The differentiation of organic from functional bowel disorders

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1 ORIGINAL ARTICLE Fecal Dimeric M2-Pyruvate Kinase () in the Differential Diagnosis of Functional and Organic Bowel Disorders Jinny Jeffery, DPhil,* Stephen J. Lewis, FRCP, and Ruth M. Ayling, FRCPath* Background: Fecal inflammatory markers have been shown to be useful as noninvasive screening tools to differentiate patients with functional from organic bowel pathology. Of these markers calprotectin has been the most intensively studied. More recently, the dimeric isoform of M2-pyruvate kinase (tumor M2-PK) has been suggested as a marker of gastrointestinal inflammation. The aim of this study was to investigate fecal tumor M2-PK in the differentiation of functional from organic bowel disease. Methods: Fecal calprotectin and tumor M2-PK were measured in 94 controls and 105 gastroenterology outpatients with a possible diagnosis of organic bowel disease. The diagnosis was made by clinical, endoscopic, and radiological criteria. Results: Organic bowel disease was diagnosed in 14 patients (13%). Median calprotectin and tumor M2-PK concentrations were 24.5 and 1 U/mL in controls, 23 and 1 U/mL in functional, and and 12.6 U/mL in organic bowel disease. Sensitivity, specificity, and positive and negative likelihood ratios for diagnosis of organic bowel disease were 93%, 92%, 11.6, and 0.07 for calprotectin and 67%, 88% 5.6, and 0.18 for tumor M2-PK, respectively. in combination with tumor M2-PK gave a sensitivity of 64%, specificity of 98%, and likelihood ratios of 32 and Elevated calprotectin or tumor M2-PK decreased specificity to 87%, but increased sensitivity to 100%. Conclusions: is able to differentiate organic from functional bowel disease but has a lower sensitivity, specificity, and predictive value than calprotectin. Further studies are required, alone or in combination with other markers, before its usefulness in this setting can be recommended. (Inflamm Bowel Dis 2009;15: ) Key Words: inflammatory bowel disease, Crohn s disease, calprotectin, tumor M2-PK Received for publication February 3, 2009; Accepted February 26, From the *Department of Clinical Biochemistry, and Department of Gastroenterology, Derriford Hospital, Plymouth, PL6 8DH UK. Reprints: Dr. Ruth M. Ayling, FRCPath, Department of Gastroenterology, Derriford Hospital, Plymouth, PL6 8DH UK ( ruthayling@ clinicalbiochemistry.org.uk) Copyright VC 2009 Crohn s & Colitis Foundation of America, Inc. DOI /ibd Published online 21 May 2009 in Wiley InterScience ( wiley.com) The differentiation of organic from functional bowel disorders is a significant issue in gastroenterology. The use of fecal inflammatory markers in diagnosis has been a focus of attention recently, particularly as such tests are noninvasive and comparatively easy and cheap to perform. Pyruvate kinase is the enzyme that catalyzes the last reaction of the glycolytic pathway from phosphoenolpyruvate to lactate. A number of isoforms of pyruvate kinase exist and their expression is related to the metabolic activity of the tissue concerned. M2-pyruvate kinase (M2-PK) is found in undifferentiated tissues and cells with rapid turnover and can oscillate between tetrameric and dimeric forms. 1 The dimeric form, often referred to as tumor M2-PK, is commonly expressed in cancer cells and can be detected in plasma. It has been found to be elevated in patients with many cancers including those of the gastrointestinal tract, 2 lung, 3 breast, 4 and kidney. 5 Plasma M2-PK has been noted to be elevated in inflammatory conditions such as rheumatoid arthritis. 6 Fecal tumor M2-PK has been investigated as a biomarker in colorectal malignancy 7 9 with a sensitivity of 91% for colorectal cancer and 60% for polyps >10 mm and specificity of 92%. 9, another protein whose fecal concentration has been investigated as a diagnostic tool in colorectal cancer, was found to have a sensitivity and specificity for colorectal cancer and polyps of 79% and 72%, respectively. 10 However, calprotectin has been more extensively studied as a marker of gastrointestinal inflammation. Its concentration has been shown to be elevated in adults 11,12 and children 13 with inflammatory bowel disease (IBD) and to be of use in distinguishing functional from organic bowel disease 12 and predicting relapse in IBD. 14 Recent studies of fecal tumor M2-PK have suggested that it has potential as a marker of intestinal inflammation in adults 15 and of IBD activity in children. 16 The aim of this study was therefore to investigate measurement of fecal M2- PK in the differentiation of functional and organic bowel disorders. MATERIALS AND METHODS Patients Participants in the study were 105 patients (62 female) attending the Gastroenterology Out Patients Department of a

2 Fecal TABLE 1. Characteristics of Study Patients Control Functional Bowel Disease Organic Bowel Disease Number Female Age (years): Mean Median Range UK university hospital for a specialist opinion regarding a diagnosis of functional or organic bowel disease. Inclusion criteria were symptoms for at least 3 months and requirement for a radiological or endoscopic procedure for clinical management. Patients were excluded if they had previous investigation or diagnosis of IBD or other gastrointestinal disease, coexisting serious illness, or medication known to be associated with intestinal inflammation 17 or intestinal infection (specifically sought by stool culture). A group of 94 healthy adults (47 female) recruited from hospital staff and families and friends of the investigators served as a control group. A full medical history was taken from all patients, including assessment of ROME III criteria, 18 and a physical examination performed. At the initial consultation a fecal sample was requested and screening blood tests were taken, including full blood count, biochemistry profile, and C-reactive protein with additional tests as dictated by clinical circumstances. All patients underwent radiology or colonoscopy according to the clinical judgment of the gastroenterologist responsible for the patient s care. A diagnosis of functional bowel disorder was made on the basis of clinical findings and the exclusion of hematological and biochemical abnormalities and normal radiology or endoscopy. Measurement of and Fecal samples were collected in disposable plastic containers and stored at 20 C until analysis. was measured using the Phical ELISA (Calpro, Norway). A total of 100 mg of feces were extracted in 5 ml of extraction buffer. The supernatant was analyzed using wells coated with polyclonal rabbit antibody and immunoaffinity-purified ALKP-labeled anticalprotectin, with detection of the enzyme reaction with a substrate at 405 nm. was quantified by enzyme-linked immunosorbent assay (ELISA; ScheBo Biotech, Giessen, Germany). A total of 100 mg of feces was extracted in 10 ml of extraction buffer. The supernatant was analyzed by an ELISA which uses wells coated with monoclonal antibody and a second biotinylated monoclonal antibody to tumor M2-PK with detection using a streptavidin-peroxidase conjugate and photometric measurement of the enzyme reaction with tetramethylbenzidine at 450 nm. Ethical Considerations Ethical approval for the study was obtained from the Cornwall and Devon Research Ethics Committee. Statistical Analysis Statistical analysis was performed using GraphPad Prism Software, v. 5.01, 2007 (San Francisco, CA) and the Analyse-it software for Excel, v. 7.1, 2003 (Leeds UK). The Kruskal Wallis test was used to assess differences between groups and receiver operating characteristic (ROC) curves to compare the tests. RESULTS A total of 105 patients was entered into the study with 94 control subjects. Their characteristics are shown in Table 1. A final diagnosis of functional bowel disorder was made in 91 patients and organic bowel disease in 14: Crohn s disease (9), ulcerative colitis (1), collagenous colitis (1), colonic cancer (1), stricture secondary to diverticulitis (1), celiac disease (1). All patients with a diagnosis of organic bowel disease received colonoscopy. Of patients with a final diagnosis of functional bowel disorder, 87 had undergone TABLE 2. Concentrations of Fecal and in Study Patients Controls Functional Bowel Disease Organic Bowel disease U/ML U/ML U/mL Mean Median Range < < <1-200 The table shows the range, mean standard error, and median concentrations of calprotectin and tumor M2-PK in controls and in patients with functional and organic bowel disease. 1631

3 Jeffery et al FIGURE 1. a: Fecal tumor M2-PK concentrations in patients with functional and organic bowel disease and in controls. Dotplots of fecal tumor M2-PK concentrations in controls (n ¼ 94) and patients with functional (n ¼ 91) and organic (n ¼ 14) bowel disease. The median tumor M2-PK concentration of controls was <1 U/mL and of patients with functional and organic bowel disease was <1 U/mL and 12.6 U/mL, respectively. b: Fecal calprotectin concentrations in functional and organic bowel disease. Dotplots of fecal calprotectin concentrations in patients with functional (n ¼ 91) and organic (n ¼ 14) bowel disease. The median calprotectin concentrations of the patients with functional and organic bowel disease were 25 and 184, respectively. colonoscopy and 4 radiology. The concentrations of calprotectin and tumor M2-PK in functional bowel disorder, organic disease, and in the control group are shown (Table 2, Fig. 1). There was no significant difference between functional bowel disease and control groups with respect to calprotectin (P ¼ 0.98) or tumor M2-PK (P ¼ 0.065). However, for both markers there were significant differences between the organic disease group and controls (P < 0.001) and between the organic and functional disease groups (P < 0.001). Using the manufacturer s cutoffs of 50 feces for calprotectin and 4 U/mL for tumor M2-PK, the sensitivity, specificity, and positive and negative predictive values of the 2 tests are shown in Table 3. The ROC curves comparing the performance of the 2 tests are shown in Figure 2. For calprotectin the area under the curve ¼ 0.96 and for tumor M2-PK ¼ DISCUSSION Differentiating between functional and organic bowel disorders, particularly IBD, can be difficult and forms up to 40% of the workload of the gastroenterology outpatient clinic. 19 The gold standard for diagnosis of intestinal inflammation is endoscopy and biopsy. 20 However, endoscopy is uncomfortable for the patient, requires a skilled operator, and is associated with clinical risk. Measurement of fecal inflammatory proteins as an alternative diagnostic tool is therefore advantageous. is the most widely accepted surrogate marker of bowel inflammation and its concentration in feces has been shown to correlate with endoscopic and histological findings. 11 Various other markers have been investigated including fecal a1-antitrypsin, lysozyme, granulocyte elastase, myeloperoxidase, and lactoferrin, of which lactoferrin has been the most studied. 21 Fecal tumor M2-1632

4 Fecal TABLE 3. Test Characteristics of Fecal and in Functional and Organic Bowel Disease Using Cutoff Values of 50 for and 4 U/mL for Sensitivity Specificity PPV NPV LRþve LR ve Tumour M2-PK and or PPV, positive predictive value; NPV, negative predictive value; LRþve, likelihood ratio associated with a positive result; LR ve, likelihood ratio associated with a positive result. PK has been shown to be a sensitive and specific marker of colorectal cancer 2 but its clinical role in this condition merits further development. More recently, fecal tumor M2-PK has received attention as a potential marker of intestinal inflammation. Fecal tumor M2-PK was observed to be increased in association with rectal abscess and ulcerative colitis during studies of colorectal carcinoma. 9 It was proposed as a screening investigation useful for predicting inflammation and severity of pouchitis 22 and subsequent studies have examined its role as an inflammatory marker. Fecal tumor M2-PK was found to be increased in IBD and was a relatively specific marker of organic gastrointestinal disease in a group of 131 newly presenting or known IBD patients, of whom 43 had IBD. 15 In this study using a predetermined cutoff of 3.7 U/mL, the marker was found to have a sensitivity of 73% and specificity of 74% with positive predictive value and negative predictive value of 89% and 57%, respectively. In our study only patients presenting to the clinic for the first time were included and the incidence of functional bowel disorder is much higher. In a group of 107 children with known IBD and 35 healthy controls tumor M2-PK was found to show better correlation with disease activity in ulcerative colitis than Crohn s disease, having a sensitivity of 94.3% in the acute phase of ulcerative colitis. 16 Our study shows that, while fecal tumor M2-PK has potential as a marker of gastrointestinal inflammation, it does not perform as well as calprotectin. An evaluation of fecal calprotectin and lactoferrin in patients with lower gastrointestinal symptoms found that calprotectin correlated better with colonic inflammation at endoscopy and lactoferrin with histological inflammation. Measurement of both tests for accurate assessment of inflammation was recommended. 23 The finding of a high likelihood ratio in the setting of a low prevalence of organic bowel disease suggests that use of calprotectin and tumor M2-PK may be particularly advantageous as a rule-out test in a clinic population with a similar disease prevalence. Further work is required to delineate the exact place of fecal tumor M2-PK in the differentiation of organic from functional bowel disorders. A combination of fecal tumor M2-PK with other surrogate markers of inflammation may be beneficial. ACKNOWLEDGMENTS The authors thank Baz Undy for statistical assistance, ScheBo Biotech for provision of the tumor M2-PK ELISA, and consultant colleagues who recruited patients to the study. FIGURE 2. Receiver operating characteristic (ROC) curves comparing performance of calprotectin and tumor M2-PK to distinguish between patients with functional and organic bowel disease. For calprotectin the area under the curve (AUC) AUC ¼ 0.96 and for tumor M2-PK the AUC ¼ REFERENCES 1. Mazurek S, Boschek CB, Hugo F, et al. Pyruvate kinase type M2 and its role in tumour growth and spreading. Semin Cancer Biol. 2005;15: Hardt PD, Ngoumou BK, Rupp J, et al. Tumor M2-pyruvate kinase: a promising tumor marker in the diagnosis of gastrointestinal cancer. Anticancer Res. 2000;20: Hoopmann M, Warm M, Mallmann P, et al. Tumor M2 pyruvate kinase: determination in breast cancer patients receiving trastuzumab therapy. Cancer Lett. 2002;187:

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