Int J Colorectal Dis DOI 10.1007/s00384-013-1817-3
ORIGINAL ARTICLE
Fecal calprotectin and lactoferrin as predictors of relapse in patients with quiescent ulcerative colitis during maintenance therapy Takayuki Yamamoto & Manabu Shiraki & Takuya Bamba & Satoru Umegae & Koichi Matsumoto
Accepted: 4 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose This prospective study was to evaluate the significance of fecal calprotectin and lactoferrin for the prediction of ulcerative colitis (UC) relapse. Methods Eighty UC patients in remission for ≥3 months on mesalamine as maintenance therapy were included. At entry, stool samples were collected for the measurement of calprotectin and lactoferrin. All patients were followed up for the following 12 months. To identify predictive factors for relapse, time-dependent analyses using the Kaplan-Meier graphs and Cox's proportional hazard model were applied. Results During the 12 months, 21 patients relapsed. Mean calprotectin and lactoferrin levels were significantly higher in patients with relapse than those in remission (calprotectin—173.7 vs 135.5 μg/g, P =0.02; lactoferrin— 165.1 vs 130.7 μg/g, P =0.03). A cutoff value of 170 μg/g for calprotectin had a sensitivity of 76 % and a specificity of 76 % to predict relapse, while a cutoff value of 140 μg/g for lactoferrin had a sensitivity of 67 % and a specificity of 68 %. In a multivariate analysis, calprotectin (≥170 μg/g) was a predictor of relapse (hazard ratio, 7.23; P =0.002). None of the following parameters were significantly associated with relapse: age, gender, duration of UC, number of UC episode, severity of the previous episode, extent of UC, extraintestinal manifestation, and lactoferrin level. Conclusions Fecal calprotectin showed a higher sensitivity and specificity than fecal lactoferrin for predicting UC relapse. Fecal calprotectin level appeared to be a significant predictor of relapse in patients with quiescent UC on mesalamine as maintenance therapy. T. Yamamoto (*) : M. Shiraki : T. Bamba : S. Umegae : K. Matsumoto Inflammatory Bowel Disease Center, Yokkaichi Social Insurance Hospital, 10-8 Hazuyamacho, Yokkaichi, Mie 510-0016, Japan e-mail: [email protected]
Keywords Calprotectin . Fecal marker . Lactoferrin . Mesalamine . Ulcerative colitis
Introduction Ulcerative colitis (UC) is a chronic disease characterized by mucosal inflammation in the colon and the rectum [1]. The course of UC is characterized by spontaneous remission, which may be followed by a relapse, and is typically unpredictable. The main goal of medical therapy in patients with UC is effective and sustained suppression of intestinal inflammation in order to induce and maintain clinical remission. Identifying patients at a significant risk of relapse during quiescent UC should help to determine a strategy for maintenance therapy during remission. Previous studies have reported that younger age, multiple previous episodes of relapse, extraintestinal manifestations, a low-fiber diet, seasonal factors, and a long-term perceived stress were risk factors for future relapse in patients with quiescent UC [2–5]. These factors are of limited value in clinical setting, and therefore more objective parameters that may predict future relapse would be desirable in our clinical practice. The presence of active intestinal inflammation in patients with UC is associated with an acute phase reaction and migration of leucocytes to the gut, and this leads to the release of several neutrophil proteins, which may be measured in stool samples [6]. Fecal markers fulfill all the criteria of being noninvasive, simple, inexpensive, sensitive, and specific markers to detect gastrointestinal inflammation. Calprotectin is a calcium-binding protein, representing up to 60 % of the cytosolic proteins in neutrophils [7]. Since calprotectin is primarily derived from neutrophils, its concentration is directly proportional to the concentration of neutrophils in the colonic/rectal mucosa [8]. It is resistant to bacterial
Int J Colorectal Dis
degradation in the gut and is stable in the stool for up to 1 week at room temperature [9, 10]. Lactoferrin is an iron-binding glycoprotein and a major component of the secondary granules of polymorphonuclear neutrophils [11, 12]. During intestinal inflammation, leucocytes invade the mucosa, which may lead to an increase in the excretion of lactoferrin into the stool. Lactoferrin can be measured by using simple and inexpensive techniques since it is stable in the stool for up to 4 days [13, 14]. Several studies [15–18] have compared fecal markers with disease activity indices and endoscopic/histological evaluations to monitor intestinal inflammation in patients with inflammatory bowel disease (IBD). Levels of fecal calprotectin and lactoferrin have a proportional correlation to the degree of inflammation of the intestinal mucosa. Fecal calprotectin and lactoferrin may have a role in monitoring disease activity in patients with IBD [19–24]. With this background in mind, the present study was designed to rigorously evaluate whether or not these neutrophil proteins are clinically relevant for predicting relapse in patients with quiescent UC. Further, we included a homogeneous group of patients who were receiving mesalamine as maintenance therapy.
Patients and methods
Table 1 Baseline characteristics of 80 eligible patients Age at entry (mean ± SE) Male/female (n) Duration of UC before entry (mean ± SE) Number of UC episode (n) 1–4 ≥5 Severity of the previous episode (n) Mild (DAI score ≤4) Moderate (5≤DAI score ≤8) Severe (DAI score ≥9) Extent of UC (n) Left-sided colitis Extensive colitisa Extraintestinal manifestations (n) Arthritis Pyoderma gangrenosum Induction therapy for the previous episode (n) Mesalamine (Pentasa 4 g/day) Corticosteroids (prednisolone 30–60 mg/day) Leukocytapheresis Immunosuppressants (Tacrolimus) Biologics (Infliximab)
54 26 28 41 11 60 20 11 3 63 47 48 10 2
UC ulcerative colitis, DAI Disease Activity Index [25] a
Study design
35.1±0.8 years 49:31 50.0±2.7 months
Involvement extends proximal to the splenic flexure
This was a prospective, single-center study undertaken at the Yokkaichi Social Insurance Hospital, a referral center treating a large number of patients with IBD in the Mie Prefecture of Japan. The study was conducted in accordance with the principle of good clinical practice and the Helsinki Declaration. Our study protocol was reviewed and approved by our Institutional Review Board.
clinical remission with medical treatment. As remission induction therapy, high-dose mesalamine (Pentasa, 4 g/day) was used in 63 patients, prednisolone (30–60 mg/day) in 47 patients, therapeutic leucocytapheresis (Adacolumn, JIMRO, Takasaki, Japan) in 48 patients, tacrolimus (Prograf) in 10 patients and infliximab (Remicade, 5 mg/kg at weeks 0, 2, and 6, and then at 8-week intervals) in 2 patients.
Patients
Remission maintenance therapy
Patient inclusion criteria were: (1) age between 20 and 75, (2) having endoscopic and histologic diagnosis of UC, (3) UC confined to the colon and the rectum, and (4) patient in clinical remission (normal stool frequency and no rectal bleeding) on oral mesalamine for ≥3 months. Exclusion criteria were: (1) patients who had received corticosteroids, immunosuppressants, or biologic agents at entry; (2) had received mesalamine enema; and (3) had received nonsteroidal anti-inflammatory drugs, antidiarrheal (loperamide or codeine), or antispasmodic medications at entry. A total of 80 patients who met the inclusion criteria were included in this study. The baseline characteristics of the 80 eligible patients are presented in Table 1. The severity of previous UC episodes was mild in 28 patients, moderate in 41 patients, and severe in 11 patients. All patients achieved
During the study period, all patients were given oral mesalamine (Pentasa, 1.5–3.0 g/day). No patient received mesalamine enema (oral, intravenous, intramuscular, or rectal) corticosteroids, immunosuppressants (tacrolimus, azathioprine, and 6-mercaptopurine), or biologic agents except patients who developed clinical relapse. Follow-up All patients were reviewed in our clinic every 2 up to 12 months after entry. Patients were advised to record their symptoms in a diary every day. At the clinic visits, patient's compliance with medication, adverse effects, general wellbeing, stool frequency, stool consistency and presence or absence of abdominal pain, tenderness, tenesmus, rectal
Int J Colorectal Dis
bleeding, and mucus in stool were recorded. Stool frequency and rectal bleeding were scored according to the Disease Activity Index (DAI) system listed in Table 2 [25]. Clinical remission was defined as normal stool frequency (= score 0) and no rectal bleeding (= score 0). When patients developed symptoms suggestive of a flare-up, endoscopic examination was immediately undertaken. Endoscopic score was according to the mucosal appearance section of the DAI. A relapse was defined as a worsening of stool frequency and/or rectal bleeding with an endoscopic score of 2 or 3 [25]. In our clinic, peripheral blood samples were collected for measurement of white blood cell count (WBC), hemoglobin (Hb), platelet count, C-reactive protein (CRP), total protein, albumin, creatinine, urea, sodium, potassium, chloride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactic dehydrogenase, total bilirubin, and cholesterol. Measurement of fecal calprotectin and lactoferrin At entry, patients provided a stool sample for the measurement of calprotectin and lactoferrin. Fecal calprotectin was measured by a quantitative enzyme immunoassay (Human Calprotectin ELISA Kit, Cell Sciences Inc., Massachusetts, USA). Lactoferrin was measured by a colloidal gold agglutination reagent (Auto Lf-Plus, Alfresa Pharma Corp., Osaka, Table 2 Disease activity index (DAI) scoring system Score Stool frequency Normal number of stools 1–2 stools/day greater than normal 3–4 stools/day greater than normal ≥5 stools/day greater than normal Rectal bleeding No blood seen in stool Streaks of blood with stools less than half the time Obvious blood with stools most of the time Blood alone passed Mucosal appearance Normal mucosa or inactive disease Mild inflammatory changes (erythema, decreased vascular patterns, mild friability) Moderate inflammatory changes (marked erythema, absent vascular patterns, friability, erosions) Severe inflammatory changes (spontaneous bleeding and ulcerations) Physician's overall assessment of disease severity Normal Mild disease Moderate disease Severe disease
0 1 2 3 0 1 2 3 0 1 2 3
0 1 2 3
Japan) by using a high-throughput discrete clinical chemistry analyzer (Hemo Techt NS-Plus C, Alfresa Pharma Corp.). As a normal control group, fecal samples were obtained from 80 age- and gender-matched healthy volunteers with no history of gastrointestinal disorder. Laboratory personnel were blinded to the clinical data. Statistical analysis Continuous data are presented as the mean±SE values. Comparisons of frequencies were done by using the chi-square test with Yates' correction. The average values between two groups are compared by unpaired t test. For comparisons involving more than two groups, the one-way analysis of variance was applied. Correlations were calculated by using the Spearman's “r ” test. The cumulative relapse rate was calculated by the Kaplan-Meier method, and was compared between the groups by using the log-rank test. Predictors of relapse were determined by multivariate analysis using Cox's proportional hazard model. P
ORIGINAL ARTICLE
Fecal calprotectin and lactoferrin as predictors of relapse in patients with quiescent ulcerative colitis during maintenance therapy Takayuki Yamamoto & Manabu Shiraki & Takuya Bamba & Satoru Umegae & Koichi Matsumoto
Accepted: 4 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose This prospective study was to evaluate the significance of fecal calprotectin and lactoferrin for the prediction of ulcerative colitis (UC) relapse. Methods Eighty UC patients in remission for ≥3 months on mesalamine as maintenance therapy were included. At entry, stool samples were collected for the measurement of calprotectin and lactoferrin. All patients were followed up for the following 12 months. To identify predictive factors for relapse, time-dependent analyses using the Kaplan-Meier graphs and Cox's proportional hazard model were applied. Results During the 12 months, 21 patients relapsed. Mean calprotectin and lactoferrin levels were significantly higher in patients with relapse than those in remission (calprotectin—173.7 vs 135.5 μg/g, P =0.02; lactoferrin— 165.1 vs 130.7 μg/g, P =0.03). A cutoff value of 170 μg/g for calprotectin had a sensitivity of 76 % and a specificity of 76 % to predict relapse, while a cutoff value of 140 μg/g for lactoferrin had a sensitivity of 67 % and a specificity of 68 %. In a multivariate analysis, calprotectin (≥170 μg/g) was a predictor of relapse (hazard ratio, 7.23; P =0.002). None of the following parameters were significantly associated with relapse: age, gender, duration of UC, number of UC episode, severity of the previous episode, extent of UC, extraintestinal manifestation, and lactoferrin level. Conclusions Fecal calprotectin showed a higher sensitivity and specificity than fecal lactoferrin for predicting UC relapse. Fecal calprotectin level appeared to be a significant predictor of relapse in patients with quiescent UC on mesalamine as maintenance therapy. T. Yamamoto (*) : M. Shiraki : T. Bamba : S. Umegae : K. Matsumoto Inflammatory Bowel Disease Center, Yokkaichi Social Insurance Hospital, 10-8 Hazuyamacho, Yokkaichi, Mie 510-0016, Japan e-mail: [email protected]
Keywords Calprotectin . Fecal marker . Lactoferrin . Mesalamine . Ulcerative colitis
Introduction Ulcerative colitis (UC) is a chronic disease characterized by mucosal inflammation in the colon and the rectum [1]. The course of UC is characterized by spontaneous remission, which may be followed by a relapse, and is typically unpredictable. The main goal of medical therapy in patients with UC is effective and sustained suppression of intestinal inflammation in order to induce and maintain clinical remission. Identifying patients at a significant risk of relapse during quiescent UC should help to determine a strategy for maintenance therapy during remission. Previous studies have reported that younger age, multiple previous episodes of relapse, extraintestinal manifestations, a low-fiber diet, seasonal factors, and a long-term perceived stress were risk factors for future relapse in patients with quiescent UC [2–5]. These factors are of limited value in clinical setting, and therefore more objective parameters that may predict future relapse would be desirable in our clinical practice. The presence of active intestinal inflammation in patients with UC is associated with an acute phase reaction and migration of leucocytes to the gut, and this leads to the release of several neutrophil proteins, which may be measured in stool samples [6]. Fecal markers fulfill all the criteria of being noninvasive, simple, inexpensive, sensitive, and specific markers to detect gastrointestinal inflammation. Calprotectin is a calcium-binding protein, representing up to 60 % of the cytosolic proteins in neutrophils [7]. Since calprotectin is primarily derived from neutrophils, its concentration is directly proportional to the concentration of neutrophils in the colonic/rectal mucosa [8]. It is resistant to bacterial
Int J Colorectal Dis
degradation in the gut and is stable in the stool for up to 1 week at room temperature [9, 10]. Lactoferrin is an iron-binding glycoprotein and a major component of the secondary granules of polymorphonuclear neutrophils [11, 12]. During intestinal inflammation, leucocytes invade the mucosa, which may lead to an increase in the excretion of lactoferrin into the stool. Lactoferrin can be measured by using simple and inexpensive techniques since it is stable in the stool for up to 4 days [13, 14]. Several studies [15–18] have compared fecal markers with disease activity indices and endoscopic/histological evaluations to monitor intestinal inflammation in patients with inflammatory bowel disease (IBD). Levels of fecal calprotectin and lactoferrin have a proportional correlation to the degree of inflammation of the intestinal mucosa. Fecal calprotectin and lactoferrin may have a role in monitoring disease activity in patients with IBD [19–24]. With this background in mind, the present study was designed to rigorously evaluate whether or not these neutrophil proteins are clinically relevant for predicting relapse in patients with quiescent UC. Further, we included a homogeneous group of patients who were receiving mesalamine as maintenance therapy.
Patients and methods
Table 1 Baseline characteristics of 80 eligible patients Age at entry (mean ± SE) Male/female (n) Duration of UC before entry (mean ± SE) Number of UC episode (n) 1–4 ≥5 Severity of the previous episode (n) Mild (DAI score ≤4) Moderate (5≤DAI score ≤8) Severe (DAI score ≥9) Extent of UC (n) Left-sided colitis Extensive colitisa Extraintestinal manifestations (n) Arthritis Pyoderma gangrenosum Induction therapy for the previous episode (n) Mesalamine (Pentasa 4 g/day) Corticosteroids (prednisolone 30–60 mg/day) Leukocytapheresis Immunosuppressants (Tacrolimus) Biologics (Infliximab)
54 26 28 41 11 60 20 11 3 63 47 48 10 2
UC ulcerative colitis, DAI Disease Activity Index [25] a
Study design
35.1±0.8 years 49:31 50.0±2.7 months
Involvement extends proximal to the splenic flexure
This was a prospective, single-center study undertaken at the Yokkaichi Social Insurance Hospital, a referral center treating a large number of patients with IBD in the Mie Prefecture of Japan. The study was conducted in accordance with the principle of good clinical practice and the Helsinki Declaration. Our study protocol was reviewed and approved by our Institutional Review Board.
clinical remission with medical treatment. As remission induction therapy, high-dose mesalamine (Pentasa, 4 g/day) was used in 63 patients, prednisolone (30–60 mg/day) in 47 patients, therapeutic leucocytapheresis (Adacolumn, JIMRO, Takasaki, Japan) in 48 patients, tacrolimus (Prograf) in 10 patients and infliximab (Remicade, 5 mg/kg at weeks 0, 2, and 6, and then at 8-week intervals) in 2 patients.
Patients
Remission maintenance therapy
Patient inclusion criteria were: (1) age between 20 and 75, (2) having endoscopic and histologic diagnosis of UC, (3) UC confined to the colon and the rectum, and (4) patient in clinical remission (normal stool frequency and no rectal bleeding) on oral mesalamine for ≥3 months. Exclusion criteria were: (1) patients who had received corticosteroids, immunosuppressants, or biologic agents at entry; (2) had received mesalamine enema; and (3) had received nonsteroidal anti-inflammatory drugs, antidiarrheal (loperamide or codeine), or antispasmodic medications at entry. A total of 80 patients who met the inclusion criteria were included in this study. The baseline characteristics of the 80 eligible patients are presented in Table 1. The severity of previous UC episodes was mild in 28 patients, moderate in 41 patients, and severe in 11 patients. All patients achieved
During the study period, all patients were given oral mesalamine (Pentasa, 1.5–3.0 g/day). No patient received mesalamine enema (oral, intravenous, intramuscular, or rectal) corticosteroids, immunosuppressants (tacrolimus, azathioprine, and 6-mercaptopurine), or biologic agents except patients who developed clinical relapse. Follow-up All patients were reviewed in our clinic every 2 up to 12 months after entry. Patients were advised to record their symptoms in a diary every day. At the clinic visits, patient's compliance with medication, adverse effects, general wellbeing, stool frequency, stool consistency and presence or absence of abdominal pain, tenderness, tenesmus, rectal
Int J Colorectal Dis
bleeding, and mucus in stool were recorded. Stool frequency and rectal bleeding were scored according to the Disease Activity Index (DAI) system listed in Table 2 [25]. Clinical remission was defined as normal stool frequency (= score 0) and no rectal bleeding (= score 0). When patients developed symptoms suggestive of a flare-up, endoscopic examination was immediately undertaken. Endoscopic score was according to the mucosal appearance section of the DAI. A relapse was defined as a worsening of stool frequency and/or rectal bleeding with an endoscopic score of 2 or 3 [25]. In our clinic, peripheral blood samples were collected for measurement of white blood cell count (WBC), hemoglobin (Hb), platelet count, C-reactive protein (CRP), total protein, albumin, creatinine, urea, sodium, potassium, chloride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactic dehydrogenase, total bilirubin, and cholesterol. Measurement of fecal calprotectin and lactoferrin At entry, patients provided a stool sample for the measurement of calprotectin and lactoferrin. Fecal calprotectin was measured by a quantitative enzyme immunoassay (Human Calprotectin ELISA Kit, Cell Sciences Inc., Massachusetts, USA). Lactoferrin was measured by a colloidal gold agglutination reagent (Auto Lf-Plus, Alfresa Pharma Corp., Osaka, Table 2 Disease activity index (DAI) scoring system Score Stool frequency Normal number of stools 1–2 stools/day greater than normal 3–4 stools/day greater than normal ≥5 stools/day greater than normal Rectal bleeding No blood seen in stool Streaks of blood with stools less than half the time Obvious blood with stools most of the time Blood alone passed Mucosal appearance Normal mucosa or inactive disease Mild inflammatory changes (erythema, decreased vascular patterns, mild friability) Moderate inflammatory changes (marked erythema, absent vascular patterns, friability, erosions) Severe inflammatory changes (spontaneous bleeding and ulcerations) Physician's overall assessment of disease severity Normal Mild disease Moderate disease Severe disease
0 1 2 3 0 1 2 3 0 1 2 3
0 1 2 3
Japan) by using a high-throughput discrete clinical chemistry analyzer (Hemo Techt NS-Plus C, Alfresa Pharma Corp.). As a normal control group, fecal samples were obtained from 80 age- and gender-matched healthy volunteers with no history of gastrointestinal disorder. Laboratory personnel were blinded to the clinical data. Statistical analysis Continuous data are presented as the mean±SE values. Comparisons of frequencies were done by using the chi-square test with Yates' correction. The average values between two groups are compared by unpaired t test. For comparisons involving more than two groups, the one-way analysis of variance was applied. Correlations were calculated by using the Spearman's “r ” test. The cumulative relapse rate was calculated by the Kaplan-Meier method, and was compared between the groups by using the log-rank test. Predictors of relapse were determined by multivariate analysis using Cox's proportional hazard model. P
