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Keywords:

  • Crohn's disease;
  • stool tests;
  • fecal biomarkers;
  • Crohn's disease index of severity;
  • tumor necrosis factor α;
  • mucosal healing

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Background: Fecal calprotectin and lactoferrin are promising noninvasive biomarkers for intestinal inflammation. In Crohn's disease (CD), during anti-TNF-alpha (TNF-α) treatment, the clinical significance of these markers has, however, been insufficiently explored.

Methods: Among CD patients receiving anti-TNF-α therapy we assessed the role of fecal calprotectin and lactoferrin as surrogate markers for mucosal healing. Before and 3 months after the beginning of anti-TNF-α induction, 15 patients underwent ileocolonoscopy with scoring of the Crohn's Disease Index of Severity (CDEIS). Fecal samples for calprotectin and for lactoferrin measurements were collected and the Crohn's Disease Activity Index (CDAI) was calculated at the time of the endoscopies and 2 and 8 weeks after the first treatment.

Results: The median CDEIS fell from 13.0 to 4.8 (P = 0.002) and CDAI from 158 to 68 (P = 0.005). Accordingly, the median fecal calprotectin concentration fell from 1173 μg/g to 130 μg/g (P = 0.001) and fecal lactoferrin from 105.0 μg/g to 2.7 μg/g (P = 0.001). Of the 15 patients, 11 (73%) showed an endoscopic response to treatment and 5 of these achieved endoscopic remission (CDEIS < 3). In those 5 patients the fecal calprotectin concentration declined from 1891 μg/g (range 813–2434) to 27 μg/g (13–130) and lactoferrin from 92.4 μg/g (35.5–235.6) to 1.9 μg/g (0.0–2.1).

Conclusions: Compared to pretreatment values, concentrations of fecal calprotectin and lactoferrin after the anti-TNF-α treatment were significantly lower. During anti-TNF-α therapy these fecal neutrophil-derived proteins may thus be useful surrogate markers for mucosal healing.

(Inflamm Bowel Dis 2008)

In Crohn's disease (CD), estimation of treatment response is widely based on changes in clinical disease activity and on nonspecific laboratory tests such as hemoglobin level and C-reactive protein (CRP). In clinical trials a change in the Crohn's Disease Activity Index (CDAI) is still almost the sole primary endpoint, although its correlation with endoscopic disease activity is weak.1 Recent studies and reviews suggest mucosal healing as becoming a therapeutic target for treatment of inflammatory bowel disease (IBD).2–5 During infliximab treatment, clinical improvement is accompanied by significant healing of endoscopic lesions and the disappearance of mucosal inflammatory infiltrate.6 Mucosal healing in CD is associated with a decreased number of disease-related procedures and hospital admissions.7 Detection of mucosal healing requires endoscopy, which is time-consuming, expensive, and unpleasant for patients. The neutrophil-derived proteins fecal calprotectin and lactoferrin correlate closely with endoscopic disease activity in ulcerative colitis (UC),8, 9 and as recently shown, also with the Crohn's Disease Index of Severity (CDEIS),10 appearing thus to be promising noninvasive surrogate markers for mucosal healing.

In IBD, fecal calprotectin concentration correlates strongly with the excretion of indium-111-labeled granulocytes, a sensitive marker of disease activity.11 In stool, both calprotectin and lactoferrin are stable12, 13 and easily measurable by commercial enzyme-linked immunosorbent assays (ELISA). These markers are sensitive for inflammation, but not disease-specific; in addition to IBD, fecal calprotectin may rise, for example in colon cancer, bacterial infections, and inflammation caused by nonsteroidal antiinflammatory drugs (NSAIDs).14–16 A normal fecal calprotectin level in clinical IBD remission seems to predict mucosal healing.17 Studies of fecal lactoferrin are fewer, but in clinically active IBD lactoferrin is significantly elevated.18, 19

Because the clinical significance of fecal markers in monitoring response to antitumor necrosis factor-alpha (TNF-α) therapy is insufficiently studied, we compared fecal calprotectin and lactoferrin to the CDEIS,20 to the CDAI, and to histologic activity before and after anti-TNF-α therapy.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Patients

Adult patients with established CD (n = 15) and scheduled for ileocolonoscopy participated in our previous cross-sectional study.10 Their ileocolonoscopy findings were scored according to the CDEIS. Based on global clinical assessment—not solely based on CDAI or CDEIS—these patients were considered to need anti-TNF-α treatment and were recruited into this prospective study. Indications for the treatment were an acute flare (n = 6), chronic active disease (6), or rapid reoccurrence of the disease postoperatively (3). Exclusion criteria were contraindication to anti-TNF-α treatment, pregnancy, history of extensive bowel resection (ileosigmoideostomy, ileorectostomy), ostomy, long-term use of NSAIDs, or perianal fistulating disease without luminal inflammation.

After the baseline endoscopy (median 7 days after, range 1–38 days), 14 patients received infliximab infusions 5 mg/kg at weeks 0 and 8. One patient received an adalimumab induction dose 80 mg subcutaneously (s.c.) at week 0, followed by 40 mg s.c. every other week until week 8. Maintenance therapy remained unchanged between the first endoscopy and introduction of the anti-TNF-α therapy. After the beginning of the anti-TNF-α treatment corticosteroids were tapered off. At the time of the first ileocolonoscopy, and 2, 8, and 12 weeks (10 weeks for the adalimumab-treated patient) after the first anti-TNF-α treatment, patients provided blood samples for serum CRP (normal value <5 mg/L), and fecal samples for measurement of calprotectin and lactoferrin. Fecal samples were stored at −40°C until analysis. Each patient underwent an endoscopic treatment response evaluation at week 12 (week 10 for the adalimumab-treated patient).

Clinical activity was assessed according to the CDAI21: a CDAI <150 indicated clinical inactivity and scores 150 to 219 mild, 220 to 450 moderate, and >450 severely active disease.22

Endoscopic Scoring

Four experienced gastroenterologists (T.S., H.N., U.T., M.F.) performed the endoscopies and graded the findings according to CDEIS.20 This score is based on the presence of superficial or deep ulcerations, proportion of affected and ulcerated surface, and presence of either ulcerated or nonulcerated stenosis in the terminal ileum and 4 segments of the colon (right, transverse, left colon and sigmoid, and rectum).20 According to our earlier data, CDEIS <3 suggests inactive disease (remission), 3 to 9 mild, ≥9–12 moderate, and ≥12 severely active disease.10 Endoscopic treatment response was defined as a 2 or 3-class improvement in CDEIS, or as achievement of remission (CDEIS <3). Partial response was defined as a 1-class improvement in CDEIS.

In addition, postoperative disease activity of the neoterminal ileum was scored according to the Rutgeerts score.23

Histology Scoring

During all endoscopies, 4 biopsy specimens—targeted at the most severely diseased areas—were taken from the ileum, right, transverse, and left colon, and rectum. If no lesions were present in the segment the biopsies were collected from random sites of each segment. Routine histology was performed on specimens stained with hematoxylin and eosin. A single experienced gastrointestinal pathologist (P.K.) scored the histological findings according to the scoring system for histological abnormalities in CD mucosal biopsy specimens24: Epithelial damage was classified as normal (score = 0), focal (score = 1), or extensive (score = 2) pathology; architectural changes as normal (score = 0), moderately (<50%) disturbed (score = 1), or severely (> 50%) disturbed (score = 2); and both infiltration of mononuclear cells in the lamina propria and polymorphonuclear cells in the lamina propria as normal (score = 0), moderate (score = 1), or severe (score = 2) increase. Polymorphonuclear cells in the epithelium were scored 1 if in the surface epithelium, 2 if cryptitis was present, and 3 if a crypt abscess was present. The presence of erosions or ulcers, or of granuloma was scored “yes” (score = 1) or “no” (score = 0). In addition, the number of biopsy specimens affected were scored none (score = 0), < 33% (score = 1), 33%–66% (score = 2), or >66% (score = 3).24 Ileal and colonic scores were counted separately; the sum of the total scores for each 4 colonic segments represented total colonic histologic activity.

Fecal Calprotectin and Lactoferrin Assays

The values quoted as normal in our laboratory were <100 μg/g of stool for fecal calprotectin,25, 26 and <7.25 μg/g of stool for fecal lactoferrin (baseline value of the manufacturer). Fecal calprotectin was measured by a quantitative enzyme immunoassay (PhiCal Test; Calpros, Oslo, Norway). Lactoferrin was measured by a quantitative enzyme immunoassay (IBD-SCAN; Inverness Medical, Princeton, NJ; Techlab, Blacksburg, VA).

In this study a fecal calprotectin <200 μg/g and a lactoferrin <10 μg/g indicated inactive disease, calprotectin 200–1000 μg/g and lactoferrin 10–70 μg/g mildly or moderately active, and calprotectin >1000 μg/g and lactoferrin >70 μg/g severely active disease.10

Ethics

All patients gave informed written consent for participation in this study which was approved by the Ethics Committee of the Helsinki University Central Hospital.

Statistics

Each value is presented as a median. For data analyses we used the SPSS for Windows software 14.0 (Chicago, IL). For correlation analyses we used nonparametric Spearman's rank order correlation (r). The Wilcoxon's signed rank test served for exploring changes between related variables. Significance was set at 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Changes in CDEIS and Fecal Markers

Of 30 endoscopies, 28 (93.3%) reached the ileum. Baseline endoscopic disease activity among the 15 patients (Table 1), according to CDEIS, was mild or moderate in 5 (33.3%) and severe in 9 (60.0%). One patient with active neoterminal ileal disease (Rutgeerts score i2) had a low baseline CDEIS of 1.8.

Table 1. Patient Characteristics
  1. TNF, tumor necrosis factor.

Age: median (range)25 (19–44) 
Disease duration in years Median (range)5.1 (0.4–27) 
 Number of patients%
Gender: female/ male6 / 940.0 / 60.0
Active disease location  
 Ileum213.3
 Colon426.7
 Ileocolon960.0
Disease type  
 Inflammatory746.7
 Stricturing533.3
 Inflammatory + perianal320.0
Prior bowel operation yes/ no4/ 1126.7/ 73.3
Smoking yes/ no7/ 846.7/ 53.3
Baseline concomitant medication  
 Azathioprine1066.7
 Methotrexate213.3
 Corticosteroids1066.7
 Mesalamine1173.3
 Salazosulphapyridine213.3
Prior anti-TNF-alpha treatment426.7

Following treatment, CDEIS (P = 0.002) and both fecal markers (P = 0.001) declined from the baseline level significantly (Table 2). Figure 1 shows fecal-marker concentrations during the study.

Table 2. Behavior of CDEIS, Fecal Markers, CDAI, and Serum CRP During Study Period
n = 15CDEISFecal calprotectin μg/gFecal lactoferrin μg/gCDAIS-CRP mg/L
  1. CDEIS, Crohn's Disease Endoscopic Index of Severity; CDAI, Crohn's Disease Activity Index.

  2. Range in brackets. Values at week 12 compared to values before treatment.

  3. Patients (n = 14) received infliximab treatment at week 0 and week 8, and 1 patient received adalimumab at weeks 0, 2, 4, 6, and 8. Endoscopies and scoring of CDEIS performed before treatment and 3 months after beginning of treatment.

Before treatment13.0 (1.8–25.3)1173 (88–15326)105.0 (4.2–1258.9)158 (49–605)9.5 (n = 14) (<5–54)
Week 2 216 (24–2349)6.7 (0.9–356.8)77 (13–197)<5 (<5–11)
Week 8 325 (n = 14) (15–20899)7.1 (n = 12) (0.0–5400.6)68 (26–192)<5 (<5–19)
Week 124.8 (0.0–11.2) P = 0.002130 (13–1419) P = 0.0012.7 (n = 14) (0.0–228.5) P = 0.00166 (24–202) P = 0.005<5 (<5–6) P = 0.005
thumbnail image

Figure 1. Fecal calprotectin and lactoferrin concentrations. Three fecal lactoferrin values at week 8 and one at week 12 were unavailable. One calprotectin value at week 8 was unavailable. Of 15 patients, 14 received infliximab 5 mg/kg infusion at week 0 and week 8. One patient had considerably high fecal-marker concentrations before the first and the second infliximab therapy (disconnected line). One patient (square symbol and gray line) received adalimumab at weeks 0, 2, 4, 6, and 8.

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When pre- and posttreatment data were combined, CDEIS correlated significantly with CDAI (r = 0.592, P = 0.001) and CRP (r = 0.608, P < 0.001), but the correlation of CDEIS was stronger with fecal calprotectin and lactoferrin (r = 0.831 and 0.865, both P < 0.001). Fecal calprotectin and lactoferrin correlated with each other significantly (r = 0.925, P < 0.001).

Based on a change in CDEIS, 11 (73%) patients responded to the treatment and 5 (33%) of these reached endoscopic remission (CDEIS <3). In these 5 patients the fecal calprotectin concentration declined from 1891 μg/g (range 813–2434) to 27 μg/g (13–130), lactoferrin from 92.4 μg/g (range 35.5–235.6) to 1.9 μg/g (0.0–2.1), CDEIS from 14.4 (range 9.8–16.5) to 1.1 (0.0–1.8), and CDAI 252 (range 61–605) to 47 (30–101), all P = 0.043.

Of 15 patients, 1 patient was a partial responder and 3 showed no endoscopic treatment response (median pretreatment CDEIS 8.1 and posttreatment CDEIS 9.6); 2 of them had unchanged active inflammation in the neoterminal ileum (Rutgeerts score i2 and i4 after treatment). In the 3 nonresponders the changes in fecal calprotectin and lactoferrin were nonsignificant (P = 0.285 and P = 0.18, respectively).

With pre- and posttreatment data combined a fecal calprotectin of 200 μg/g gave a sensitivity of 87%, a specificity of 100%, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 70% in predicting endoscopically active disease (CDEIS ≥3). Accordingly, a fecal lactoferrin concentration of 10 μg/g gave a sensitivity, specificity, PPV, and NPV of 77%, 100%, 100%, and 58%, respectively.

Changes in CDAI

Prior to treatment, of 15 CDAI values 8 (53.3%) were ≥150 (active disease), but in 14 (93.3%) fecal calprotectin and 13 (86.7%) lactoferrin concentrations were elevated. Although baseline fecal calprotectin indicated severe disease (>1000 μg/g) in 12 (80.0%), and lactoferrin (>70 μg/g) in 10 (66.7%) patients, only 1 CDAI did so.

Pre- and posttreatment CDAI in responders or partial responders (n = 12) was 166 (range 49–605) and 51 (24–202) (P = 0.008). In nonresponders (n = 3), CDAI was at baseline 106 (75–222) and after therapy was 81 (66–112) (P = 0.285).

In predicting endoscopically active disease, CDAI >150 gave a sensitivity of 39%, a specificity of 100%, a PPV of 100%, and a NPV of 33%.

Changes in Histology Score

Colonic pre- and posttreatment biopsy specimens were available of 15 patients and ileal specimens of 14 patients. During treatment the total histologic score for colon biopsy specimens declined significantly from 23.0 (range 0–38) to 11.0 (0–25), P = 0.002. Ileal histologic activity score declined from 7.0 (range 0–11) to 4.0 (range 0–9), P = 0.022. Correlations of changes in histologic scores with changes in endoscopic, clinical, and fecal variables were, however, all nonsignificant (Table 3).

Table 3. Correlations of Changes (Δ) in Endoscopic Activity from Baseline to Posttreatment Level with Fecal Markers, CDAI, and Histologic Measures of Disease Activity
 Δ CalprotectinΔ LactoferrinΔ CDAIΔ Ileal Histologic ScoreΔ Colonic Histologic Score
  • CDEIS, Crohn's Disease Index of Severity; CDAI, Crohn's Disease Activity Index.

  • Spearman's rank-order correlation (2-tailed).

  • *

    Significant, P < 0.05.

Δ CDEIS0.561*0.600*0.679*0.0580.369
 P = 0.030P = 0.023P = 0.005P = 0.845P = 0.090
Δ calprotectin0.749*0.300− 0.2910.297
  P = 0.002P = 0.277P = 0.313P = 0.177
Δ lactoferrin0.749*0.1780.0330.361
 P = 0.002 P = 0.543P = 0.914P = 0.204
Δ CDAI0.3000.1780.0600.367
 P = 0.277P = 0.543 P = 0.839P = 0.179

The pretreatment total colonic histologic score correlated significantly with CDEIS, CDAI (Spearman's r = 0.522 and 0.518, P = 0.046 and 0.048), and fecal calprotectin (r = 0.522, P = 0.046) but not with fecal lactoferrin (r = 0.482, P = 0.069). Posttreatment CDEIS, CDAI, and fecal markers failed to correlate significantly with colonic histologic score (all P > 0.05). The ileal histologic score correlated with CDEIS, CDAI, or fecal markers nonsignificantly (all P > 0.05). In the 5 patients in endoscopic remission after the therapy, the changes of ileal and colonic histology scores were nonsignificant (both P > 0.05).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Endoscopic and Clinical Activity and Fecal Markers

Our prospective study showed that both fecal calprotectin and lactoferrin correlated closely with endoscopic activity during anti-TNF-α therapy, and that their elevated concentration was a highly specific surrogate marker of endoscopically active disease. The concentration of calprotectin and lactoferrin in stool declined in treatment responders significantly from the baseline level and normalized in almost all those who reached endoscopic remission; these fecal proteins thus seem to serve well as surrogate markers for mucosal healing.

Prospective studies designed to explore fecal granulocyte proteins as surrogate markers for endoscopic findings following anti-TNF-α therapy— or other medical therapies—are limited. A study including 5 pediatric patients with clinically active CD showed fecal lactoferrin to be a sensitive and specific biomarker of intestinal inflammation, and thus to be a helpful noninvasive diagnostic tool for monitoring infliximab treatment response.27 Following infliximab therapy, a transient decrease in fecal calprotectin concentrations in 2 CD patients corresponded to an improvement in clinical disease activity, but endoscopic activity evaluation was not performed.28 In pediatric patients, during glucocorticoid therapy, fecal calprotectin declined in line with clinical improvement, but seldom fell within the normal range—suggesting ongoing inflammation in clinically silent disease.25 Also in pediatric IBD patients being weaned from steroid therapy, fecal lactoferrin was higher than in clinically inactive disease, but lower than in patients with clinically active disease not receiving steroids.29 In IBD patients with clinical remission, a normal calprotectin level seemed to be a good surrogate marker for mucosal healing.17 From a previous active CD episode, 6 samples were available showing the calprotectin concentration to be significantly higher during active disease than during remission.17

Although in our study CDAI correlated with endoscopic activity and with fecal markers, it remained low in half the patients whose endoscopic findings and fecal markers indicated active disease. It is known that in clinical CD remission mucosal inflammation is often ongoing.30 For evaluation of treatment response with biologicals, CDAI is less sensitive than is either fecal marker. Fecal calprotectin correlated closely and significantly with fecal lactoferrin. Both serve well as surrogate markers for mucosal healing, making combination of these markers probably unnecessary. In a recent study of chronic diarrhea patients, calprotectin appeared to represent the most accurate marker in differentiation of IBD from irritable bowel syndrome, and simultaneous determination of several fecal biomarkers compared with each individual neutrophil-derived fecal protein failed to improve diagnostic power.31

A specific CDEIS cutoff value to define remission remains to be determined.32 A recent study of 28 CD patients with elevated CDAI but normal CRP failed to find a significant correlation of CDEIS (cutoff 6 for mild disease) with fecal calprotectin.33 In that study, however, more than half the patients had ileal disease and overall endoscopic disease activity remained low (median CDEIS 3.4). In the present study, representing a different and most probably more selected study population, the median CDEIS was higher and only 2 patients had purely ileal disease. Only 3 patients with clinically active disease at baseline had normal CRP, but in each of them both fecal markers and CDEIS indicated active disease. Corresponding to our findings, another study showed fecal calprotectin and lactoferrin significantly higher in patients with CDAI >150 than in those in clinical remission and also in moderate-to-severe endoscopically active CD than in mildly active disease.34

As our study was designed to closely resemble routine practice, we determined the need for anti-TNF-α therapy by global clinical assessment, not solely by clinical activity or CDEIS scoring. Induction with infliximab usually comprises 3 infusions (at 0, 2, and 6 weeks),35 but in this study the induction was modified to 0- and 8-week intervals.

A small number of participating patients was a limitation of the present study. The decline of fecal markers, however, was apparent in each treatment responder, and thus we consider that, during anti-TNF-α therapy, both these fecal markers are promising surrogate markers of mucosal healing and may reduce the need for endoscopic treatment response evaluation. Future larger studies are warranted to confirm our findings before widely adapting this approach to clinical practice. The present research was designed to explore fecal markers only during the induction therapy and left the behavior of these markers during maintenance treatment unanswered. A third of the patients in this study had a stricturing disease type. At baseline, median CDAI was only mildly increased. The endoscopic findings, however, indicated baseline active disease in all studied patients—including those with a stricturing disease—and based on calprotectin level, 80% had severe disease activity.

Histology

In UC patients histological grading correlates closely with fecal calprotectin concentration.8 In IBD patients increased fecal calprotectin strongly associated with advancing histologic grades of colorectal inflammation.36, 37 In the present study, with a small study population and only CD patients, only the pretreatment histologic activity score for the colon correlated with clinical and endoscopic activity and with fecal calprotectin. Ileal score correlated neither with fecal markers nor with clinical or endoscopic activity. This finding is in part similar to that of Geboes et al,38 in which baseline CDEIS correlated significantly with global colonic histology score, and changes in CDAI and CDEIS following infliximab treatment correlated at week 10 with the histologic score of the colon, but not with the ileal score. In all endoscopies we targeted the biopsies at the most diseased areas. Probably our posttreatment results—with lower and more focal inflammation activity than in pretreatment findings—are biased by sampling error: biopsy specimens may represent histologic changes in a solitary minor aphthous ulcer in otherwise healed mucosa. Due to uncertain clinical relevance, the histologic disease activity assessment cannot be recommended as a treatment endpoint.32 Based on our findings, routine histologic biopsy specimens only for detection of CD activity seem unnecessary. Compared to histology, both fecal calprotectin and lactoferrin seem to be better surrogate markers for global disease activity.

CONCLUSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

In conclusion, both fecal calprotectin and lactoferrin correlated significantly with CDEIS. Compared to their pretreatment levels, concentrations of these fecal markers after anti-TNF-α therapy were significantly lower. In patients on anti-TNF-α therapy these fecal neutrophil-derived proteins thus would be useful surrogate markers for mucosal healing.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES
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