Role of Inflammatory Markers as Predictors of Clinical Activity and Endoscopic Severity in Crohn ’ s Disease

Objective: We aim to assess the role of inflammatory markers as predictors of clinical activity and endoscopic severity in Crohn’s disease (CD). Methods: Patients attending for colonoscopy with known or suspected CD were recruited. Clinical disease activity was recorded as per the Crohn’s Disease Activity Index (CDAI) and endoscopic activity was recorded using the simple endoscopic score (SES) for CD. Receiver operating characteristic analysis (ROC) determined the predictive value and optimal predictive thresholds for the inflammatory markers. Results: The folllowing parameters were significantly different between clinically active CD and CD in remission: SES, C-reactive protein (CRP), white blood cell count (WBC), neutrophil count, hemoglobin, platelet count, albumin and fecal calprotectin (FC). And the following parameters were significantly different between patients with mild (SES<7) and moderate to severe (SES≥7) endoscopic activity: CDAI, WBC, CRP, neutrophil count, albumin and FC. The cut-off value for CRP for the detection of moderate to severe endoscopic activity in CD patients was calculated as ≥11.5 mg/L using ROC analysis [Sensitivity: 70%, specificity: 100%, AUC: 0.790 (0.567-1.013), P=0.028] whereas this cut-off value was 487 μg/g for FC [Sensitivity: 70%, specificity: 90%, AUC: 0.805 (0.598-1.01), P=0.028]. All of the 6 patients with FC >487 μg/g and CRP>11.5 mg/L were found to have moderate to severe endoscopic activity. Conclusion: Increased concentrations of either CRP or FC are predictive of clinical and endoscopic disease activity.


INTRODUCTION
Crohn's disease (CD) is a chronic, debilitating subset of inflammatory bowel diseases (IBD) that necessitates a life-long follow-up.The disease has a dynamic behaviour and disease activity often shows fluctuations (1,2).Approximately 13% of CD patients have chronically active disease, 73% have a chronic intermittent course, while only 10% remain in prolonged remission over many years (2).Monitoring disease activity is a critical subject to optimize management of CD patients.Symptom assessment should be used in each patient.The CD activity index (CDAI) has evolved as the gold standard for clinical trials (3).But analysis of data from a large, prospective, well-defined subpopulation of the SONIC trial (4) showed that CDAI was not accurate to predict C-reactive protein (CRP) normalisation and/or mucosal healing in CD as half the patients with CD in clinical remission had endoscopic and/or CRP evidence of residually active CD (5).Objective outcome measures of CD activity, in addition to the assessment of clinical symptoms, are obviously needed to evaluate disease activity in CD.
In this study, we aim to assess the role of inflammatory markers as predictors of clinical activity and endoscopic severity in Crohn's disease.

METHODS
Patients presenting to the Gastroenterology Department of our institution with gastrointestinal symptoms suggestive of CD or a previous diagnosis of CD and who had colonoscopic examination between March 2011 and March 2015 were retrospectively enrolled into this study.Patients were recruited 2 weeks before their appointment to allow fort h collection of fecal samples before bowel preparation.Basic demographic and disease-specific data were collected at the time of colonoscopy.Colonoscopy was performed by specialist staff experienced in the diagnosis of IBD.The diagnosis of CD was made using standard criteria based on endoscopic, histological, and radiological appearances.The clinical activity was evaluated before colonoscopy by means of Crohn's Disease Activity Index (CDAI) and remission was defined as a reduction of CDAI to a score below 150 points (6).Endoscopic severity was assessed using the simple endoscopic score for CD (SES) (7).Accordingly SES-CD 3 to 6, mild disease; SES-CD 7 to 15, moderate; SES-CD 16, severe.
The study protocol was approved by the ethics committee of Gazi University, and the study was carried out in accordance with the principles of the Declaration ofHelsinki.

Statistical Analyses
All statistical analyses were performed with SPSS version 15.0 for Windows (Chicago, IL).Normality was assessed using the Kolmogorov-Smirnov test.Accordingly data were expressed as median (min-max) or mean±SD.Demographic and clinical characteristics were compared among study groups using analysis of variance for continuous variables and Chi square analysis for dichotomous variables.Receiver operating characteristic (ROC) curve analysis was used to determine the overall diagnostic utility of a test for differentiating between active disease and remission in regard to CDAI index and SES score.A two sided p value of 0.05 or less was considered statistically significant.

RESULTS
The clinical and demographic characteristics of CD patients were presented in Table 1.The comparison of inflammatory markers between CD patients with remission and active disease in regard to CDAI index and SES score were shown in table 2 and 3. Accordingly the following parameters were significantly different between the active CD patients and CD patients in remission: SES, CRP, WBC, neutrophil count, hemoglobin, platelet count, albumin and FC.And the following parameters were significantly different between patients with mild (SES<7) and moderate to severe (SES≥7) endoscopic activity: CDAI, WBC, CRP, neutrophil count, albumin and FC.ROC curve analysis results that show which inflammatory markers had a significant ability to differentiate between clinically active disease vs remission and moderate to severe (SES≥7) vs mild (SES≤7) mucosal involvement were demonstrated in table 1.And ROC curves for FC and CRP for the diagnosis of clinically active Crohn's disease and moderate to severe endoscopic activity were shown in figure 1 and 2. All of the 6 patients who had a FC > 487 µg/g and CRP>11.5 mg/dL were found to have moderate to severe mucosal involvement.

DISCUSSION
Achieving endoscopic healing after medical therapy has been associated with a better disease outcome (8).Mucosal activity assessed by ileocolonoscopy is predictive of requiring abdominal surgery (9) and hospitalisation under under anti-Tumor necrosis factor (TNF) therapy (10) and risk of relapse upon anti-TNF withdrawal (11).Mucosal activity is also associated with postoperative recurrence (12).Endoscopic examination, however, is neither available nor desirable for routine disease assessment, and standard clinical assessment is inaccurate in this respect.Consequently, objective markers of disease activity are needed to provide a noninvasive means of disease assessment.
FC and CRP represent objective markers of active inflammation.Fecal calprotectin is a reliable marker in predicting CD activity as it has been already proven for UC patients (13).Monitoring of CD with blood CRP and fecal calprotectin is predictive of disease relapse under or after medical therapy, response to anti-TNF treatment and mucosal healing (14)(15)(16)(17).These two biomarkers may be combined to improve prediction (14).It should, however, be kept in mind that they are not perfectly correlated with mucosal healing and predictive threshold values for these markers have not fully been established yet (8).  3.   Assessment of prognosis with laboratory values generated much interest in the past decade.Currently the three commonly used parameters for this purpose are CRP, ESR and FC.In order to predict disease relapse by biologic markers GETAID group evaluated 71 CD pateints in whom 38 had a relapse (18).Multivariate analysis defined two markers predictive of relapse: CRP >20 mg/L and ESR >15 mm.Patients positive for both markers had an 8-fold elevated risk for relapse, with a negative predictive value of 97%.Calprotectin as a fecal biomarker for assessing and monitoring disease activity in patients with IBD, can also be used as a prognostic index in CD.In a landmark study by Tibble et al [20] 43 CD patients were prospectively followed over 12 months.Twenty-five (58%) patients with CD had a relapse and median calprotectin levels in the relapse groups (122 mg/L for CD) differed significantly (P<0.0001)from those of the nonrelapse groups (41.5 mg/L).At a level of 50 mg/L, the sensitivity and specificity of calprotectin for predicting relapse was reported to be %90 in CD patients (14).
Our results showed that among all the paramters studied CRP and FC were the only paremeters that were predictors for both clinically active disease and endoscopic severity (Table 4).The cut-off value for CRP for the detection of moderate to severe mucosal disease in CD patients was calculated as ≥ 11.5 mg/dL using ROC analysis [Sensitivity: 70%, specificity: 100%, AUC: 0.790 (0.567-1.013),P=0.028] whereas this cut-off value was 487 µg/g for FC [Sensitivity: 70%, specificity: 90%, AUC: 0.805 (0.598-1.01),P=0.028].And none of the patients with FC > 487 µg/g and CRP>11.5 mg/dL had mild mucosal involvement.Although CDAI and neutrophil count also significantly predicted the presence of moderate to severe mucosal disease the cutt-off values for these parameters were within normal range.For this reason it is not proper to use these last two parameters in the clinical practice especially when the limitations of CDAI is considered.
The major limitations of our study were the small sample size, the retrospective nature and the absence of serial assessments during follow up of the patients.However our study was powerful enough to determine cut-off values for CRP and FC for the detection of moderate to severe mucosal involvement according to SES.

CONCLUSION
Biomarkers are an important facet of disease assessment in IBD.The current study found that abnormal concentrations of either CRP or FC are predictive of clinical and endoscopic disease activity.Further investigation is required to confirm our results and to identify novel and better biomarkers.

Figure 1 .
Figure 1.Receiver operating characteristic curve for fecal calprotectin (FC) and C-reactive protein (CRP) for the diagnosis of clinically active Crohn's disease.

Figure 2 .
Figure 2. Receiver operating characteristic curve for fecal calprotectin (FC) and C-reactive protein (CRP) for the diagnosis of more severe endoscopic activity (Simple endoscopic score≥7) within the study group of patients with Crohn's disease.

Table 1 .
Clinical characteristics of patients with Crohn's disease.

Table 2 .
Inflammatory markers and endoscopic severity according to clinical activity of Crohn's disease.

Table 3 .
Inflammatory markers according to endoscopic severity of Crohn's disease.

Table 4 .
Receiver operating characteristics analysis results that show which inflammatory markers have a significant ability to differentiate between clinically active disease vs remission and moderate to severe (SES≥7) vs mild (SES≤7) mucosal involvement.Simple endoscopic score.Remission: Crohn's Disease Activity Index (CDAI) < 150, Clinically Active Disease: CDAI ≥ 150.CRP: C-reactive protein.WBC: White blood cell count.N/L: Neutrophil/Lymphocyte ratio.CP: Cut off point.AUC: Area under curve.CI: Confidence interval.Sens: Sensitivity.Spec: Specificity.All units of measurements are as shown in Table