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Diagnostic Accuracy of Fecal Calprotectin Concentration in Evaluating Therapeutic Outcomes of Patients With Ulcerative Colitis

Open AccessPublished:August 12, 2020DOI:https://doi.org/10.1016/j.cgh.2020.08.019

      Background & Aims

      Histologic features of inflammation (histologic inflammation) are associated with clinical relapse in patients with ulcerative colitis (UC). Concentration of fecal calprotectin (FC) can be used to identify patients with mucosal inflammation. We aimed to assess the accuracy of FC measurements in identifying patients with histologic inflammation and to develop a model to predict outcomes of therapy.

      Methods

      We performed a post hoc analysis of data from a phase 4 trial of the efficacy of multimatrix mesalamine in patients with mild to moderate UC (the MOMENTUM trial). We obtained clinical, endoscopic, and histologic data from week 8 (n = 639) and week 52 (n = 373) of the trial. We used area under the receiver operating characteristic curves to determine the accuracy and optimal cut-off values of FC in identifying patients with different therapeutic outcomes (clinical remission, endoscopic healing, deep remission, or histologic remission) at week 8 and week 52. We performed multivariable logistic regression analyses to identify factors associated with these outcomes.

      Results

      Median FC concentrations were lower in patients who achieved outcomes of clinical remission, endoscopic healing, deep remission, or histologic remission vs patients who did not. FC concentrations identified patients with endoscopic healing and histologic remission with area under the receiver operating characteristic curve values of 0.77 and 0.76 at week 8, and 0.79 and 0.80 at week 52, respectively. The optimal FC cut-off concentrations for identification of patients with histologic remission were 75 μg/g at week 8 and 99 μg/g at week 52. In the subpopulation with an endoscopy score of 0, median FC concentrations were lower in patients with histologic remission than in patients with microscopic inflammation at week 8 (30 vs 140 μg/g; area under the receiver operating characteristic, 0.72) and week 52 (21.5 vs 134.5 μg/g; area under the receiver operating characteristic, 0.71). At both time points, the optimal FC cut-off concentration was approximately 75 μg/g. Our final prediction model for week 52 histologic remission comprised endoscopic score at week 8, FC concentration at week 8, and histologic activity at baseline and week 8.

      Conclusions

      A post hoc analysis of data from a phase 4 trial found that, even in patients with complete endoscopic healing of UC, FC concentration can be used to discriminate patients with ongoing microscopic inflammation from patients with histologic remission. The optimal cut-off concentration of FC is between 75 and 100 μg/g. ClinicalTrials.gov no: NCT01124149.

      Keywords

      Abbreviations used in this paper:

      AUC (area under the curve), FC (fecal calprotectin), IQR (interquartile range), ROC (receiver operating characteristic), UC (ulcerative colitis)

       Background

      Histologic features of inflammation are associated with clinical relapse in patients with ulcerative colitis (UC). Concentration of fecal calprotectin (FC) can be used to identify patients with mucosal inflammation.

       Findings

      A post hoc analysis of data from a phase 4 clinical trial found that even in patients with UC with complete endoscopic healing, FC concentration can be used to discriminate patients with ongoing microscopic inflammation from patients with histologic features of remission (histologic remission).

       Implications for patient care

      The optimal cut-off concentration of FC is between 75 and 100 μg/g for identification of patients with UC who are in histologic remission.
      Over the past decade there has been an evolution in therapeutic targets in inflammatory bowel disease. Treatment success now is defined by a combination of both symptom control and healing of the intestinal mucosa.
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      An accurate and noninvasive surrogate marker of histologic inflammation therefore would be of great benefit.
      The diagnostic accuracy of fecal calprotectin (FC) for endoscopic inflammation in UC is relatively high.
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      Furthermore, some studies have shown an acceptable correlation between FC values and histologic disease activity, however, with great heterogeneity in design, patient numbers, scoring indices, and definitions of remission.
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      Recently, a large post hoc analysis on the data from different clinical trials showed that FC levels are associated with the levels of different histologic grading indices and are related to the presence of neutrophils.
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      Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levels.
      Mesalamines are the first-line treatment for patients with mild-to-moderate UC.
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      • et al.
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      In the MOMENTUM trial, adult patients with mild-to-moderate UC who responded to multimatrix mesalamine 4.8 g/d at week 8 received 12 months of multimatrix mesalamine 2.4 g/d maintenance treatment.
      • Rubin D.T.
      • Bradette M.
      • Gabalec L.
      • et al.
      ulcerative colitis remission status after induction with mesalazine predicts maintenance outcomes: the MOMENTUM trial.
      In this post hoc analysis of the MOMENTUM trial, we aimed to investigate the diagnostic accuracy of FC for endoscopic and histologic remission, and develop a prediction model for different levels of remission after 1 year of treatment.

      Materials and Methods

      This was a post hoc analysis of the previously published prospective clinical trial MOMENTUM (ClinicalTrials.gov identifier: NCT01124149).
      • Rubin D.T.
      • Bradette M.
      • Gabalec L.
      • et al.
      ulcerative colitis remission status after induction with mesalazine predicts maintenance outcomes: the MOMENTUM trial.
      Data are reported according to the Standards for Reporting of Diagnostic Accuracy Studies checklist and the Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis checklist.
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      • Reitsma J.B.
      • Bruns D.E.
      • et al.
      Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative.
      ,
      • Collins G.S.
      • Reitsma J.B.
      • Altman D.G.
      • et al.
      Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis (TRIPOD): the TRIPOD statement.
      All authors had access to the study data and reviewed and approved the final manuscript. This trial was conducted with approval by the Institutional Review Board at each site and in accordance with current applicable regulations, International Conference on Harmonisation Good Clinical Practice, and local ethical and legal requirements, and in line with the principles of the Declaration of Helsinki. All participating patients provided written informed consent before screening.
      Inclusion and exclusion criteria for study participants and study treatments are detailed in the Supplementary Methods section.

       Procedures

      Fecal samples were collected at screening and at weeks 8 and 52 or at early withdrawal for the measurement of FC concentration. FC was measured by a quantitative enzyme-linked immunosorbent assay, using in-house kits. The upper detection limit was 2500 μg/g.
      Sigmoidoscopies were performed at screening and at weeks 8 and 52 or at early withdrawal. As predefined in the original study protocol, 2 biopsy specimens were taken from the rectum (≤15 cm from the anal verge, avoiding the center of ulcers) during all endoscopies. If the worst inflamed area was not the rectum, 2 additional biopsy specimens were taken from the worst inflamed area, usually the sigmoid, avoiding the center of ulceration (ie, a total of 4 biopsy specimens).

       Histology Scoring

      Biopsy specimens were fixed in neutral buffered formalin. Deidentified biopsy specimens then were paraffin-embedded at a central histopathology laboratory (GdH). Serial sections (5-μm thickness) were stained with H&E and scored using a grading system based on the Geboes et al
      • Geboes K.
      • Riddell R.
      • Ost A.
      • et al.
      A reproducible grading scale for histological assessment of inflammation in ulcerative colitis.
      grading scale by 2 independent histopathologists (G.d.H. and J.R.T.) who were blinded to the time point, participant’s clinical course, and endoscopic findings. Participants without histologically active disease at baseline were excluded from the study. If a discrepancy between the score of both histopathologists existed, the score of the lead histopathologist (G.d.H.) was used. To evaluate the correlation between FC and histologic disease activity, the histology data were transformed to an ordinal Geboes score, ranging from 0 to 13. The ordinal score was calculated as the sum of the grading values within each of the Geboes score ≥2B parameters (lamina propria neutrophils, neutrophils in epithelium, crypt destruction, and erosion/ulceration) (Supplementary Table 1). Parameters related to chronic inflammation (architectural disturbance, mononuclear cell infiltrate, and eosinophils) were scored as 0. Where multiple biopsies were performed at a visit, the largest (maximum) score was used in the analyses.

       Outcomes

      End points analyzed at the end of the induction (week 8) and maintenance (week 52) phases for the current post hoc analyses were clinical remission (rectal bleeding and stool frequency score of 0), endoscopic healing (modified endoscopic Mayo score ≤1, with complete healing defined as a score of 0), deep remission (combination of clinical remission and endoscopic healing), and histologic remission (absence of acute histologic inflammation, ie, neutrophils with a decrease in ordinal transformed Geboes score from >0 at baseline to 0 postbaseline).

       Statistical Analyses

      The post hoc analyses focused mainly on the diagnostic accuracy of FC and the development of a prediction model for predefined outcomes at week 52; statistical analyses methods are detailed in the Supplementary Materials.

      Results

       Patients

      Detailed descriptions of baseline patient characteristics and patient disposition through the study were reported previously and are summarized in Supplementary Table 2 and Supplementary Figure 1.
      • Rubin D.T.
      • Bradette M.
      • Gabalec L.
      • et al.
      ulcerative colitis remission status after induction with mesalazine predicts maintenance outcomes: the MOMENTUM trial.

       Diagnostic Accuracy of Fecal Calprotectin

       Correlation between fecal calprotectin and clinical remission and deep remission

      Data on clinical remission and deep remission were available for 605 of 639 patients (94.7%) and 593 of 639 patients (92.8%), respectively, at week 8, and for 310 of 373 patients (83.1%) and 305 of 373 patients (81.8%), respectively, at week 52. The median and interquartile range (IQR) FC values were lower in patients in clinical remission and deep remission than in patients with ongoing disease activity (Figure 1). The accuracy of FC to predict clinical remission and deep remission was assessed by plotting a receiver operating characteristic (ROC) curve (Supplementary Figure 2). The diagnostic accuracy (area under the curve [AUC]) of this assay was low at both week 8 (0.58 [95% CI, 0.54–0.63] for clinical remission and 0.60 [95% CI, 0.55–0.64] for deep remission) and week 52 (0.66 [95% CI, 0.59–0.73] for clinical remission and 0.67 [95% CI, 0.60–0.73] for deep remission). FC test characteristics at different predefined cut-off values for both clinical remission and deep remission are shown in Table 1.
      Figure thumbnail gr1
      Figure 1Median fecal calprotectin (FC) values at week 8 and week 52 by our predefined outcomes. Whiskers show the interquartile range.
      Table 1FC Test Characteristics for Several Therapeutic Outcomes at Different FC Cut-Off Values
      Week 8Week 52
      FC, μg/gSensitivitySpecificityPPVNPVYouden IndexFC, μg/gSensitivitySpecificityPPVNPVYouden Index
      Clinical remission
      500.460.700.560.600.15500.530.720.810.410.25
       750.500.640.540.600.14760.580.650.780.410.23
       1000.550.560.520.590.12990.660.630.800.460.29
       1500.630.460.500.590.091470.710.560.780.470.27
       2020.680.400.490.590.072040.750.440.750.440.19
       2510.740.330.490.600.072450.800.400.750.480.20
       3040.780.310.490.620.092960.810.360.740.470.17
       3480.810.300.500.650.113510.830.330.730.470.16
       4000.830.270.500.650.104030.860.320.740.520.18
       4450.840.250.490.640.084470.870.300.730.520.17
       4980.850.240.490.650.095040.890.290.730.540.18
      Endoscopic healing
       500.420.880.950.200.30500.520.910.980.230.43
       750.480.870.960.220.35760.580.850.960.240.43
       1000.550.850.960.240.40990.630.810.960.250.44
       1500.630.780.940.260.411470.680.720.940.260.41
       2020.700.730.940.290.432040.740.660.930.280.40
      2510.770.690.940.340.462450.790.620.930.310.40
       3040.790.630.930.340.422960.810.620.930.330.43
       3480.810.590.920.350.413510.830.570.930.340.40
       4000.830.550.920.360.384030.850.530.920.360.38
       4450.840.480.900.330.314470.870.510.920.370.38
       4980.850.440.900.330.295040.880.490.920.380.37
      Deep remission
      500.470.700.570.610.17500.540.740.790.460.28
       750.510.640.550.610.16760.590.650.760.460.25
       1000.570.570.530.610.14990.670.630.770.500.29
       1500.640.480.500.610.111470.710.540.740.500.25
       2020.690.410.490.610.102040.750.430.710.480.18
       2510.760.340.490.630.102450.800.390.710.520.20
       3040.790.320.490.650.112960.820.360.700.510.17
       3480.830.310.500.680.143510.830.320.690.510.15
       4000.840.280.490.680.124030.870.310.700.560.18
       4450.850.250.490.670.104470.880.300.700.570.18
       4980.860.240.490.680.105040.890.280.700.580.17
      Histologic remission
       500.590.820.740.690.41500.640.830.870.580.47
      750.650.770.710.710.42760.710.790.850.620.50
       1000.700.700.670.730.40990.770.750.840.660.52
       1500.770.590.630.740.361470.800.650.790.660.46
       2020.820.530.610.770.352040.830.560.760.670.39
       2510.860.430.570.780.292450.870.500.740.690.37
       3040.880.400.570.790.282960.880.470.730.700.35
       3480.910.390.570.830.303510.900.430.720.710.32
       4000.920.340.550.820.264030.910.390.710.730.31
       4450.920.320.550.820.244470.930.370.710.750.30
       4980.930.310.540.840.245040.930.340.700.750.27
      NOTE. Bolded values indicate optimal FC cut-off values. The closest data points are presented if cut-off values at 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, and 500 were missing in the data.
      FC, fecal calprotectin; NPV, negative predictive value; PPV, positive predictive value.

       Correlation between fecal calprotectin and endoscopic healing

      Data on endoscopic healing were available for 595 of 639 patients (93.1%) at week 8 and for 351 of 373 patients (94.1%) at week 52. A low positive correlation was observed between FC and the modified endoscopic Mayo score at week 8 (r = 0.359; R2 = 12.9%) and at week 52 (r = 0.427; R2 = 18.2%). The distribution of FC across the different endoscopic scores is shown in Figure 2. The median FC values were lower in patients with endoscopic healing than in patients without endoscopic healing at week 8 (median, 82.0 μg/g [IQR, 19.0–229.0 μg/g] vs 412.0 μg/g [IQR, 189.0–1442.0 μg/g]) and at week 52 (43.0 μg/g [IQR, 16.0–216.0 μg/g] vs 493.0 μg/g [IQR, 134.0–1125.0 μg/g]) (Figure 1). At week 8, the AUC value for FC and endoscopic healing was 0.77 (95% CI, 0.72–0.83) (Figure 3A). The optimal FC cut-off value was 251 μg/g (sensitivity, specificity, positive predictive value, negative predictive value, and Youden Index for each outcome are shown in Table 1). At week 52, the AUC value was 0.79 (95% CI, 0.72–0.86) (Figure 3B), with an optimal FC cut-off value of 99 μg/g (Table 1).
      Figure thumbnail gr2
      Figure 2Box plot of fecal calprotectin (FC) values at week 8 and week 52 by endoscopic disease activity. The middle line and hinges of the box represent the median FC value with 25th and 75th percentiles, respectively. The whiskers represent the minimum and maximum values.
      Figure thumbnail gr3
      Figure 3Receiver operating characteristic graphs of endoscopic healing by fecal calprotectin (FC) at (A) week 8 and (B) week 52, and histologic remission by FC at (C) week 8 and (D) week 52.

       Correlation between fecal calprotectin and histologic disease activity

      Data on histologic remission were available for 604 of 639 (94.5%) patients at week 8 and for 355 of 373 (95.2%) patients at week 52. The interobserver agreement between the 2 independent gastrointestinal pathologists (G.D.H. and J.H.T.) was moderate (Cohen’s weighted κ coefficient, 0.6; 95% CI, 0.33–0.87).
      • Landis J.R.
      • Koch G.G.
      The measurement of observer agreement for categorical data.
      A moderate positive correlation was observed between FC and the ordinal transformed Geboes score at week 8 (r = 0.510; R2 = 26.0%) and week 52 (r = 0.533; R2 = 28.4%) (Supplementary Figure 3). Median FC values were lower in patients with histologic remission than in patients with ongoing histologic inflammation at week 8 (median, 32.0 μg/g [IQR, 16.0–127.5 μg/g] vs 219.5 μg/g [IQR, 81.5–618.0 μg/g]) and week 52 (24.5 μg/g [IQR, 16.0–96.0 μg/g] vs 244.0 μg/g [IQR, 101.0–747.0 μg/g]) (Figure 1).
      The accuracy of FC to predict the presence of histologic remission was assessed by plotting a ROC curve (Figure 3C and D). At week 8, the AUC value was 0.76 (95% CI, 0.72–0.80) and the optimal FC cut-off value was 75 μg/g (Table 1). At week 52, the AUC was 0.80 (95% CI, 0.75–0.85), and the optimal FC cut-off value was 99 μg/g (Table 1).

       Correlation between FC and histologic disease activity in the subpopulation of patients with an endoscopy score of 0

      Interestingly, even when the analysis was limited to the subpopulation of patients with complete endoscopic healing, median FC values were lower in patients with histologic remission compared with patients with ongoing histologic inflammation at week 8 (median, 30.0 μg/g [IQR, 16.0–112.0 μg/g] vs 140.0 μg/g [IQR, 61.0–232.0 μg/g]) and week 52 (21.5 μg/g [IQR, 16.0–88.0 μg/g] vs 134.5 μg/g [IQR, 30.0–418.0 μg/g]). The AUC was 0.72 (95% CI, 0.64–0.80) at week 8 and 0.71 (95% CI, 0.60–0.82) at week 52 (Supplementary Figure 3). The optimal cut-off value of FC to predict histologic remission (in patients with an endoscopy score of 0) was 73 μg/g at week 8 and 76 μg/g at week 52 (Supplementary Table 3 and Supplementary Figure 4).

       Multivariable Prediction Model

      Prediction models for both clinical remission and deep remission are presented in Supplementary Tables 4 and 5.

       Prediction model for endoscopic healing at weeks 8 and 52

      During induction, 476 of 556 patients with available endoscopy data achieved endoscopic healing (86%). During the 12-month follow-up period, 304 of 351 patients with available data during the follow-up period achieved/maintained endoscopic healing (86.6%). The final prediction model for endoscopic healing at week 8 comprised the number of acute episodes in the year before enrollment and the baseline stool frequency and endoscopy and histologic disease activity scores (Table 2). The model for week 52 comprised the endoscopic score at week 8, the FC concentration at week 8, and male sex (Table 3). The predictors were associated with a lower probability of achieving endoscopic healing (odds ratios and 95% CIs shown in Tables 2 and 3). The ability of the model to discriminate between achieving or failing complete endoscopic healing at weeks 8 and 52 was assessed by ROC curve (AUC, 0.75; 95% CI, 0.69–0.81 at week 8; AUC, 0.71; 95% CI, 0.64–0.78 at week 52).
      Table 2Logistic Regression Analysis of Outcomes at Week 8: Endoscopic Healing and Histologic Remission
      Endoscopic healing at week 8Histologic healing at week 8
      CovariatesOR (95% CI)P valueCovariatesOR (95% CI)P value
      Univariable regression
       Age, y0.99 (0.97–1.00).1254Age, y1.01 (1.00–1.02).2481
       Sex, male/female0.65 (0.41–1.02).0643Sex, male/female0.96 (0.70–1.32).8056
       Disease extent/locationa0.86 (0.52–1.45).5512Disease extent/locationa0.67 (0.45–0.99).0462
       Disease duration at baseline, y0.98 (0.37–2.87).9736Disease duration at baseline, y1.42 (0.69–2.92).3443
       Number of acute episodes in preceding year0.86 (0.75–0.97).0215Number of acute episodes in preceding year0.97 (0.87–1.08).6340
       Endoscopic score at baseline, 0–30.31 (0.20–0.49)<.001Endoscopic score at baseline, 0–30.50 (0.36–0.68)<.001
       SF score at baseline,b 0–3 continuous0.53 (0.40–0.71)<.001SF score at baseline,b 0–3 continuous0.57 (0.46–0.70)<.001
       RB score at baseline,b 0–3 continuous0.68 (0.50–0.92).0118RB score at baseline,b 0–3 continuous0.71 (0.57–0.89).0027
       Histologic activity at baseline, 0–130.82 (0.76–0.87)<.001Histologic activity at baseline, 0–130.80 (0.76–0.84)<.001
       FC at baseline, 100 μg/g0.96 (0.94–0.99).0025FC at baseline, 100 μg/g0.94 (0.92–0.96)<.001
      Final model
       Number of acute episodes in preceding year0.85 (0.74–0.96).0103FC at baseline (100 μg/g)0.97 (0.95–0.99).0047
       Endoscopic score at baseline (0–3)0.47 (0.27–0.79).0052SF score at baseline (0–3 continuous)0.68 (0.54–0.86).0011
       SF score at baseline (0–3 continuous)0.63 (0.45–0.87).0062Histologic activity at baseline (0–13)0.83 (0.79–0.87)<.001
       Histologic activity at baseline (0–13)0.86 (0.79–0.92)<.001
      CI, confidence interval; FC, fecal calprotectin; OR, odds ratio; RB, rectal bleeding; SF, stool frequency.
      Table 3Logistic Regression Analysis of Outcomes at Week 52: Endoscopic Healing and Histologic Remission
      Endoscopic healing at week 52Histologic healing at week 52
      CovariatesOR (95% CI)P valueCovariatesOR (95% CI)P value
      Univariable regression
       Age, y0.99 (0.97–1.02).551Age, y1.01 (0.99–1.02).511
       Sex, male/female0.43 (0.22–0.81).012Sex, male/female0.75 (0.49–1.14).176
       Disease extent/location0.62 (0.33–1.24).162Disease extent/location1.22 (0.74–2.07).445
       Disease duration at baseline, y1.01 (0.28–4.25).991Disease duration at baseline, y0.85 (0.32–2.25).732
       Number of acute episodes in preceding year0.84 (0.65–0.99).103Number of acute episodes in preceding year1.00 (0.86–1.18).991
       Endoscopic score at baseline, 0–30.58 (0.33–1.00).053Endoscopic score at baseline, 0–30.56 (0.37–0.83).004
       Endoscopic score at week 8, 0–30.31 (0.16–0.58)<.001Endoscopic score at week 8, 0–30.36 (0.23–0.55)<.001
       SF score at baseline, 0–3 continuous0.82 (0.55–1.22).321SF score at baseline, 0–3 continuous0.77 (0.58–1.03).083
       RB score at baseline, 0–3 continuous1.09 (0.71–1.71).685RB score at baseline, 0–3 continuous0.90 (0.67–1.23).519
       SF score at week 8, 0–3 continuous0.68 (0.33–1.46).302SF score at week 8, 0–3 continuous0.67 (0.39–1.16).152
       RB score at week 8, 0–3 continuous19.79 (0.71–71,524.8).244RB score at week 8, 0–3 continuous0.76 (0.19–3.25).697
       Histologic activity at baseline, 0–130.89 (0.82–0.97).005Histologic activity at baseline, 0–130.88 (0.83–0.93)<.001
       Histologic activity at week 8, 0–130.87 (0.79–0.95).003Histologic activity at week 8, 0–130.80 (0.74–0.86)<.001
       FC at baseline, 100 μg/g0.96 (0.93–1.00).034FC at baseline, 100 μg/g0.99 (0.96–1.01).363
       FC at week 8, 100 μg/g0.89 (0.84–0.94)<.001FC at week 8, 100 μg/g0.87 (0.81–0.93)<.001
       Δ FC between baseline and week 8, 100 μg/g0.99 (0.95–1.03).718Δ FC between baseline and week 8, 100 μg/g0.98 (0.95–1.01).128
      Final model
       Endoscopic score at week 8, 0–30.40 (0.20–0.75)Endoscopic score at week 8, 0–30.52 (0.32–0.82)
       FC at week 8, 100 μg/g0.91 (0.85–0.96)FC at week 8, 100 μg/g0.91 (0.85–0.97)
       Sex, male/female0.53 (0.26–1.04)Histologic activity at baseline, 0–130.92 (0.86–0.98)
      Histologic activity at week 8, 0–130.89 (0.81–0.97)
      CI, confidence interval; FC, fecal calprotectin; OR, odds ratio; RB, rectal bleeding; SF, stool frequency.

       Prediction model for histologic remission at weeks 8 and 52

      During induction, 283 of 615 patients with available histology data achieved histologic remission at week 8 (46%). During subsequent follow-up evaluation, 228 of 355 (64.2%) patients with available data achieved histologic remission at week 52, of which 222 had nonmissing FC data at week 52. The final prediction model for histologic remission at week 8 comprised FC concentration, stool frequency score, and histologic disease activity at baseline (Table 2). The model for week 52 comprised endoscopic severity at week 8, FC level at week 8, and histologic disease activity at both baseline and at week 8. All predictors were associated with a lower probability of achieving histologic remission (Tables 2 and 3). The ability of the model to predict histologic remission at weeks 8 and 52 was assessed by ROC curve (AUC, 0.75; 95% CI, 0.71–0.78 at week 8; AUC, 0.72; 95% CI, 0.67–0.78 at week 52).

      Discussion

      In this post hoc analysis of a phase 3b/4, 58-week clinical trial in patients with UC receiving multimatrix mesalamine, median FC values were lower in patients who achieved clinical remission, endoscopic healing, deep remission, and histologic remission than in patients who did not meet these end points. The AUC varied according to therapeutic target and time point. There was poor diagnostic accuracy of FC for prediction of clinical remission and deep remission at both week 8 and week 52. Interestingly, when clinical remission was defined as the absence of rectal bleeding alone (disregarding the severity of diarrhea), the accuracy of FC increased markedly (data not shown). It appears that in terms of clinical symptoms, rectal bleeding is the key driver of FC increase. This is not surprising because the sensitivity to predict quiescent endoscopic disease is higher for the absence of rectal bleeding than a normalization of stool frequency.
      • Restellini S.
      • Chao C.Y.
      • Martel M.
      • et al.
      Clinical parameters correlate with endoscopic activity of ulcerative colitis: a systematic review.
      As expected, performance of FC concentration for the prediction of endoscopic and histologic disease activity was better. This corroborates the moderate correlation between symptoms and severity of mucosal inflammation, which has been reported consistently.
      • Peyrin-Biroulet L.
      • Reinisch W.
      • Colombel J.F.
      • et al.
      Clinical disease activity, C-reactive protein normalisation and mucosal healing in Crohn's disease in the SONIC trial.
      ,
      • Rutgeerts P.
      • Sandborn W.J.
      • Feagan B.G.
      • et al.
      Infliximab for induction and maintenance therapy for ulcerative colitis.
      With an AUC of 0.77 at week 8 and 0.79 at week 52, FC had fair diagnostic accuracy for predicting endoscopic healing. Optimal cut-off values varied according to the chosen time point: 251 μg/g at week 8 and 99 μg/g at week 52. Remarkably, the sensitivity for endoscopic healing remained low with concordant low negative predictive value (ie, that an FC value greater than the determined cut-off value indeed meant there was no endoscopic healing). A considerable proportion of patients with an increased FC value greater than the optimal cut-off value had endoscopically quiescent disease. Previous studies have shown the inability of endoscopy to visualize residual histologic inflammation in UC.
      • Magro F.
      • Lopes S.
      • Coelho R.
      • et al.
      Accuracy of faecal calprotectin and neutrophil gelatinase B-associated lipocalin in evaluating subclinical inflammation in UlceRaTIVE Colitis-the ACERTIVE study.
      ,
      • Bessissow T.
      • Lemmens B.
      • Ferrante M.
      • et al.
      Prognostic value of serologic and histologic markers on clinical relapse in ulcerative colitis patients with mucosal healing.
      ,
      • Kim D.B.
      • Lee K.M.
      • Lee J.M.
      • et al.
      Correlation between histological activity and endoscopic, clinical, and serologic activities in patients with ulcerative colitis.
      Hence, a possible explanation would be the presence of residual inflammation at a microscopic (histologic) level. Indeed, in the subpopulation of patients with complete endoscopic healing (modified Mayo endoscopy subscore, 0), the FC level remained higher in patients with ongoing histologic inflammation compared with patients in histologic remission and with AUC values of 0.76 and 0.80 at weeks 8 and 52, respectively, FC had fair to good diagnostic accuracy for the detection of histologic inflammation. When limited to the population of patients with an endoscopy score of 0, the accuracy of FC to predict ongoing histologic inflammation remained fair (week 8: AUC, 0.72; week 52: AUC, 0.71). Despite limited large prospective data, there is growing evidence to support the benefit of achieving histologic remission in addition to endoscopic healing.
      • Ponte A.
      • Pinho R.
      • Fernandes S.
      • et al.
      Impact of histological and endoscopic remissions on clinical recurrence and recurrence-free time in ulcerative colitis.
      • Frieri G.
      • Galletti B.
      • Di Ruscio M.
      • et al.
      The prognostic value of histology in ulcerative colitis in clinical remission with mesalazine.
      • Zenlea T.
      • Yee E.U.
      • Rosenberg L.
      • et al.
      Histology grade is independently associated with relapse risk in patients with ulcerative colitis in clinical remission: a prospective study.
      • Park S.
      • Abdi T.
      • Gentry M.
      • et al.
      Histological disease activity as a predictor of clinical relapse among patients with ulcerative colitis: systematic review and meta-analysis.
      • Bryant R.V.
      • Winer S.
      • Travis S.P.
      • et al.
      Systematic review: histological remission in inflammatory bowel disease. Is 'complete' remission the new treatment paradigm? An IOIBD initiative.
      • Gordon I.O.
      • Agrawal N.
      • Willis E.
      • et al.
      Fibrosis in ulcerative colitis is directly linked to severity and chronicity of mucosal inflammation.
      • Lobaton T.
      • Bessissow T.
      • Ruiz-Cerulla A.
      • et al.
      Prognostic value of histological activity in patients with ulcerative colitis in deep remission: a prospective multicenter study.
      In a recent observational study of 377 UC patients by Magro et al,
      • Magro F.
      • Lopes J.
      • Borralho P.
      • et al.
      Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levels.
      the Geboes Score, Nancy Index, and Robarts Histopathology Index predicted FC levels accurately. Overall, the evidence from the available literature and the present study suggest that FC correlates well with histologic disease activity and that the optimal cut-off value is between 75 and 150 μg/g.
      • Patel A.
      • Panchal H.
      • Dubinsky M.C.
      Fecal calprotectin levels predict histological healing in ulcerative colitis.
      • Zittan E.
      • Kelly O.B.
      • Kirsch R.
      • et al.
      Low fecal calprotectin correlates with histological remission and mucosal healing in ulcerative colitis and colonic Crohn's disease.
      • Theede K.
      • Holck S.
      • Ibsen P.
      • et al.
      Fecal calprotectin predicts relapse and histological mucosal healing in ulcerative colitis.
      ,
      • Magro F.
      • Lopes J.
      • Borralho P.
      • et al.
      Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levels.
      Even though the clinical utility of FC as a surrogate marker of inflammation generally is accepted, some limitations should be acknowledged. The number of proposed cut-off values in the literature is considerable.
      • Mosli M.H.
      • Zou G.
      • Garg S.K.
      • et al.
      C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis.
      This is explained in part by different FC measurement tools with distinct underlying mechanisms. A recent cross-sectional study in 371 patients with UC showed considerable differences in mean and median FC values according to which FC quantification tool (Quantum Blue vs EliA) was used.
      • Magro F.
      • Lopes S.
      • Coelho R.
      • et al.
      Accuracy of faecal calprotectin and neutrophil gelatinase B-associated lipocalin in evaluating subclinical inflammation in UlceRaTIVE Colitis-the ACERTIVE study.
      Another limitation and potential explanation for the wide range in reported cut-off values was the large interindividual and intraindividual variability in FC values.
      • Sandborn W.J.
      • Panes J.
      • Zhang H.
      • et al.
      Correlation between concentrations of fecal calprotectin and outcomes of patients with ulcerative colitis in a phase 2 trial.
      In the majority of patients with UC, mesalamines are recommended as first-line treatment.
      • Harbord M.
      • Eliakim R.
      • Bettenworth D.
      • et al.
      Third European evidence-based consensus on diagnosis and management of ulcerative colitis. Part 2: current management.
      With an increasing focus on identifying the right drug for the right patient, ideally, patients who are most likely to benefit from mesalamines would be identified before or early in treatment.
      • Harbord M.
      • Eliakim R.
      • Bettenworth D.
      • et al.
      Third European evidence-based consensus on diagnosis and management of ulcerative colitis. Part 2: current management.
      The present study aimed to identify predictors associated with therapeutic outcomes at both week 8 (initial remission) and week 52 (maintained remission). The stool frequency score and histologic disease activity at baseline were associated inversely with the likelihood of achieving both endoscopic healing and histologic remission after induction treatment. Interestingly, baseline FC concentration was associated inversely with achieving histologic remission, but not with endoscopic healing at week 8. This perhaps could be explained, in part, by the low number of patients who did not achieve endoscopic healing at week 8. There was a strong inverse association between the week 8 stool frequency score and the week 52 therapeutic outcomes that involved clinical assessment (clinical remission and deep remission). Endoscopic severity at week 8 consistently was associated inversely with week 52 therapeutic outcomes (endoscopic healing, deep remission, and histologic remission), and histologic disease activity at both baseline and week 8 was associated independently with achieving histologic remission at week 52.
      A few limitations to the present study should be acknowledged. The results do not necessarily apply to patients with severe or long-standing UC because this population has not been studied. Endoscopic disease activity assessment was performed by local investigators without the use of central reading. In each center, FC measurements were performed using in-house assays. Although different assays show comparable performance, there still might be considerable quantitative differences that could influence optimal cut-off determination.
      • Labaere D.
      • Smismans A.
      • Van Olmen A.
      • et al.
      Comparison of six different calprotectin assays for the assessment of inflammatory bowel disease.
      Furthermore, although this was a multicenter clinical trial with a large sample size, these analyses were completed post hoc with limitations in their study design. The aim of the present analyses was not to develop a prediction algorithm suited for predictions on the individual patient level and no formal methods for model validation have been applied. Finally, the Geboes score was transformed to an ordinal score. For the analyses of the correlation between FC and histologic disease activity, only parameters 2b to 5 of the Geboes score were evaluated. Because parameters 0 to 2A reflect chronic changes rather than active inflammation, chronic inflammatory infiltrates and lamina propria eosinophils were disregarded.
      In conclusion, in mild-to-moderate UC, even in the presence of complete endoscopic healing, FC can aid in discriminating between patients with ongoing microscopic inflammation and patients in histologic remission. The optimal FC cut-off value to predict histologic remission varied between 75 and 100 μg/g. If histologic remission is adopted as a therapeutic target in UC, FC target values should be considered within this window rather than the commonly accepted 150 to 250 μg/g.
      • Sandborn W.J.
      • Panes J.
      • Zhang H.
      • et al.
      Correlation between concentrations of fecal calprotectin and outcomes of patients with ulcerative colitis in a phase 2 trial.
      ,
      • D'Haens G.
      • Ferrante M.
      • Vermeire S.
      • et al.
      Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease.
      Furthermore, endoscopic disease activity after the induction phase of mesalamine treatment was the only independent predictor associated with several important therapeutic outcomes (endoscopic healing, deep remission, and histologic remission) at week 52.
      We developed a prediction model for short-term (week 8) and long-term (week 52) histologic remission in patients with mild-to-moderate UC treated with multimatrix mesalamine. These models comprised the baseline FC, stool frequency, and histologic disease activity score for short-term response and the endoscopic score at week 8, FC concentration at week 8, and histologic disease activity at both baseline and week 8 for long-term response. These findings have considerable importance for evolving treatment end points.

      CRediT Authorship Contributions

      Toer W. Stevens, MD (Conceptualization: Lead; Formal analysis: Lead; Methodology: Lead; Validation: Lead; Writing – original draft: Lead; Writing – review & editing: Lead);
      Krisztina Gecse, MD, PhD (Investigation: Supporting; Methodology: Supporting; Supervision: Lead; Validation: Supporting; Visualization: Supporting; Writing – review & editing: Supporting);
      Jerrold R. Turner, MD, PhD (Investigation: Supporting; Methodology: Supporting; Supervision: Lead; Validation: Supporting; Visualization: Supporting; Writing – review & editing: Supporting);
      Gert de Hertogh, MD, PhD (Investigation: Supporting; Methodology: Supporting; Supervision: Lead; Validation: Supporting; Visualization: Supporting; Writing – review & editing: Supporting);
      David T. Rubin, MD (Investigation: Supporting; Methodology: Supporting; Supervision: Lead; Validation: Supporting; Visualization: Supporting; Writing – review & editing: Supporting);
      Geert R. D'Haens, MD, PhD (Conceptualization: Lead; Data curation: Lead; Investigation: Lead; Methodology: Lead; Supervision: Lead; Writing – review & editing: Supporting). The authors thank Y. Xie for assistance with statistical analyses.

      Supplementary Material

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