Accepted Article
Serological Markers Associated with Disease Behavior and Response to Anti-TNF Therapy in Ulcerative Colitis1
Short Title: IBD Serology and Ulcerative Colitis D Kevans, MD1,2, M Waterman, MD1,2, R Milgrom, MD1, W Xu, PhD3, G Van Assche, PhD1,2, MS Silverberg, PhD1,2
1. Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital, Toronto
2. Division of Gastroenterology, Department of Medicine, University of Toronto 3. Dalla Lana School of Public Health, University of Toronto
Corresponding Author David Kevans, MD, Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital, 60 Murray Street, Toronto, Ontario ON M5T 3L9 Tel: 416-586-4800 ext. 8349 Fax: 416-586-5932 [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12661
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Abstract
Background & Aim Information is limited on the relationship between serological markers and disease behavior and anti-tumor necrosis factor-α(anti-TNF) therapy response in ulcerative colitis(UC). This
study aimed to determine the association between serological markers and unfavorable UC behavior defined as need for colectomy or UC-related hospitalization. The association between serological markers and requirement for and outcome of anti-TNF therapy was also evaluated. Methods
230 patients were studied. Requirement for colectomy, UC-related hospitalization, and antiTNF therapy were documented. Response to anti-TNF therapy at 1 year and rates of therapy discontinuation were recorded. Titers
of
peri-nuclear anti-neutrophil
cytoplasmic
antibodies(pANCAs), anti-Saccharomyces Cerevisiae antibody(ASCA) and antibody to Escherichia Coli outer membrane porin(anti-OmpC) were determined. Antibody reference ranges were used to dichotomize subjects into seropositive and seronegative groups. Where multiple tests were performed p values were Bonferroni corrected (pcorr). Results
Extensive colitis was associated with requirement for colectomy and UC-related hospitalization, HR 7.7 (95%CI 1.9 – 32.2) pcorr=0.03 and HR 2.7 (95%CI 1.5 – 4.6),
pcorr=0.006 respectively. No serological variable was associated with unfavorable UC behavior. Anti-OmpC positivity was associated with a lack of response to anti-TNF therapy at 1 year [OR 0.14 (95% CI 0.03 - 0.60), pcorr= 0.04] and increased likelihood of therapy discontinuation [HR 2.2 (95% CI 1.1 – 4.7), p=0.03].
Conclusions Extensive colitis is associated with unfavorable disease course in UC. Anti-OmpC holds promise as a biomarker of anti-TNF therapy response in UC however prospective studies are required before it can be incorporated into routine clinical practice. Keywords: UC; pANCA; ASCA; Anti-OmpC; anti-TNF therapy
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Introduction Inflammatory bowel diseases (IBD) encompass a range of inflammatory disorders affecting the intestinal tract, with the two main entities being ulcerative colitis (UC) and Crohn’s disease (CD). While its etiology remains unknown, IBD is thought to occur due to an aberrant immune response to intestinal bacteria in a genetically susceptible host. This theory is supported by the presence of a number of antibodies, in UC and CD, which are directed against self and microbial antigens. Anti-neutrophil cytoplasmic antibodies (ANCAs) occur in the sera of patients with a number of other chronic inflammatory disorders including UC. Reported positivity rates for ANCAs in UC, CD and primary sclerosing cholangitis (PSC) are 60 - 80%, 5 - 25% and 88% respectively.1-4 Anti-Saccharomyces Cerevisiae antibody (ASCA),1, 5, 6 and an antibody directed against Escherichia coli outer membrane porin (antiOmpC) have more recently been found to occur in CD and less commonly UC.1,
6
While
these IBD-associated antibodies can be a useful aid in classifying IBD subtypes and in predicting disease course in CD, data are few regarding their relationship with disease behavior in UC. Reports regarding the association between anti-tumor necrosis factor-α (TNF) therapy response, in UC and CD, and seropositivity for various IBD-associated
antibodies have also been few and conflicting.7-9 We therefore aimed to determine the association between clinical variables, pANCA, ASCA and anti-OmpC and unfavorable UC disease course, defined as need for colectomy or UC-related hospitalization. We also assessed the association between these variables and the requirement for, response to, and rates of discontinuation of anti-TNF therapy.
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Methods
Patient Population & Definitions Subjects with a diagnosis of UC were identified from an institutional database with those with a prior colectomy at the time of serum collection excluded. Of the 264 eligible subjects with UC; 20 were excluded due the lack of an available serum sample while a further 14 were excluded due to a lack of clinical follow up or as they were unclassified IBD (IBD-U);10 resulting in an eligible study cohort of 230 UC subjects. UC was diagnosed by the accepted clinical, endoscopic, radiological and pathological criteria.10 Information on clinical variables including age at diagnosis, disease extent, UCrelated hospitalizations, previous surgery, medication use, and response to anti-TNF therapy was obtained by review of medical records. Disease extent was categorized, based on the maximal extent observed during endoscopic follow up into proctitis, left-sided colitis and extensive colitis.10 UC-related hospitalization was defined as any hospitalization for the management of UC disease activity. Indication for colectomy was determined in each case and only those performed for medically refractory disease were considered in analyses. The study received approval from the institutional research ethics board and all included subjects gave informed consent.
Definitions of outcome of Anti-TNF therapy Patients receiving Infliximab therapy (n=59) were administered an initial infusion of infliximab at a dose of 5mg/Kg; the majority (n=50) received conventional 3 dose induction, a minority (n=7) received 1 or 2 dose induction; while a small number (n=2) had no data available on the induction regime received. Thereafter maintenance therapy was administered as appropriate and dose escalation was allowed whenever clinically indicated. Subjects were
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excluded from analyses where there was uncertainty regarding their anti-TNF therapy response at 1 year or requirement for therapy discontinuation; N=54 subjects had available data on therapy response while n=55 subjects had data available on requirement for therapy discontinuation. Anti-TNF response was categorized by review of clinical notes and was defined at 1 year as the absence of UC-related symptoms, no systemic corticosteroid use, no requirement for regular rectal corticosteroid or 5ASA therapy, ongoing Infliximab therapy and no requirement for colectomy. Rates and time to discontinuation of anti-TNF therapy were also documented with subjects discontinuing therapy for any reason included in analyses.
IBD-Associated Antibody Estimation Serum was separated from venous whole blood and stored at -70°C. Thawed samples were tested at Prometheus Laboratories (San Diego, CA) in a manner previously described.11 Due
to the retrospective nature of this study, serum samples were processed on two different serological platforms depending on their time of collection 129 samples were analyzed using the IBD Diagnostics System - Generation II (Prometheus Laboratories, San Diego, CA) while a further 101 samples were analyzed using the IBD SGI Platform (Prometheus Laboratories, San Diego, CA). Sera were tested for the presence of the following IBD-associated antibodies: ANCA, ASCA (IgA and IgG) and anti-OmpC. The two Prometheus platforms differ in the reference ranges applied for each IBD-associated antibody. For the IBD Diagnostics System – Generation II, antibodies were considered positive if they exceeded the following titers: ASCA IgA > 20EU/mL, ASCA IgG > 40 EU/mL, and anti-OmpC > 16.4 EU/mL. pANCA positivity was defined as an ANCA enzyme linked immunosorbent assay (ELISA) titer > 12.1 EU/mL and pANCA immunofluorescence assay (IFA) reactive / deoxyribonuclease (DNAse) sensitive. For the IBD SGI Diagnostic Platform, antibodies were
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considered positive if they exceeded the following titers: ASCA IgA ≥ 8.5 EU/mL, ASCA IgG ≥ 17.8 EU/mL and anti-OmpC ≥ 10.9 EU/mL. pANCA positivity was defined as an ANCA ELISA titer ≥ 19.8 EU/mL and pANCA IFA reactive / DNAse sensitive. For both panels subjects were considered ASCA positive if either ASCA IgA or IgG was positive. Given the differences in the reference ranges between the two IBD serology platforms utilized, actual antibody titers were not used in the analysis but rather subjects were categorized as positive or negative for each IBD-associated antibody.
Study Endpoints Clinical variables (age, gender and colitis extent) and IBD associated antibodies (pANCA, ASCA and anti-OmpC) were associated with study endpoints. Two primary endpoints were selected to represent unfavorable UC disease behavior: need for colectomy and requirement for 1 or more UC-related hospitalization (referred to hereafter as “UC-related hospitalization”). Secondary endpoints assessed were the requirement for anti-TNF therapy, anti-TNF therapy response at 1 year, and rates of discontinuation of anti-TNF therapy.
Statistical Analysis Continuous data were presented as medians and ranges. Age at diagnosis and colitis extent at diagnosis were dichotomized into two groups for survival analyses: greater versus less than 17 years & extensive colitis versus left-sided colitis respectively. As anti-TNF agents only entered routine clinical practice around the year 2000, only n=93 subjects diagnosed on or after the year 2000 were included in the analysis of time to requirement for anti-TNF therapy. Univariate Cox proportional hazard regression was used to explore associations between variables and time to colectomy, UC-related hospitalization and anti-TNF therapy. Variables associated with response to anti-TNF therapy were evaluated using univariate binary logistic
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regression. For each endpoint assessed, in univariate analysis, Bonferroni correction was applied to p values (pcorr). Where more than one variable demonstrated a significant
association (pcorr < 0.1) with a given endpoint in univariate analysis, these variables were tested in a multivariate model (uncorrected p values reported). For the endpoints “1 year antiTNF response” & “anti-TNF therapy discontinuation” there was an exception to this approach with concomitant immunomodulator use at the time of anti-TNF therapy (which has been associated with improved efficacy of anti-TNF therapy) included in multivariate models irrespective of the significance of its association with a given endpoint in univariate analysis. Kaplan-Meier survival curves were constructed for variables and differences between groups evaluated using the logrank test (uncorrected p values reported). Statistical analysis was performed by using SAS (version 9.1.2; SAS Institute, Cary, North Carolina, USA) and Statistical Package for the Social Sciences (IBM, New York, USA)
Results
Baseline Characteristics The baseline characteristics of the cohort are summarized TABLE 1. 230 subjects were studied; the median age at diagnosis was 25.5 years (range 8.7 – 59.6). 51% of subjects were
female. Median follow up duration from diagnosis and time of serum collection were 12.1 years (range 0 – 45.6) and 3.7 years (0 – 9.6) respectively. Of the cohort, 3% had proctitis,
32% had left-sided colitis and 65% had extensive colitis. 6% of subjects were current smokers. The proportion of subjects exposed to 5-ASA, thiopurine, oral prednisone (at least one course) and Infliximab therapy was: 100%, 39%, 76%; and 26%, respectively. During the
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follow up period, UC-related hospitalization and colectomy occurred in 46% (n=105) and 16% (n=37) of subjects respectively [FIGURE 1A].
IBD-Associated Antibodies Profiles and Relationship with Demographic Factors Positivity for pANCA, ASCA and anti-OmpC occurred in 57% (n=131), 11% (n=26), and 18% (n=41) of the cohort respectively [FIGURE 2]. There was no significant association demonstrated between IBD-associated antibody positivity and gender, younger age at diagnosis (age less than 17 years), colitis extent or current smoking status.
Association between Clinical Variables and Colectomy, UC-related Hospitalization and Requirement for Anti-TNF therapy The association of colectomy, UC-related hospitalization and requirement for anti-TNF therapy with the following clinical variables was assessed: younger age at diagnosis, gender and colitis extent. The only clinical variable associated with the study endpoints was colitis extent. In a univariate analysis extensive colitis was significantly associated with requirement for colectomy and UC-related hospitalization HR 7.7 (95% CI: 1.9 – 32.2), pcorr=0.03 and HR 2.7 (95% CI: 1.5 – 4.6), pcorr=0.006 respectively.[TABLE 2 and FIGURE 1 B & C] As
extensive colitis was the only clinical or serological variable significantly associated with colectomy or requirement for UC-related hospitalization a multivariate analysis was not performed. There was no association between clinical variables and requirement for anti-TNF therapy.
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Association between IBD-Associated Antibodies and Requirement for Colectomy, UCRelated Hospitalization and Anti-TNF Therapy In a univariate analysis pANCA positivity showed no association with requirement for colectomy HR 1.3 (95% CI 0.7 – 2.6), pcorr=1.0.[FIGURE 1D] ASCA and anti-OmpC were
not associated with requirement for colectomy either.[TABLE 2] There was no significant association
between
IBD-associated
antibodies
and
requirement
for
UC-related
hospitalization or anti-TNF therapy.[TABLE 2] As no serological variable demonstrated an association with any of the study endpoints a multivariate analysis was not performed.
Association of Clinical Variables and Serological Variables with Outcome of Anti-TNF Therapy
In a univariate analysis (n=54 subjects) anti-OmpC was the only clinical or serologic variable associated with anti-TNF therapy response.[TABLE 3] Anti-OmpC positivity was associated with a lack of response to anti-TNF therapy at 1 year, Odds ratio (OR) 0.14 (95% CI 0.03 0.6), pcorr = 0.04; with 3 of 16 (19%) anti-OmpC positive subjects versus 24 of 38 (63%) of anti-OmpC negative subjects responding to anti-TNF therapy at 1 year.[Figure 3A] The use of concomitant immunomodulators in combination with anti-TNF therapy was not associated with improved 1 year therapy response, OR 1.4 (95% CI 0.4 – 4.6), pcorr = 1.0.[TABLE 3] In a multivariate model, which included concomitant immunomodulator use, anti-OmpC positivity remained independently associated with a decreased 1 year anti-TNF therapy response, OR 0.14 (95% CI 0.03 – 0.6), p=0.006. The probability of discontinuation of anti-
TNF therapy was significantly higher in anti-OmpC positive versus negative subjects (median time free of anti-TNF therapy discontinuation, 1.1 versus 6.0 years respectively); HR (for discontinuation of anti-TNF therapy) 2.2 (95% CI 1.1 – 4.7), p=0.03.[Figure 3B] The use of concomitant immunomodulators in combination with anti-TNF therapy was not associated
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with a decreased rate of therapy discontinuation, HR 0.8 (95% CI 0.4 – 1.9), p=0.64. In a
multivariate model, which included concomitant immunomodulator use, anti-OmpC positivity remained independently associated with likelihood of anti-TNF therapy discontinuation, HR 2.3 (95% CI 1.0 – 5.0), p=0.04.
Discussion
Consistent with previous reports we have shown that extensive colitis is associated with adverse disease behavior in UC.12,
13
We did not find an association between pANCA
positivity and disease behavior or anti-TNF therapy response in this cohort. We demonstrated a novel association between anti-OmpC positivity and lack of response to anti-TNF therapy at 1 year and increased likelihood of anti-TNF therapy discontinuation.
ANCAs describe a class of autoantibodies directed against antigens in the cytoplasm of neutrophils and monocytes. By indirect immunofluorescent microscopy, ANCA activity has been divided into two broad categories: cytoplasmic staining (cANCA) and perinuclear staining (pANCA). Another pattern of immunofluorescence in the setting of detectable ANCA has been observed characterized by a broad inhomogeneous rim-like staining of the nuclear periphery. These so called “atypical” pANCAs have been observed in UC, primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH).1 The pANCA seropositivity rate of 57% in our cohort was similar to that previously reported in UC where 41 - 73% of individuals have been found to be pANCA seropositive.14
A number of antibodies against microbial species have been described in IBD. Increased ASCA concentrations have been found in 44 - 62% of patients with CD and 10 - 15% of patients with UC.1,
6, 11
Outer membrane porin C (OmpC) is an Escherichia Coli protein
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which regulates bacterial metabolite and toxin transport. Anti-OmpC has been demonstrated to be an IBD-associated antibody occurring in 24 - 42% of CD patients, 11 - 28% of UC patients and 3 - 5% of healthy controls.6, 11 ASCA and anti-OmpC sero-positivity rates in our
cohort of 11% and 18% respectively, were consistent with these reports.
The association between IBD-associated antibodies and disease behavior has been best demonstrated in CD and in individuals with an ileal pouch-anal anastomosis performed in the setting of IBD.1,
15-17
Positivity for more than one IBD-associated antibody as well as the
actual antibody titer may also be important as several studies have shown that higher titers of multiple anti-microbial antibodies are associated with a more aggressive CD disease behavior.18, 19 Data on the association of IBD-associated antibodies with disease behavior in UC are few and have generally been restricted to the evaluation of pANCA and ASCA. There have been discordant reports regarding the association of pANCA and disease course in UC. Vecchi et al. demonstrated pANCA positivity to be associated with unfavourable disease behaviour,20 while a second larger multicentre study demonstrated increased risk of disease
relapse, but not colectomy to be significantly associated with pANCA and ASCA sero-
positivity.21 On the other hand a report by Seow et al. showed no association between pANCA positivity and either clinical remission or requirement for colectomy.22 In our cohort we did not find an association between pANCA positivity and UC disease behavior expressed as requirement for colectomy, UC related hospitalization or anti-TNF therapy.
To our knowledge only two previous studies have evaluated the association between antiOmpC and disease behaviour in UC. A study by Elkadri et al. demonstrated an association between anti-OmpC positivity and requirement for colectomy in UC.6 While a report by Papp
et al., utilizing an anti-OmpC assay which detected antibodies against multiple bacterial
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proteins derived from two species of intestinal bacteria (neither of which were Escherichia
coli), showed no association between anti-OmpC serotype and UC disease course.23, 24 We did no demonstrate any association between anti-OmpC positivity and adverse disease behavior in UC in our cohort.
The utility of pANCA as a predictor of anti-TNF response in UC and CD has been assessed in a number of studies with differing results._ENREF_257-9 Data on the utility of anti-OmpC
as predictor of response to anti-TNF therapy is very limited with only one previous study assessing this issue. Papp et al. evaluated the utility of anti-OmpC in CD as a predictor of response to anti-TNF therapy and found no association. As previously mentioned this study utilized an anti-OmpC assay which differed significantly from that used in the current study.25 Anti-OmpC has not previously been evaluated as a predictor of anti-TNF response in
UC. We demonstrated a novel association between anti-OmpC positivity and lack of response
to anti-TNF therapy at 1 year. In addition we demonstrated a significantly greater probability of discontinuing anti-TNF therapy in anti-OmpC positive versus negative subjects. These
data suggest that anti-OmpC is a potential biomarker of anti-TNF response in UC. It may be a that an intestinal microbial profile associated with anti-TNF therapy resistance in UC, results in anti-OmpC reactivity, however clearly this hypothesis needs to be formally evaluated in future studies. An important caveat to these data is the relatively small number of subjects included in our analysis of variables associated with anti-TNF response, therefore while this association is potentially important it requires validation in independent cohorts.
We acknowledge that this report is based on a referral center cohort with the inherent limitations associated with this type of study design. Like many other studies which have assessed the association between serology and IBD disease behavior, this study was limited
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by its cross sectional rather than prospective design, with serum sampling occurring later in subject’s disease course rather than at or soon after diagnosis. To address this limitation
subjects were only included if clinical follow up was available at the time of, or subsequent to their serum draw, therefore some prospective follow up was available for the majority of subjects. If IBD-associated antibodies are to be used in routine clinical practice they most
likely need to provide prognostic information early in UC disease course. An important question therefore is whether IBD-associated antibody profiles estimated in subjects with established UC, as performed in this study, reflect those estimated near the time of UC diagnosis. Information in this regard is provided in a report by Rieder et al. which demonstrated that, while substantial changes in anti-glycan antibody levels (such as ASCA) occur over time, antibody positivity / negativity remained stable in the vast majority of UC and CD patients.19 P-ANCA status, on the other hand has been found to change from positive to negative after colectomy.26 As we excluded subjects who had undergone a prior colectomy
at the time of serum collection and performed our analysis considering only positivity / negativity for each IBD-associated antibody, we believe that while this study characterized
antibody profiles in subjects with established UC, these profiles are likely to be representative of those estimated earlier in disease course.
In summary, consistent with previous reports, we have shown extensive colitis to be associated with adverse disease behavior in UC. We did not demonstrate an association between pANCA positivity and UC behavior or anti-TNF therapy response. We demonstrated a novel association between anti-OmpC positivity and lack of response to anti-TNF therapy at 1 year and increased probability of anti-TNF therapy discontinuation. Anti-OmpC holds promise as biomarker of anti-TNF therapy response in UC however larger prospective studies are required before this marker can be incorporated into routine clinical practice.
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Acknowledgements These data were presented at Digestive Disease Week 2013, Orlando, Florida, USA. David Kevans is a recipient of a CIHR/CAG/Abbvie IBD Fellowship and honoraria from Abbvie; Gert Van Assche recieves honoraria and/or research support from Abbvie, Janssen, MSD, Ferring, Aptatlis, Warner-Chilcott, Shire, UCB, Novartis, Chiesi and BMS. Mark Silverberg is partially supported by the Gale and Graham Wright Research Chair in Digestive Disease and by grants from CCFC and NIDDK (DK-062423). Mark Silverberg receives speaker fees, consulting fees and research support from Prometheus Laboratories. The authors have no conflicts to disclose.
References
1. X. Bossuyt. Serologic markers in inflammatory bowel disease. Clin Chem 2006; 52: 17181.
2. A. Saxon, F. Shanahan, C. Landers, T. Ganz and S. Targan. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990; 86: 202-10.
3. J. A. Rump, J. Scholmerich, V. Gross, et al. A new type of perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) in active ulcerative colitis but not in Crohn's disease. Immunobiology 1990; 181: 406-13. 4. B. Terjung and H. J. Worman. Anti-neutrophil antibodies in primary sclerosing cholangitis. Best Pract Res Clin Gastroenterol 2001; 15: 629-42. 5. C. J. Landers, O. Cohavy, R. Misra, et al. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123: 689-99. 6. A. A. Elkadri, J. M. Stempak, T. D. Walters, et al. Serum antibodies associated with complex inflammatory bowel disease. Inflamm Bowel Dis 2013; 19: 1499-505. 7. M. Ferrante, S. Vermeire, K. H. Katsanos, et al. Predictors of early response to infliximab in patients with ulcerative colitis. Inflamm Bowel Dis 2007; 13: 123-8. 8. N. Esters, S. Vermeire, S. Joossens, et al. Serological markers for prediction of response to anti-tumor necrosis factor treatment in Crohn's disease. Am J Gastroenterol 2002; 97: 145862. 9. M. Jurgens, R. P. Laubender, F. Hartl, et al. Disease activity, ANCA, and IL23R genotype status determine early response to infliximab in patients with ulcerative colitis. Am J Gastroenterol 2010; 105: 1811-9. 14 This article is protected by copyright. All rights reserved.
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10. M. S. Silverberg, J. Satsangi, T. Ahmad, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005; 19 Suppl A: 5A-36A. 11. A. Zholudev, D. Zurakowski, W. Young, A. Leichtner and A. Bousvaros. Serologic testing with ANCA, ASCA, and anti-OmpC in children and young adults with Crohn's disease and ulcerative colitis: diagnostic value and correlation with disease phenotype. Am J Gastroenterol 2004; 99: 2235-41.
12. L. E. Targownik, H. Singh, Z. Nugent and C. N. Bernstein. The epidemiology of colectomy in ulcerative colitis: results from a population-based cohort. Am J Gastroenterol 2012; 107: 1228-35. 13. G. Bresci, G. Parisi and A. Capria. Duration of remission and long-term prognosis according to the extent of disease in patients with ulcerative colitis on continuous mesalamine treatment. Colorectal Dis 2008; 10: 814-7. 14. J. H. Sellin and R. R. Shah. The promise and pitfalls of serologic testing in inflammatory bowel disease. Gastroenterol Clin North Am 2012; 41: 463-82. 15. D. K. Amre, S. E. Lu, F. Costea and E. G. Seidman. Utility of serological markers in predicting the early occurrence of complications and surgery in pediatric Crohn's disease patients. Am J Gastroenterol 2006; 101: 645-52. 16. W. S. Mow, E. A. Vasiliauskas, Y. C. Lin, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126: 414-24. 17. A. D. Tyler, R. Milgrom, W. Xu, et al. Antimicrobial antibodies are associated with a Crohn's disease-like phenotype after ileal pouch-anal anastomosis. Clin Gastroenterol Hepatol 10: 507-12 e1. 18. I. D. Arnott, C. J. Landers, E. J. Nimmo, et al. Sero-reactivity to microbial components in Crohn's disease is associated with disease severity and progression, but not NOD2/CARD15 genotype. Am J Gastroenterol 2004; 99: 2376-84. 19. F. Rieder, S. Schleder, A. Wolf, et al. Association of the novel serologic anti-glycan antibodies anti-laminarin and anti-chitin with complicated Crohn's disease behavior. Inflamm Bowel Dis 2010; 16: 263-74. 20. M. Vecchi, M. B. Bianchi, C. Calabresi, G. Meucci, M. Tatarella and R. de Franchis. Long-term observation of the perinuclear anti-neutrophil cytoplasmic antibody status in ulcerative colitis patients. Scand J Gastroenterol 1998; 33: 170-3. 21. O. Hoie, G. Aamodt, S. Vermeire, et al. Serological markers are associated with disease course in ulcerative colitis. A study in an unselected population-based cohort followed for 10 years. J Crohns Colitis 2008; 2: 114-22.
15 This article is protected by copyright. All rights reserved.
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22. C. H. Seow, A. Newman, S. P. Irwin, A. H. Steinhart, M. S. Silverberg and G. R. Greenberg. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut 2010; 59: 49-54.
23. M. Papp, I. Altorjay, N. Dotan, et al. New serological markers for inflammatory bowel disease are associated with earlier age at onset, complicated disease behavior, risk for surgery, and NOD2/CARD15 genotype in a Hungarian IBD cohort. Am J Gastroenterol 2008; 103: 665-81. 24. M. Papp, G. L. Norman, Z. Vitalis, et al. Presence of anti-microbial antibodies in liver cirrhosis - a tell-tale sign of compromised immunity? PLoS One 2010; 5: e12957. 25. M. Papp, I. Altorjay, G. L. Norman, et al. Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients. Inflamm Bowel Dis 2007; 13: 984-92. 26. J. A. Rump, I. Worner, M. Roth, J. Scholmerich, M. Hansch and H. H. Peter. p-ANCA of undefined specificity in ulcerative colitis: correlation to disease activity and therapy. Adv Exp Med Biol 1993; 336: 507-13.
Figure Legend
Figure 1. Kaplan Meir curves of clinical and serological variables significantly associated with requirement for colectomy, UC-related hospitalization and anti-TNF therapy.
Panel A. Cumulative colectomy-free survival in the study cohort (n=230); Panel B. Colectomy free survival segregated by colitis extent; Panel C. Survival free of UC-related hospitalization segregated by colitis extent; Panel D. Colectomy-free survival segregated by pANCA serotype.
Figure 2. Prevalence of IBD-associated antibodies in cohort (n=230).
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Figure 3. Anti-OmpC as a predictor of outcome of anti-TNF therapy.
Panel A demonstrates that a lesser proportion of patients achieved anti-TNF therapy response at 1 year in anti-OmpC positive compared with anti-OmpC negative groups. Panel B displays a Kaplan Meir survival curve comparing survival free of anti-TNF therapy discontinuation in
anti-OmpC positive versus anti-OmpC negative groups. Anti-OmpC positive versus negative subjects had a greater probability of earlier anti-TNF therapy discontinuation (median time free of anti-TNF therapy discontinuation, 1.1 years versus 6.0 years respectively); HR (for discontinuation of Anti-TNF therapy 2.2 (95% CI 1.1 – 4.7), p = 0.03.
*Univariate binary logistic regression (bonferroni corrected p value) **Log rank test (uncorrected p value)
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25.5 (8.7 – 59.6)
Accepted Article
Age at Diagnosis (years) † Gender Female Male Colitis Extent at Diagnosis Proctitis (E1) Left-sided Colitis (E2) Extensive Colitis (E3) Primary Sclerosing Cholangitis No Yes Smoking Status Non-Smoker Ex-Smoker Current Smoker Follow up (years) † From diagnosis From time of serum collection UC-Related Hospitalization No Yes Colectomy ‡ No Yes 5ASA Therapy Yes Oral Prednisone § No Yes Thiopurine therapy No Yes Infliximab Therapy No Yes
118 (51%) 112 (49%) 8 (3%) 73 (32%) 148 (65%) 217 (96%) 8 (4%) 143 (63%) 69 (31%) 14 (6%) 12.1 (0 – 45.6) 3.7 (0 – 9.6) 122 (54%) 105 (46%) 195 (84%) 37 (16%) 230 (100%) 54 (24%) 175 (76%) 141 (61%) 89 (39%) 171 (74%) 59 (26%)
Table 1. Baseline Characteristics of the Study Cohort (n=230).
†Continuous variables presented as median (range) ‡Colectomy for medically refractory disease
§Exposure to at least one course of oral prednisone Where totals in each row do not sum to 230 data was unavailable or unknown
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Colectomy UC-Related Hospitalization Anti-TNF Therapy† HR (95% CI) Pcorr HR (95% CI) Pcorr HR (95% CI) PCorr 1.1 (0.5 – 2.7) 1.0 0.7 (0.4 – 1.2) 1.0 0.9 (0.4 – 1.9) 1.0 Age > 17 years 1.2 (0.6 – 2.2) 1.0 0.7 (0.5 – 1.1) 0.54 0.8 (0.4 – 1.7) 1.0 Male gender 7.7 (1.9 – 32.2) 2.7 (1.5 – 4.6) 0.006 4.4 (1.3 – 14.3) 0.12 Extensive Colitis 0.03 1.3 (0.7 – 2.6) 1.0 1.2 (0.8 - 1.8) 1.0 1.6 (0.8 – 3.5) 1.0 pANCA+ ve 0.4 (0.1 – 1.7) 1.0 1.3 (0.7 - 2.3) 1.0 1.7 (0.8 – 4.0) 1.0 ASCA+ve 1.6 (0.8 – 3.4) 1.0 1.3 (0.8 – 2.0) 1.0 1.7 (0.8 – 3.9) 1.0 Anti-OmpC+ve Table 2. Univariate analysis evaluating the association between clinical and serologic variables and the requirement for colectomy, UCRelated Hospitalization and anti-TNF therapy.
† N=93 subjects diagnosed on or after the year 2000 and with available data on therapy commencement date were included in analysis Pcorr represents p value corrected for multiple tests performed by Bonferroni method
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Age > 17 years Male gender Extensive colitis Concomitant IMM use pANCA +ve ASCA +ve Anti-OmpC +ve
Anti-TNF Therapy Response Pcorr† OR (95% CI) 1.5 (0.4 – 5.6) 1.0 1.6 (0.5 – 4.7) 1.0 0.3 (0.03 – 3.2) 1.0 1.4 (0.4 – 4.6) 1.0 0.7 (0.2 – 2.2) 1.0 1.0 (0.2 – 4.5) 1.0 0.14 (0.03 - 0.60) 0.04
Table 3. Univariate analysis of clinical and serological variables associated with antiTNF therapy response at 1 year (n=54 subjects). †Pcorr represents p values corrected for multiple testing by Bonferroni correction method Anti-TNF therapy constituted Infliximab in all cases Concomitant immunomodulator (IMM) use at commencement of anti-TNF therapy (n=16 subjects): thiopurines (n=14), tacrolimus (n=1), methotrexate (n=1) Infliximab response at 1 year was defined as an absence of symptoms related to UC activity, no requirement for systemic corticosteroids, no requirement for regular topical rectal corticosteroid or 5ASA therapy and continuation of Infliximab therapy.
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A
B Left-sided Colitis (n=69) Study cohort (n=230)
Extensive Colitis (n=161)
p = 0.001
C
Figure 1
D pANCA -ve (n=99) Left sided Colitis (n=65) Left-sided pANCA +ve (n=131)
Extensive Colitis (n=164)
p < 0.001
p = 0.42
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57%
pANCA+
ASCA+
11%
Anti-OmpC
Figure 2
18%
0
10
20
30
40
Percentage of cohort
50
60
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A
100
pcorr* = 0.04
80
63%
60 40
19%
20
24/38
3/16
Anti-T TNF discontinuation ffree probability
B
Anti-OmpC –ve (n=39)
A ti O C +ve (n=16) Anti-OmpC ( 16) p** = 0.03
0
1 Year Infliximab Response Anti-OmpC +ve
Figure 3
Anti-OmpC -ve
Time on anti-TNF therapy (years)
Serological Markers Associated with Disease Behavior and Response to Anti-TNF Therapy in Ulcerative Colitis1
Short Title: IBD Serology and Ulcerative Colitis D Kevans, MD1,2, M Waterman, MD1,2, R Milgrom, MD1, W Xu, PhD3, G Van Assche, PhD1,2, MS Silverberg, PhD1,2
1. Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital, Toronto
2. Division of Gastroenterology, Department of Medicine, University of Toronto 3. Dalla Lana School of Public Health, University of Toronto
Corresponding Author David Kevans, MD, Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital, 60 Murray Street, Toronto, Ontario ON M5T 3L9 Tel: 416-586-4800 ext. 8349 Fax: 416-586-5932 [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.12661
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Abstract
Background & Aim Information is limited on the relationship between serological markers and disease behavior and anti-tumor necrosis factor-α(anti-TNF) therapy response in ulcerative colitis(UC). This
study aimed to determine the association between serological markers and unfavorable UC behavior defined as need for colectomy or UC-related hospitalization. The association between serological markers and requirement for and outcome of anti-TNF therapy was also evaluated. Methods
230 patients were studied. Requirement for colectomy, UC-related hospitalization, and antiTNF therapy were documented. Response to anti-TNF therapy at 1 year and rates of therapy discontinuation were recorded. Titers
of
peri-nuclear anti-neutrophil
cytoplasmic
antibodies(pANCAs), anti-Saccharomyces Cerevisiae antibody(ASCA) and antibody to Escherichia Coli outer membrane porin(anti-OmpC) were determined. Antibody reference ranges were used to dichotomize subjects into seropositive and seronegative groups. Where multiple tests were performed p values were Bonferroni corrected (pcorr). Results
Extensive colitis was associated with requirement for colectomy and UC-related hospitalization, HR 7.7 (95%CI 1.9 – 32.2) pcorr=0.03 and HR 2.7 (95%CI 1.5 – 4.6),
pcorr=0.006 respectively. No serological variable was associated with unfavorable UC behavior. Anti-OmpC positivity was associated with a lack of response to anti-TNF therapy at 1 year [OR 0.14 (95% CI 0.03 - 0.60), pcorr= 0.04] and increased likelihood of therapy discontinuation [HR 2.2 (95% CI 1.1 – 4.7), p=0.03].
Conclusions Extensive colitis is associated with unfavorable disease course in UC. Anti-OmpC holds promise as a biomarker of anti-TNF therapy response in UC however prospective studies are required before it can be incorporated into routine clinical practice. Keywords: UC; pANCA; ASCA; Anti-OmpC; anti-TNF therapy
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Introduction Inflammatory bowel diseases (IBD) encompass a range of inflammatory disorders affecting the intestinal tract, with the two main entities being ulcerative colitis (UC) and Crohn’s disease (CD). While its etiology remains unknown, IBD is thought to occur due to an aberrant immune response to intestinal bacteria in a genetically susceptible host. This theory is supported by the presence of a number of antibodies, in UC and CD, which are directed against self and microbial antigens. Anti-neutrophil cytoplasmic antibodies (ANCAs) occur in the sera of patients with a number of other chronic inflammatory disorders including UC. Reported positivity rates for ANCAs in UC, CD and primary sclerosing cholangitis (PSC) are 60 - 80%, 5 - 25% and 88% respectively.1-4 Anti-Saccharomyces Cerevisiae antibody (ASCA),1, 5, 6 and an antibody directed against Escherichia coli outer membrane porin (antiOmpC) have more recently been found to occur in CD and less commonly UC.1,
6
While
these IBD-associated antibodies can be a useful aid in classifying IBD subtypes and in predicting disease course in CD, data are few regarding their relationship with disease behavior in UC. Reports regarding the association between anti-tumor necrosis factor-α (TNF) therapy response, in UC and CD, and seropositivity for various IBD-associated
antibodies have also been few and conflicting.7-9 We therefore aimed to determine the association between clinical variables, pANCA, ASCA and anti-OmpC and unfavorable UC disease course, defined as need for colectomy or UC-related hospitalization. We also assessed the association between these variables and the requirement for, response to, and rates of discontinuation of anti-TNF therapy.
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Methods
Patient Population & Definitions Subjects with a diagnosis of UC were identified from an institutional database with those with a prior colectomy at the time of serum collection excluded. Of the 264 eligible subjects with UC; 20 were excluded due the lack of an available serum sample while a further 14 were excluded due to a lack of clinical follow up or as they were unclassified IBD (IBD-U);10 resulting in an eligible study cohort of 230 UC subjects. UC was diagnosed by the accepted clinical, endoscopic, radiological and pathological criteria.10 Information on clinical variables including age at diagnosis, disease extent, UCrelated hospitalizations, previous surgery, medication use, and response to anti-TNF therapy was obtained by review of medical records. Disease extent was categorized, based on the maximal extent observed during endoscopic follow up into proctitis, left-sided colitis and extensive colitis.10 UC-related hospitalization was defined as any hospitalization for the management of UC disease activity. Indication for colectomy was determined in each case and only those performed for medically refractory disease were considered in analyses. The study received approval from the institutional research ethics board and all included subjects gave informed consent.
Definitions of outcome of Anti-TNF therapy Patients receiving Infliximab therapy (n=59) were administered an initial infusion of infliximab at a dose of 5mg/Kg; the majority (n=50) received conventional 3 dose induction, a minority (n=7) received 1 or 2 dose induction; while a small number (n=2) had no data available on the induction regime received. Thereafter maintenance therapy was administered as appropriate and dose escalation was allowed whenever clinically indicated. Subjects were
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excluded from analyses where there was uncertainty regarding their anti-TNF therapy response at 1 year or requirement for therapy discontinuation; N=54 subjects had available data on therapy response while n=55 subjects had data available on requirement for therapy discontinuation. Anti-TNF response was categorized by review of clinical notes and was defined at 1 year as the absence of UC-related symptoms, no systemic corticosteroid use, no requirement for regular rectal corticosteroid or 5ASA therapy, ongoing Infliximab therapy and no requirement for colectomy. Rates and time to discontinuation of anti-TNF therapy were also documented with subjects discontinuing therapy for any reason included in analyses.
IBD-Associated Antibody Estimation Serum was separated from venous whole blood and stored at -70°C. Thawed samples were tested at Prometheus Laboratories (San Diego, CA) in a manner previously described.11 Due
to the retrospective nature of this study, serum samples were processed on two different serological platforms depending on their time of collection 129 samples were analyzed using the IBD Diagnostics System - Generation II (Prometheus Laboratories, San Diego, CA) while a further 101 samples were analyzed using the IBD SGI Platform (Prometheus Laboratories, San Diego, CA). Sera were tested for the presence of the following IBD-associated antibodies: ANCA, ASCA (IgA and IgG) and anti-OmpC. The two Prometheus platforms differ in the reference ranges applied for each IBD-associated antibody. For the IBD Diagnostics System – Generation II, antibodies were considered positive if they exceeded the following titers: ASCA IgA > 20EU/mL, ASCA IgG > 40 EU/mL, and anti-OmpC > 16.4 EU/mL. pANCA positivity was defined as an ANCA enzyme linked immunosorbent assay (ELISA) titer > 12.1 EU/mL and pANCA immunofluorescence assay (IFA) reactive / deoxyribonuclease (DNAse) sensitive. For the IBD SGI Diagnostic Platform, antibodies were
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considered positive if they exceeded the following titers: ASCA IgA ≥ 8.5 EU/mL, ASCA IgG ≥ 17.8 EU/mL and anti-OmpC ≥ 10.9 EU/mL. pANCA positivity was defined as an ANCA ELISA titer ≥ 19.8 EU/mL and pANCA IFA reactive / DNAse sensitive. For both panels subjects were considered ASCA positive if either ASCA IgA or IgG was positive. Given the differences in the reference ranges between the two IBD serology platforms utilized, actual antibody titers were not used in the analysis but rather subjects were categorized as positive or negative for each IBD-associated antibody.
Study Endpoints Clinical variables (age, gender and colitis extent) and IBD associated antibodies (pANCA, ASCA and anti-OmpC) were associated with study endpoints. Two primary endpoints were selected to represent unfavorable UC disease behavior: need for colectomy and requirement for 1 or more UC-related hospitalization (referred to hereafter as “UC-related hospitalization”). Secondary endpoints assessed were the requirement for anti-TNF therapy, anti-TNF therapy response at 1 year, and rates of discontinuation of anti-TNF therapy.
Statistical Analysis Continuous data were presented as medians and ranges. Age at diagnosis and colitis extent at diagnosis were dichotomized into two groups for survival analyses: greater versus less than 17 years & extensive colitis versus left-sided colitis respectively. As anti-TNF agents only entered routine clinical practice around the year 2000, only n=93 subjects diagnosed on or after the year 2000 were included in the analysis of time to requirement for anti-TNF therapy. Univariate Cox proportional hazard regression was used to explore associations between variables and time to colectomy, UC-related hospitalization and anti-TNF therapy. Variables associated with response to anti-TNF therapy were evaluated using univariate binary logistic
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regression. For each endpoint assessed, in univariate analysis, Bonferroni correction was applied to p values (pcorr). Where more than one variable demonstrated a significant
association (pcorr < 0.1) with a given endpoint in univariate analysis, these variables were tested in a multivariate model (uncorrected p values reported). For the endpoints “1 year antiTNF response” & “anti-TNF therapy discontinuation” there was an exception to this approach with concomitant immunomodulator use at the time of anti-TNF therapy (which has been associated with improved efficacy of anti-TNF therapy) included in multivariate models irrespective of the significance of its association with a given endpoint in univariate analysis. Kaplan-Meier survival curves were constructed for variables and differences between groups evaluated using the logrank test (uncorrected p values reported). Statistical analysis was performed by using SAS (version 9.1.2; SAS Institute, Cary, North Carolina, USA) and Statistical Package for the Social Sciences (IBM, New York, USA)
Results
Baseline Characteristics The baseline characteristics of the cohort are summarized TABLE 1. 230 subjects were studied; the median age at diagnosis was 25.5 years (range 8.7 – 59.6). 51% of subjects were
female. Median follow up duration from diagnosis and time of serum collection were 12.1 years (range 0 – 45.6) and 3.7 years (0 – 9.6) respectively. Of the cohort, 3% had proctitis,
32% had left-sided colitis and 65% had extensive colitis. 6% of subjects were current smokers. The proportion of subjects exposed to 5-ASA, thiopurine, oral prednisone (at least one course) and Infliximab therapy was: 100%, 39%, 76%; and 26%, respectively. During the
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follow up period, UC-related hospitalization and colectomy occurred in 46% (n=105) and 16% (n=37) of subjects respectively [FIGURE 1A].
IBD-Associated Antibodies Profiles and Relationship with Demographic Factors Positivity for pANCA, ASCA and anti-OmpC occurred in 57% (n=131), 11% (n=26), and 18% (n=41) of the cohort respectively [FIGURE 2]. There was no significant association demonstrated between IBD-associated antibody positivity and gender, younger age at diagnosis (age less than 17 years), colitis extent or current smoking status.
Association between Clinical Variables and Colectomy, UC-related Hospitalization and Requirement for Anti-TNF therapy The association of colectomy, UC-related hospitalization and requirement for anti-TNF therapy with the following clinical variables was assessed: younger age at diagnosis, gender and colitis extent. The only clinical variable associated with the study endpoints was colitis extent. In a univariate analysis extensive colitis was significantly associated with requirement for colectomy and UC-related hospitalization HR 7.7 (95% CI: 1.9 – 32.2), pcorr=0.03 and HR 2.7 (95% CI: 1.5 – 4.6), pcorr=0.006 respectively.[TABLE 2 and FIGURE 1 B & C] As
extensive colitis was the only clinical or serological variable significantly associated with colectomy or requirement for UC-related hospitalization a multivariate analysis was not performed. There was no association between clinical variables and requirement for anti-TNF therapy.
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Association between IBD-Associated Antibodies and Requirement for Colectomy, UCRelated Hospitalization and Anti-TNF Therapy In a univariate analysis pANCA positivity showed no association with requirement for colectomy HR 1.3 (95% CI 0.7 – 2.6), pcorr=1.0.[FIGURE 1D] ASCA and anti-OmpC were
not associated with requirement for colectomy either.[TABLE 2] There was no significant association
between
IBD-associated
antibodies
and
requirement
for
UC-related
hospitalization or anti-TNF therapy.[TABLE 2] As no serological variable demonstrated an association with any of the study endpoints a multivariate analysis was not performed.
Association of Clinical Variables and Serological Variables with Outcome of Anti-TNF Therapy
In a univariate analysis (n=54 subjects) anti-OmpC was the only clinical or serologic variable associated with anti-TNF therapy response.[TABLE 3] Anti-OmpC positivity was associated with a lack of response to anti-TNF therapy at 1 year, Odds ratio (OR) 0.14 (95% CI 0.03 0.6), pcorr = 0.04; with 3 of 16 (19%) anti-OmpC positive subjects versus 24 of 38 (63%) of anti-OmpC negative subjects responding to anti-TNF therapy at 1 year.[Figure 3A] The use of concomitant immunomodulators in combination with anti-TNF therapy was not associated with improved 1 year therapy response, OR 1.4 (95% CI 0.4 – 4.6), pcorr = 1.0.[TABLE 3] In a multivariate model, which included concomitant immunomodulator use, anti-OmpC positivity remained independently associated with a decreased 1 year anti-TNF therapy response, OR 0.14 (95% CI 0.03 – 0.6), p=0.006. The probability of discontinuation of anti-
TNF therapy was significantly higher in anti-OmpC positive versus negative subjects (median time free of anti-TNF therapy discontinuation, 1.1 versus 6.0 years respectively); HR (for discontinuation of anti-TNF therapy) 2.2 (95% CI 1.1 – 4.7), p=0.03.[Figure 3B] The use of concomitant immunomodulators in combination with anti-TNF therapy was not associated
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with a decreased rate of therapy discontinuation, HR 0.8 (95% CI 0.4 – 1.9), p=0.64. In a
multivariate model, which included concomitant immunomodulator use, anti-OmpC positivity remained independently associated with likelihood of anti-TNF therapy discontinuation, HR 2.3 (95% CI 1.0 – 5.0), p=0.04.
Discussion
Consistent with previous reports we have shown that extensive colitis is associated with adverse disease behavior in UC.12,
13
We did not find an association between pANCA
positivity and disease behavior or anti-TNF therapy response in this cohort. We demonstrated a novel association between anti-OmpC positivity and lack of response to anti-TNF therapy at 1 year and increased likelihood of anti-TNF therapy discontinuation.
ANCAs describe a class of autoantibodies directed against antigens in the cytoplasm of neutrophils and monocytes. By indirect immunofluorescent microscopy, ANCA activity has been divided into two broad categories: cytoplasmic staining (cANCA) and perinuclear staining (pANCA). Another pattern of immunofluorescence in the setting of detectable ANCA has been observed characterized by a broad inhomogeneous rim-like staining of the nuclear periphery. These so called “atypical” pANCAs have been observed in UC, primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH).1 The pANCA seropositivity rate of 57% in our cohort was similar to that previously reported in UC where 41 - 73% of individuals have been found to be pANCA seropositive.14
A number of antibodies against microbial species have been described in IBD. Increased ASCA concentrations have been found in 44 - 62% of patients with CD and 10 - 15% of patients with UC.1,
6, 11
Outer membrane porin C (OmpC) is an Escherichia Coli protein
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which regulates bacterial metabolite and toxin transport. Anti-OmpC has been demonstrated to be an IBD-associated antibody occurring in 24 - 42% of CD patients, 11 - 28% of UC patients and 3 - 5% of healthy controls.6, 11 ASCA and anti-OmpC sero-positivity rates in our
cohort of 11% and 18% respectively, were consistent with these reports.
The association between IBD-associated antibodies and disease behavior has been best demonstrated in CD and in individuals with an ileal pouch-anal anastomosis performed in the setting of IBD.1,
15-17
Positivity for more than one IBD-associated antibody as well as the
actual antibody titer may also be important as several studies have shown that higher titers of multiple anti-microbial antibodies are associated with a more aggressive CD disease behavior.18, 19 Data on the association of IBD-associated antibodies with disease behavior in UC are few and have generally been restricted to the evaluation of pANCA and ASCA. There have been discordant reports regarding the association of pANCA and disease course in UC. Vecchi et al. demonstrated pANCA positivity to be associated with unfavourable disease behaviour,20 while a second larger multicentre study demonstrated increased risk of disease
relapse, but not colectomy to be significantly associated with pANCA and ASCA sero-
positivity.21 On the other hand a report by Seow et al. showed no association between pANCA positivity and either clinical remission or requirement for colectomy.22 In our cohort we did not find an association between pANCA positivity and UC disease behavior expressed as requirement for colectomy, UC related hospitalization or anti-TNF therapy.
To our knowledge only two previous studies have evaluated the association between antiOmpC and disease behaviour in UC. A study by Elkadri et al. demonstrated an association between anti-OmpC positivity and requirement for colectomy in UC.6 While a report by Papp
et al., utilizing an anti-OmpC assay which detected antibodies against multiple bacterial
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proteins derived from two species of intestinal bacteria (neither of which were Escherichia
coli), showed no association between anti-OmpC serotype and UC disease course.23, 24 We did no demonstrate any association between anti-OmpC positivity and adverse disease behavior in UC in our cohort.
The utility of pANCA as a predictor of anti-TNF response in UC and CD has been assessed in a number of studies with differing results._ENREF_257-9 Data on the utility of anti-OmpC
as predictor of response to anti-TNF therapy is very limited with only one previous study assessing this issue. Papp et al. evaluated the utility of anti-OmpC in CD as a predictor of response to anti-TNF therapy and found no association. As previously mentioned this study utilized an anti-OmpC assay which differed significantly from that used in the current study.25 Anti-OmpC has not previously been evaluated as a predictor of anti-TNF response in
UC. We demonstrated a novel association between anti-OmpC positivity and lack of response
to anti-TNF therapy at 1 year. In addition we demonstrated a significantly greater probability of discontinuing anti-TNF therapy in anti-OmpC positive versus negative subjects. These
data suggest that anti-OmpC is a potential biomarker of anti-TNF response in UC. It may be a that an intestinal microbial profile associated with anti-TNF therapy resistance in UC, results in anti-OmpC reactivity, however clearly this hypothesis needs to be formally evaluated in future studies. An important caveat to these data is the relatively small number of subjects included in our analysis of variables associated with anti-TNF response, therefore while this association is potentially important it requires validation in independent cohorts.
We acknowledge that this report is based on a referral center cohort with the inherent limitations associated with this type of study design. Like many other studies which have assessed the association between serology and IBD disease behavior, this study was limited
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by its cross sectional rather than prospective design, with serum sampling occurring later in subject’s disease course rather than at or soon after diagnosis. To address this limitation
subjects were only included if clinical follow up was available at the time of, or subsequent to their serum draw, therefore some prospective follow up was available for the majority of subjects. If IBD-associated antibodies are to be used in routine clinical practice they most
likely need to provide prognostic information early in UC disease course. An important question therefore is whether IBD-associated antibody profiles estimated in subjects with established UC, as performed in this study, reflect those estimated near the time of UC diagnosis. Information in this regard is provided in a report by Rieder et al. which demonstrated that, while substantial changes in anti-glycan antibody levels (such as ASCA) occur over time, antibody positivity / negativity remained stable in the vast majority of UC and CD patients.19 P-ANCA status, on the other hand has been found to change from positive to negative after colectomy.26 As we excluded subjects who had undergone a prior colectomy
at the time of serum collection and performed our analysis considering only positivity / negativity for each IBD-associated antibody, we believe that while this study characterized
antibody profiles in subjects with established UC, these profiles are likely to be representative of those estimated earlier in disease course.
In summary, consistent with previous reports, we have shown extensive colitis to be associated with adverse disease behavior in UC. We did not demonstrate an association between pANCA positivity and UC behavior or anti-TNF therapy response. We demonstrated a novel association between anti-OmpC positivity and lack of response to anti-TNF therapy at 1 year and increased probability of anti-TNF therapy discontinuation. Anti-OmpC holds promise as biomarker of anti-TNF therapy response in UC however larger prospective studies are required before this marker can be incorporated into routine clinical practice.
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Acknowledgements These data were presented at Digestive Disease Week 2013, Orlando, Florida, USA. David Kevans is a recipient of a CIHR/CAG/Abbvie IBD Fellowship and honoraria from Abbvie; Gert Van Assche recieves honoraria and/or research support from Abbvie, Janssen, MSD, Ferring, Aptatlis, Warner-Chilcott, Shire, UCB, Novartis, Chiesi and BMS. Mark Silverberg is partially supported by the Gale and Graham Wright Research Chair in Digestive Disease and by grants from CCFC and NIDDK (DK-062423). Mark Silverberg receives speaker fees, consulting fees and research support from Prometheus Laboratories. The authors have no conflicts to disclose.
References
1. X. Bossuyt. Serologic markers in inflammatory bowel disease. Clin Chem 2006; 52: 17181.
2. A. Saxon, F. Shanahan, C. Landers, T. Ganz and S. Targan. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990; 86: 202-10.
3. J. A. Rump, J. Scholmerich, V. Gross, et al. A new type of perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) in active ulcerative colitis but not in Crohn's disease. Immunobiology 1990; 181: 406-13. 4. B. Terjung and H. J. Worman. Anti-neutrophil antibodies in primary sclerosing cholangitis. Best Pract Res Clin Gastroenterol 2001; 15: 629-42. 5. C. J. Landers, O. Cohavy, R. Misra, et al. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123: 689-99. 6. A. A. Elkadri, J. M. Stempak, T. D. Walters, et al. Serum antibodies associated with complex inflammatory bowel disease. Inflamm Bowel Dis 2013; 19: 1499-505. 7. M. Ferrante, S. Vermeire, K. H. Katsanos, et al. Predictors of early response to infliximab in patients with ulcerative colitis. Inflamm Bowel Dis 2007; 13: 123-8. 8. N. Esters, S. Vermeire, S. Joossens, et al. Serological markers for prediction of response to anti-tumor necrosis factor treatment in Crohn's disease. Am J Gastroenterol 2002; 97: 145862. 9. M. Jurgens, R. P. Laubender, F. Hartl, et al. Disease activity, ANCA, and IL23R genotype status determine early response to infliximab in patients with ulcerative colitis. Am J Gastroenterol 2010; 105: 1811-9. 14 This article is protected by copyright. All rights reserved.
Accepted Article
10. M. S. Silverberg, J. Satsangi, T. Ahmad, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005; 19 Suppl A: 5A-36A. 11. A. Zholudev, D. Zurakowski, W. Young, A. Leichtner and A. Bousvaros. Serologic testing with ANCA, ASCA, and anti-OmpC in children and young adults with Crohn's disease and ulcerative colitis: diagnostic value and correlation with disease phenotype. Am J Gastroenterol 2004; 99: 2235-41.
12. L. E. Targownik, H. Singh, Z. Nugent and C. N. Bernstein. The epidemiology of colectomy in ulcerative colitis: results from a population-based cohort. Am J Gastroenterol 2012; 107: 1228-35. 13. G. Bresci, G. Parisi and A. Capria. Duration of remission and long-term prognosis according to the extent of disease in patients with ulcerative colitis on continuous mesalamine treatment. Colorectal Dis 2008; 10: 814-7. 14. J. H. Sellin and R. R. Shah. The promise and pitfalls of serologic testing in inflammatory bowel disease. Gastroenterol Clin North Am 2012; 41: 463-82. 15. D. K. Amre, S. E. Lu, F. Costea and E. G. Seidman. Utility of serological markers in predicting the early occurrence of complications and surgery in pediatric Crohn's disease patients. Am J Gastroenterol 2006; 101: 645-52. 16. W. S. Mow, E. A. Vasiliauskas, Y. C. Lin, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126: 414-24. 17. A. D. Tyler, R. Milgrom, W. Xu, et al. Antimicrobial antibodies are associated with a Crohn's disease-like phenotype after ileal pouch-anal anastomosis. Clin Gastroenterol Hepatol 10: 507-12 e1. 18. I. D. Arnott, C. J. Landers, E. J. Nimmo, et al. Sero-reactivity to microbial components in Crohn's disease is associated with disease severity and progression, but not NOD2/CARD15 genotype. Am J Gastroenterol 2004; 99: 2376-84. 19. F. Rieder, S. Schleder, A. Wolf, et al. Association of the novel serologic anti-glycan antibodies anti-laminarin and anti-chitin with complicated Crohn's disease behavior. Inflamm Bowel Dis 2010; 16: 263-74. 20. M. Vecchi, M. B. Bianchi, C. Calabresi, G. Meucci, M. Tatarella and R. de Franchis. Long-term observation of the perinuclear anti-neutrophil cytoplasmic antibody status in ulcerative colitis patients. Scand J Gastroenterol 1998; 33: 170-3. 21. O. Hoie, G. Aamodt, S. Vermeire, et al. Serological markers are associated with disease course in ulcerative colitis. A study in an unselected population-based cohort followed for 10 years. J Crohns Colitis 2008; 2: 114-22.
15 This article is protected by copyright. All rights reserved.
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22. C. H. Seow, A. Newman, S. P. Irwin, A. H. Steinhart, M. S. Silverberg and G. R. Greenberg. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut 2010; 59: 49-54.
23. M. Papp, I. Altorjay, N. Dotan, et al. New serological markers for inflammatory bowel disease are associated with earlier age at onset, complicated disease behavior, risk for surgery, and NOD2/CARD15 genotype in a Hungarian IBD cohort. Am J Gastroenterol 2008; 103: 665-81. 24. M. Papp, G. L. Norman, Z. Vitalis, et al. Presence of anti-microbial antibodies in liver cirrhosis - a tell-tale sign of compromised immunity? PLoS One 2010; 5: e12957. 25. M. Papp, I. Altorjay, G. L. Norman, et al. Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients. Inflamm Bowel Dis 2007; 13: 984-92. 26. J. A. Rump, I. Worner, M. Roth, J. Scholmerich, M. Hansch and H. H. Peter. p-ANCA of undefined specificity in ulcerative colitis: correlation to disease activity and therapy. Adv Exp Med Biol 1993; 336: 507-13.
Figure Legend
Figure 1. Kaplan Meir curves of clinical and serological variables significantly associated with requirement for colectomy, UC-related hospitalization and anti-TNF therapy.
Panel A. Cumulative colectomy-free survival in the study cohort (n=230); Panel B. Colectomy free survival segregated by colitis extent; Panel C. Survival free of UC-related hospitalization segregated by colitis extent; Panel D. Colectomy-free survival segregated by pANCA serotype.
Figure 2. Prevalence of IBD-associated antibodies in cohort (n=230).
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Accepted Article
Figure 3. Anti-OmpC as a predictor of outcome of anti-TNF therapy.
Panel A demonstrates that a lesser proportion of patients achieved anti-TNF therapy response at 1 year in anti-OmpC positive compared with anti-OmpC negative groups. Panel B displays a Kaplan Meir survival curve comparing survival free of anti-TNF therapy discontinuation in
anti-OmpC positive versus anti-OmpC negative groups. Anti-OmpC positive versus negative subjects had a greater probability of earlier anti-TNF therapy discontinuation (median time free of anti-TNF therapy discontinuation, 1.1 years versus 6.0 years respectively); HR (for discontinuation of Anti-TNF therapy 2.2 (95% CI 1.1 – 4.7), p = 0.03.
*Univariate binary logistic regression (bonferroni corrected p value) **Log rank test (uncorrected p value)
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25.5 (8.7 – 59.6)
Accepted Article
Age at Diagnosis (years) † Gender Female Male Colitis Extent at Diagnosis Proctitis (E1) Left-sided Colitis (E2) Extensive Colitis (E3) Primary Sclerosing Cholangitis No Yes Smoking Status Non-Smoker Ex-Smoker Current Smoker Follow up (years) † From diagnosis From time of serum collection UC-Related Hospitalization No Yes Colectomy ‡ No Yes 5ASA Therapy Yes Oral Prednisone § No Yes Thiopurine therapy No Yes Infliximab Therapy No Yes
118 (51%) 112 (49%) 8 (3%) 73 (32%) 148 (65%) 217 (96%) 8 (4%) 143 (63%) 69 (31%) 14 (6%) 12.1 (0 – 45.6) 3.7 (0 – 9.6) 122 (54%) 105 (46%) 195 (84%) 37 (16%) 230 (100%) 54 (24%) 175 (76%) 141 (61%) 89 (39%) 171 (74%) 59 (26%)
Table 1. Baseline Characteristics of the Study Cohort (n=230).
†Continuous variables presented as median (range) ‡Colectomy for medically refractory disease
§Exposure to at least one course of oral prednisone Where totals in each row do not sum to 230 data was unavailable or unknown
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Accepted Article
Colectomy UC-Related Hospitalization Anti-TNF Therapy† HR (95% CI) Pcorr HR (95% CI) Pcorr HR (95% CI) PCorr 1.1 (0.5 – 2.7) 1.0 0.7 (0.4 – 1.2) 1.0 0.9 (0.4 – 1.9) 1.0 Age > 17 years 1.2 (0.6 – 2.2) 1.0 0.7 (0.5 – 1.1) 0.54 0.8 (0.4 – 1.7) 1.0 Male gender 7.7 (1.9 – 32.2) 2.7 (1.5 – 4.6) 0.006 4.4 (1.3 – 14.3) 0.12 Extensive Colitis 0.03 1.3 (0.7 – 2.6) 1.0 1.2 (0.8 - 1.8) 1.0 1.6 (0.8 – 3.5) 1.0 pANCA+ ve 0.4 (0.1 – 1.7) 1.0 1.3 (0.7 - 2.3) 1.0 1.7 (0.8 – 4.0) 1.0 ASCA+ve 1.6 (0.8 – 3.4) 1.0 1.3 (0.8 – 2.0) 1.0 1.7 (0.8 – 3.9) 1.0 Anti-OmpC+ve Table 2. Univariate analysis evaluating the association between clinical and serologic variables and the requirement for colectomy, UCRelated Hospitalization and anti-TNF therapy.
† N=93 subjects diagnosed on or after the year 2000 and with available data on therapy commencement date were included in analysis Pcorr represents p value corrected for multiple tests performed by Bonferroni method
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Age > 17 years Male gender Extensive colitis Concomitant IMM use pANCA +ve ASCA +ve Anti-OmpC +ve
Anti-TNF Therapy Response Pcorr† OR (95% CI) 1.5 (0.4 – 5.6) 1.0 1.6 (0.5 – 4.7) 1.0 0.3 (0.03 – 3.2) 1.0 1.4 (0.4 – 4.6) 1.0 0.7 (0.2 – 2.2) 1.0 1.0 (0.2 – 4.5) 1.0 0.14 (0.03 - 0.60) 0.04
Table 3. Univariate analysis of clinical and serological variables associated with antiTNF therapy response at 1 year (n=54 subjects). †Pcorr represents p values corrected for multiple testing by Bonferroni correction method Anti-TNF therapy constituted Infliximab in all cases Concomitant immunomodulator (IMM) use at commencement of anti-TNF therapy (n=16 subjects): thiopurines (n=14), tacrolimus (n=1), methotrexate (n=1) Infliximab response at 1 year was defined as an absence of symptoms related to UC activity, no requirement for systemic corticosteroids, no requirement for regular topical rectal corticosteroid or 5ASA therapy and continuation of Infliximab therapy.
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Accepted Article
A
B Left-sided Colitis (n=69) Study cohort (n=230)
Extensive Colitis (n=161)
p = 0.001
C
Figure 1
D pANCA -ve (n=99) Left sided Colitis (n=65) Left-sided pANCA +ve (n=131)
Extensive Colitis (n=164)
p < 0.001
p = 0.42
Accepted Article
57%
pANCA+
ASCA+
11%
Anti-OmpC
Figure 2
18%
0
10
20
30
40
Percentage of cohort
50
60
Accepted Article
A
100
pcorr* = 0.04
80
63%
60 40
19%
20
24/38
3/16
Anti-T TNF discontinuation ffree probability
B
Anti-OmpC –ve (n=39)
A ti O C +ve (n=16) Anti-OmpC ( 16) p** = 0.03
0
1 Year Infliximab Response Anti-OmpC +ve
Figure 3
Anti-OmpC -ve
Time on anti-TNF therapy (years)
