin A seroreactivity in CD (54% of patients), but infrequent seroreactivity in patients with ulcerative colitis, other inflammatory enteric diseases, or normals (10%, 19%, and 4%, respectively; P<0.001 to 0.00001). CONCLUSIONS: These findings relate CD to a novel lesion-localized and immunologically associated bacterial sequence, suggesting that the microorganism expressing the I2 gene product may be related to CD pathogenesisBACKGROUND & AIMS: Enteric microorganisms are implicated in the pathogenesis of Crohn's disease (CD), but no clear bacterial or viral species has been identified. In this study, representational difference analysis (RDA) was used to isolate DNA segments preferentially abundant in lamina propria mononuclear cells of lesional mucosa vs. adjacent uninvolved mucosa. METHODS: Two RDA-derived microbial sequences were isolated (I1 and I2) and identified as novel homologues of the ptxR and tetR bacterial transcription-factor families. RESULTS: Quantitative competitive polymerase chain reaction of paraffin-embedded intestinal specimens from 212 patients showed that I2 DNA was present in many CD colonic lesions (43%), but was infrequent in other colonic specimens (9% of ulcerative colitis lesions and 5% of non-inflammatory bowel disease diseases; P<0.0001). I2 was prevalent in ileal specimens, regardless of disease status (43%-54%). Enzyme-linked immunosorbent assay analysis of 150 individuals with an I2 glutathione-S-transferase fusion protein showed frequent immunoglobulin A seroreactivity in CD (54% of patients), but infrequent seroreactivity in patients with ulcerative colitis, other inflammatory enteric diseases, or normals (10%, 19%, and 4%, respectively; P<0.001 to 0.00001). CONCLUSIONS: These findings relate CD to a novel lesion-localized and immunologically associated bacterial sequence, suggesting that the microorganism expressing the I2 gene product may be related to CD pathogenesis

11. Can Serological Markers Differentiate IBD from Non-IBD? pANCA and ASCA are specific for and have high positive predictive value for UC and CD respectively Rule in disease The low sensitivity and negative predictive value preclude them as a screening test Cannot rule out disease Potential application in pediatric disease to avoid invasive work up

12. Why Use Serological Markers in Clinical Practice? Differentiate IBD from functional bowel disorders Accurately diagnose Crohn�s or UC in a patient with: Severe colitis Indeterminate colitis Predict disease course or complications in IBD CD phenotype Severity of disease Risk of pouchitis

13. Criteria for Indeterminate Colitis No evidence of small bowel involvement, fistula, or perianal disease Absence of diagnostic criteria for CD or UC by microscopy

14. Presentation of Ulcerative Colitis Classic presentation Bloody diarrhea! Never or former smoker Tenesmus (dry heaves of the rectum) Red Flags Active smoker Perianal disease Abdominal mass on examination

15. DISTINGUISHING FEATURES OF CROHN�S DISEASE 105. DISTINGUISHING FEATURES OF CROHN�S DISEASE In most cases, Crohn�s disease of the colon is readily distinguished from ulcerative colitis by clinical, radiologic, endoscopic, and pathologic features. The great majority of cases (70-85%) of Crohn�s colitis have small bowel involvement and relative sparing of the rectum. Although most patients have some gross rectal bleeding, a substantial minority (25-30%) note no blood; this absence of bleeding, by contrast, is almost never reported in ulcerative colitis. Except for small rectovaginal fistulae, which are sometimes seen in ulcerative colitis, major perianal lesions in IBD are a feature exclusively of Crohn�s disease. Other characteristics of the lesions of Crohn�s disease, as distinguished from ulcerative colitis, are locality in the setting of relatively normal intervening mucosa, segmental rather than diffuse or continuous distribution, and asymmetric involvement of different parts of the wall within given segments of bowel. Fistulization into surrounding tissues and organs occurs only in Crohn�s disease as opposed to ulcerative colitis. Granulomas or �microgranulomas� may be found on careful study of Crohn�s disease rectal biopsies in 25-30%, and in surgical specimens in up to 50-75% of cases; they are not necessary to establish a diagnosis of Crohn�s disease, but when present they are virtually pathognomonic. � Lockhart-Mummery HE, Morson BC. Crohn�s disease (regional enteritis) of the large intestine and its distinction from ulcerative colitis. Gut 1960;1:87-105. � Wolf BS, Marshak RH. Granulomatous colitis (Crohn�s disease of the colon): Roentgen features. Am J Roentgenol 1962;88:662-670. � Lindner AE, Marshak RH, Wolf BS et al. Granulomatous colitis�a clinical study. N Engl J Med 1963;269:379-385. � Marshak RH, LindnerAE, Janowitz HD. Granulomatous ileecolitis. Gut 1966;7:258-264. � Farmer RG, Hawk WA, Turnbull RB. Regional enteritis of the colon: Clinical and pathological comparison with ulcerative colitis. Am J Dig Dis 1968;13:501-14. � Lennard-Jones JE, Lockhart-Mummery HE, Morson BC. Clinical and pathological differentiation of Crohn�s disease and proctocolitis. Gastroenterology 1968;64:1162-1170. � Cook MG, Dixon MF. An analysis of the reliability of detection and diagnostic value of various patholegic features in Crohn�s disease and ulcerative colitis. Gut 1973;14:255-62. � Waye JD. The role of colonoscopy in the differential diagnosis of inflammatory bowel disease. Gastrointest Endosc 1977;23:150-4.105. DISTINGUISHING FEATURES OF CROHN�S DISEASE In most cases, Crohn�s disease of the colon is readily distinguished from ulcerative colitis by clinical, radiologic, endoscopic, and pathologic features. The great majority of cases (70-85%) of Crohn�s colitis have small bowel involvement and relative sparing of the rectum. Although most patients have some gross rectal bleeding, a substantial minority (25-30%) note no blood; this absence of bleeding, by contrast, is almost never reported in ulcerative colitis. Except for small rectovaginal fistulae, which are sometimes seen in ulcerative colitis, major perianal lesions in IBD are a feature exclusively of Crohn�s disease. Other characteristics of the lesions of Crohn�s disease, as distinguished from ulcerative colitis, are locality in the setting of relatively normal intervening mucosa, segmental rather than diffuse or continuous distribution, and asymmetric involvement of different parts of the wall within given segments of bowel. Fistulization into surrounding tissues and organs occurs only in Crohn�s disease as opposed to ulcerative colitis. Granulomas or �microgranulomas� may be found on careful study of Crohn�s disease rectal biopsies in 25-30%, and in surgical specimens in up to 50-75% of cases; they are not necessary to establish a diagnosis of Crohn�s disease, but when present they are virtually pathognomonic. � Lockhart-Mummery HE, Morson BC. Crohn�s disease (regional enteritis) of the large intestine and its distinction from ulcerative colitis. Gut 1960;1:87-105. � Wolf BS, Marshak RH. Granulomatous colitis (Crohn�s disease of the colon): Roentgen features. Am J Roentgenol 1962;88:662-670. � Lindner AE, Marshak RH, Wolf BS et al. Granulomatous colitis�a clinical study. N Engl J Med 1963;269:379-385. � Marshak RH, LindnerAE, Janowitz HD. Granulomatous ileecolitis. Gut 1966;7:258-264. � Farmer RG, Hawk WA, Turnbull RB. Regional enteritis of the colon: Clinical and pathological comparison with ulcerative colitis. Am J Dig Dis 1968;13:501-14. � Lennard-Jones JE, Lockhart-Mummery HE, Morson BC. Clinical and pathological differentiation of Crohn�s disease and proctocolitis. Gastroenterology 1968;64:1162-1170. � Cook MG, Dixon MF. An analysis of the reliability of detection and diagnostic value of various patholegic features in Crohn�s disease and ulcerative colitis. Gut 1973;14:255-62. � Waye JD. The role of colonoscopy in the differential diagnosis of inflammatory bowel disease. Gastrointest Endosc 1977;23:150-4.

16. Crohn�s Disease Red Flags Onset after stopping smoking Bleeding only Diverticulosis Atherosclerosis Prolapse

17. INDETERMINATE COLITIS 36. INDETERMINATE COLITIS It is uncertain to what extent �indeterminate colitis� represents an inevitable subset of cases in which it is simply difficult to establish a clear differential diagnosis between ulcerative and Crohn�s colitis, as opposed to a specific clinicopathological entity or set of entities distinct from either of the other two �classical� forms if IBD. At least part of the confusion, however, stems from an over-reliance on certain features that �conventional wisdom� incorrectly considers absolutely pathognomonic of either ulcerative colitis or Crohn�s disease. For example, a number of �typical� characteristics of Crohn�s disease that sometimes occur in ulcerative colitis include relative rectal sparing, an isolated patch of inflammation in the cecum over-interpreted as a �skip lesion,� and granulomas that are actually cryptolytic (or �mucinophagic�) rather than truly epithelioid. Likewise, the �typical� ulcerative colitis pattern of inflammation confined to the mucosa may occasionally be seen in Crohn�s disease as well. Moreover, the �typical� appearance of ulcerative colitis may be modified�often sufficiently to resemble Crohn�s disease�as a consequence of treatment-induced alterations. � Sachar DB. Is indeterminate colitis a problem of classification or is it an entity? Drugs of Today 2001;37(Supplement E): 63-6.36. INDETERMINATE COLITIS It is uncertain to what extent �indeterminate colitis� represents an inevitable subset of cases in which it is simply difficult to establish a clear differential diagnosis between ulcerative and Crohn�s colitis, as opposed to a specific clinicopathological entity or set of entities distinct from either of the other two �classical� forms if IBD. At least part of the confusion, however, stems from an over-reliance on certain features that �conventional wisdom� incorrectly considers absolutely pathognomonic of either ulcerative colitis or Crohn�s disease. For example, a number of �typical� characteristics of Crohn�s disease that sometimes occur in ulcerative colitis include relative rectal sparing, an isolated patch of inflammation in the cecum over-interpreted as a �skip lesion,� and granulomas that are actually cryptolytic (or �mucinophagic�) rather than truly epithelioid. Likewise, the �typical� ulcerative colitis pattern of inflammation confined to the mucosa may occasionally be seen in Crohn�s disease as well. Moreover, the �typical� appearance of ulcerative colitis may be modified�often sufficiently to resemble Crohn�s disease�as a consequence of treatment-induced alterations. � Sachar DB. Is indeterminate colitis a problem of classification or is it an entity? Drugs of Today 2001;37(Supplement E): 63-6.

18. Sensitivity, Specificity, and Positive and Negative Predictive Value of ASCA

19. Results of ASCA and pANCA in the Study Population BACKGROUND & AIMS: In the absence of pathognomonic markers for Crohn's disease (CD) and ulcerative colitis (UC), the diagnosis of inflammatory bowel disease depends on a compendium of clinical, radiographic, endoscopic, and histologic criteria that bears imperfect specificity to the individual disorders. In 10% of cases of colitis, no differentiation can be made between CD and UC; these patients are diagnosed with indeterminate colitis (IC). We evaluated the value of anti-Saccharomyces cerevisiae antibodies (ASCA) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) to increase diagnostic accuracy in categorizing IC. METHODS: Since 1996, 97 patients with IC from 3 centers (Leuven, Lille, and Vienna) were enrolled, analyzed for pANCA and ASCA, and followed up prospectively. RESULTS: A definitive diagnosis has been reached for 31 of 97 patients (32%). In these patients, ASCA+/pANCA- correlated with CD in 8 of 10 patients, whereas ASCA-/pANCA+ correlated with UC in 7 of 11 patients. The remaining 4 cases became CD, clinically behaving as UC-like CD. Almost half of the patients (47 of 97 [48.5%]) were negative for ASCA and pANCA, and 40 remain diagnosed with IC to date. Only 7 seronegative cases (14.9%) became CD or UC compared with 48% (24 of 50) of seropositive patients (P < 0.001). CONCLUSIONS: Results so far show that ASCA+/pANCA- predicts CD in 80% of patients with IC and ASCA-/pANCA+ predicts UC in 63.6%. Interestingly, 48.5% of patients do not show antibodies against ASCA or pANCA. Most of these patients remain diagnosed with IC during their further clinical course, perhaps reflecting a distinct clinicoserological entity.BACKGROUND & AIMS: In the absence of pathognomonic markers for Crohn's disease (CD) and ulcerative colitis (UC), the diagnosis of inflammatory bowel disease depends on a compendium of clinical, radiographic, endoscopic, and histologic criteria that bears imperfect specificity to the individual disorders. In 10% of cases of colitis, no differentiation can be made between CD and UC; these patients are diagnosed with indeterminate colitis (IC). We evaluated the value of anti-Saccharomyces cerevisiae antibodies (ASCA) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) to increase diagnostic accuracy in categorizing IC. METHODS: Since 1996, 97 patients with IC from 3 centers (Leuven, Lille, and Vienna) were enrolled, analyzed for pANCA and ASCA, and followed up prospectively. RESULTS: A definitive diagnosis has been reached for 31 of 97 patients (32%). In these patients, ASCA+/pANCA- correlated with CD in 8 of 10 patients, whereas ASCA-/pANCA+ correlated with UC in 7 of 11 patients. The remaining 4 cases became CD, clinically behaving as UC-like CD. Almost half of the patients (47 of 97 [48.5%]) were negative for ASCA and pANCA, and 40 remain diagnosed with IC to date. Only 7 seronegative cases (14.9%) became CD or UC compared with 48% (24 of 50) of seropositive patients (P < 0.001). CONCLUSIONS: Results so far show that ASCA+/pANCA- predicts CD in 80% of patients with IC and ASCA-/pANCA+ predicts UC in 63.6%. Interestingly, 48.5% of patients do not show antibodies against ASCA or pANCA. Most of these patients remain diagnosed with IC during their further clinical course, perhaps reflecting a distinct clinicoserological entity.

20. Relationship Between Marker Antibodies and CD Cohort BACKGROUND & AIMS: Previous studies in Crohn's disease suggest global loss of tolerance with sonicated bacteria preparations containing hundreds of antigens. Monoassociation studies show that a solitary bacterium can induce colitis in one animal model, whereas another is responsible in other models. Among patients with Crohn's disease, serum responses have been documented to microbial and autoantigens (antibodies to the Escherichia coli outer-membrane porin C and the Pseudomonas fluorescens-associated sequence I2, antisaccharomyces cerevisiae antibody (ASCA), and perinuclear antineutrophil cytoplasmic antibodies). Our aim was to determine whether there are heterogeneous responses to these specific antigens. METHODS: Sera from 330 Crohn's patients were analyzed. Immunoglobulin A enzyme-linked immunosorbent assays to ASCA, outer-membrane porin C, or I2 and immunoglobulin G enzyme-linked immunosorbent assay to ASCA and ANCA determined the presence and level of antibodies. Perinuclear antineutrophil cytoplasmic antibodies were determined by immunofluorescence. RESULTS: ASCA was detected in 56% of patients; 55% were seroreactive to outer-membrane porin C, 50% were seroreactive to I2, and 23% were perinuclear antineutrophil cytoplasmic antibody positive. Eighty-five percent responded to at least 1 antigen; only 4% responded to all 4. Among microbial antigens, 78% responded to at least 1, and 57% were double positive, but only 26% responded to all 3. The level of response was stable over time and with change in disease activity. Among patients with the same qualitative antigen-response profiles, quantitative response differed. Cluster analysis of these antibody responses yielded 4 groups: ASCA, outer-membrane porin C/I2, perinuclear antineutrophil cytoplasmic antibodies, or no/low response. CONCLUSIONS: Rather than global loss of tolerance, there seem to be patient subsets with differing responses to selected microbial and autoantigensBACKGROUND & AIMS: Previous studies in Crohn's disease suggest global loss of tolerance with sonicated bacteria preparations containing hundreds of antigens. Monoassociation studies show that a solitary bacterium can induce colitis in one animal model, whereas another is responsible in other models. Among patients with Crohn's disease, serum responses have been documented to microbial and autoantigens (antibodies to the Escherichia coli outer-membrane porin C and the Pseudomonas fluorescens-associated sequence I2, antisaccharomyces cerevisiae antibody (ASCA), and perinuclear antineutrophil cytoplasmic antibodies). Our aim was to determine whether there are heterogeneous responses to these specific antigens. METHODS: Sera from 330 Crohn's patients were analyzed. Immunoglobulin A enzyme-linked immunosorbent assays to ASCA, outer-membrane porin C, or I2 and immunoglobulin G enzyme-linked immunosorbent assay to ASCA and ANCA determined the presence and level of antibodies. Perinuclear antineutrophil cytoplasmic antibodies were determined by immunofluorescence. RESULTS: ASCA was detected in 56% of patients; 55% were seroreactive to outer-membrane porin C, 50% were seroreactive to I2, and 23% were perinuclear antineutrophil cytoplasmic antibody positive. Eighty-five percent responded to at least 1 antigen; only 4% responded to all 4. Among microbial antigens, 78% responded to at least 1, and 57% were double positive, but only 26% responded to all 3. The level of response was stable over time and with change in disease activity. Among patients with the same qualitative antigen-response profiles, quantitative response differed. Cluster analysis of these antibody responses yielded 4 groups: ASCA, outer-membrane porin C/I2, perinuclear antineutrophil cytoplasmic antibodies, or no/low response. CONCLUSIONS: Rather than global loss of tolerance, there seem to be patient subsets with differing responses to selected microbial and autoantigens

21. Conclusions-2 pANCA and ASCA have low sensitivity in CD and UC pANCA and ASCA have good specificity and PPV in CD and UC In patients with indeterminate colitis, available serological markers do not accurately predict the subsequent disease course Is indeterminate colitis a different form of IBD? Will performance of serological markers improve with introduction of other markers?

22. Why Use Serological Markers in Clinical Practice? Differentiate functional from organic disorders Differentiate type of IBD Implications for medical and surgical therapy Predict disease course or complications in IBD CD phenotype Severity of disease Pouchitis

23. Antibody Expression Stratifies Homogeneous Subgroups with Distinct Clinical Characteristics BACKGROUND: Perinuclear antineutrophil cytoplasmic antibodies (pANCA) have been detected in a clinically distinct Crohn's disease subpopulation. Antibodies to Saccharomyces cerevisiae (ASCA) have been demonstrated in the majority of patients with Crohn's disease. AIMS: To examine the relationship between selective marker antibody expression in Crohn's disease and disease onset, location, and clinical behaviour patterns. METHODS: Sera from 156 consecutive patients with established Crohn's disease were evaluated in a blinded fashion for the presence of ASCA and ANCA. Clinical profiles were generated by investigators blinded to immune marker status. RESULTS: Using multiple regression analyses, higher ASCA levels were shown to be independently associated with early age of disease onset as well as both fibrostenosing and internal penetrating disease behaviours. Higher ANCA levels were associated with later age of onset and ulcerative colitis-like behaviour. Substratification of the Crohn's disease population using selective ANCA and ASCA expression (high levels of a single marker antibody): (1) distinguished homogeneous subgroups that manifested similar disease location and behaviours; and (2) identified patients with more aggressive small bowel disease. CONCLUSIONS: The findings suggest that by taking into account the magnitude of the host immune response, Crohn's disease can now be stratified on an immunological basis into more homogeneous clinically distinct subgroups, characterised by greater uniformity among anatomical distribution of disease and disease behaviour.BACKGROUND: Perinuclear antineutrophil cytoplasmic antibodies (pANCA) have been detected in a clinically distinct Crohn's disease subpopulation. Antibodies to Saccharomyces cerevisiae (ASCA) have been demonstrated in the majority of patients with Crohn's disease. AIMS: To examine the relationship between selective marker antibody expression in Crohn's disease and disease onset, location, and clinical behaviour patterns. METHODS: Sera from 156 consecutive patients with established Crohn's disease were evaluated in a blinded fashion for the presence of ASCA and ANCA. Clinical profiles were generated by investigators blinded to immune marker status. RESULTS: Using multiple regression analyses, higher ASCA levels were shown to be independently associated with early age of disease onset as well as both fibrostenosing and internal penetrating disease behaviours. Higher ANCA levels were associated with later age of onset and ulcerative colitis-like behaviour. Substratification of the Crohn's disease population using selective ANCA and ASCA expression (high levels of a single marker antibody): (1) distinguished homogeneous subgroups that manifested similar disease location and behaviours; and (2) identified patients with more aggressive small bowel disease. CONCLUSIONS: The findings suggest that by taking into account the magnitude of the host immune response, Crohn's disease can now be stratified on an immunological basis into more homogeneous clinically distinct subgroups, characterised by greater uniformity among anatomical distribution of disease and disease behaviour.

24. Relative Contribution of Antibody Responses and Complicated Small Bowel Disease BACKGROUND & AIMS: Crohn's disease patients can be characterized by antibody responses against Crohn's disease-related bacterial sequence, Escherichia coli outer membrane porin C, Saccharomyces cerevisiae (oligomannan), and neutrophil nuclear antigens. Our aim was to determine whether expression of antibodies against Crohn's disease-related bacterial sequence and Escherichia coli outer membrane porin C is associated with distinct phenotypic manifestations. METHODS: Sera from 303 patients were tested for antibodies to the Crohn's disease-related bacterial sequence (I2), anti-Escherichia coli outer membrane porin C, anti-Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibodies and for 3 Crohn's disease-associated variants of the NOD2 gene (R702W, G908R, and 1007fs) and compared with clinical data. RESULTS: Patients expressing I2 were more likely to have fibrostenosing Crohn's disease (64.4% vs. 40.7%; P < 0.001) and to require small bowel surgery (62.2% vs. 37.4%; P < 0.001). Patients with anti-Escherichia coli outer membrane porin C were more likely to have internal perforating disease (50.0% vs. 30.7%; P = 0.001) and to require small bowel surgery (61.4% vs. 44.2%; P = 0.003). Anti-Crohn's disease-related bacterial sequence was independently associated with fibrostenosis (P = 0.027) and small bowel surgery (P = 0.01), whereas anti-Escherichia coli outer membrane porin C was independently associated with internal perforations (P < 0.006). Patients positive for I2, anti-Escherichia coli outer membrane porin C, and anti-Saccharomyces cerevisiae were the most likely to have undergone small bowel surgery (72.0%; odds ratio, 8.6; P < 0.001) compared with patients without reactivity (23.0%). When the presence and magnitude of antibody responses were considered, 90% of patients with small bowel disease who required surgery had high levels of I2, Escherichia coli outer membrane porin C, and oligomannan antibodies, compared with only 18.2% with low-titer responses (P < 0.001). CONCLUSIONS: I2 and anti-Escherichia coli outer membrane porin C are associated with Crohn's disease phenotypes, and patients with the highest level of serum reactivity toward an increasing number of microbiota have the greatest frequency of strictures, internal perforations, and small bowel surgery.BACKGROUND & AIMS: Crohn's disease patients can be characterized by antibody responses against Crohn's disease-related bacterial sequence, Escherichia coli outer membrane porin C, Saccharomyces cerevisiae (oligomannan), and neutrophil nuclear antigens. Our aim was to determine whether expression of antibodies against Crohn's disease-related bacterial sequence and Escherichia coli outer membrane porin C is associated with distinct phenotypic manifestations. METHODS: Sera from 303 patients were tested for antibodies to the Crohn's disease-related bacterial sequence (I2), anti-Escherichia coli outer membrane porin C, anti-Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibodies and for 3 Crohn's disease-associated variants of the NOD2 gene (R702W, G908R, and 1007fs) and compared with clinical data. RESULTS: Patients expressing I2 were more likely to have fibrostenosing Crohn's disease (64.4% vs. 40.7%; P < 0.001) and to require small bowel surgery (62.2% vs. 37.4%; P < 0.001). Patients with anti-Escherichia coli outer membrane porin C were more likely to have internal perforating disease (50.0% vs. 30.7%; P = 0.001) and to require small bowel surgery (61.4% vs. 44.2%; P = 0.003). Anti-Crohn's disease-related bacterial sequence was independently associated with fibrostenosis (P = 0.027) and small bowel surgery (P = 0.01), whereas anti-Escherichia coli outer membrane porin C was independently associated with internal perforations (P < 0.006). Patients positive for I2, anti-Escherichia coli outer membrane porin C, and anti-Saccharomyces cerevisiae were the most likely to have undergone small bowel surgery (72.0%; odds ratio, 8.6; P < 0.001) compared with patients without reactivity (23.0%). When the presence and magnitude of antibody responses were considered, 90% of patients with small bowel disease who required surgery had high levels of I2, Escherichia coli outer membrane porin C, and oligomannan antibodies, compared with only 18.2% with low-titer responses (P < 0.001). CONCLUSIONS: I2 and anti-Escherichia coli outer membrane porin C are associated with Crohn's disease phenotypes, and patients with the highest level of serum reactivity toward an increasing number of microbiota have the greatest frequency of strictures, internal perforations, and small bowel surgery.

25. Disease Characteristics in Patients with Antibodies to Multiple Microbial Antigens

26. Incidence of Pouchitis in pANCA+ and pANCA- Patients

27. Conclusions-3 Antibody profiles can predict disease behavior in IBD ASCA and I2 generally predict small bowel disease, fibrostenotic behavior, and need for surgery Multiple antibodies associated with an even higher risk pANCA predicts �UC-like� behavior pANCA+ associated with risk of pouchitis after IPAA

28. Summary pANCA and ASCA are specific for UC and CD respectively Neither pANCA nor ASCA are sensitive enough to exclude IBD In patients with IC, available serological markers do not accurately predict the subsequent disease course Antibody profiles can predict disease behavior in IBD