Annals of Clinical & Laboratory Science 36:294-298 (2006)
© 2006 Association of Clinical Scientists
Rapid Fecal Cytokeratin-19 Test and Fecal Occult Blood Test in Screening for Gastrointestinal Diseases
Hyunjung Kim1,
Yonggoo Kim1,
Sangsoon Yoon3,
Jihyang Lim1,
Myungshin Kim1,
Soonwook Lee1,
Sunghan Kang1,
Eun Jung Lee2,
Chang Suk Kang2 and
Kyungja Han1
1 Departments of Laboratory Medicine and 2 Hospital Pathology, Catholic University Medical College, Seoul; 3 DiNonA Research Institute, Seoul, Korea
Address correspondence to Kyungja Han, M.D., Department of Clinical Pathology, Catholic University Medical College, St. Mary’s Hospital, Youngdeungpo-gu, Youido-dong 62, Seoul, Korea (South) 150-713; tel 822 3779 1297; fax 822 783 6648; e-mail hankja{at}catholic.ac.kr.
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Abstract
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To evaluate the screening power of the fecal cytokeratin-19 test (CK-19) and the fecal occult blood test (FOBT), we performed rapid fecal CK-19 and FOBT tests on 515 stool samples from patients with various GI diseases and 814 stool samples from control patients. The rapid fecal CK-19 test (developed by DiNonA Research Institute, Seoul, Korea) is based on gold immunochromatography and has a sensitivity of 1 ng/ml. The positive rate of the FOBT was 2.1% in controls, 14.0% in GI cancer patients, 3.5% in GI inflammation patients, 11.7% in bone marrow transplant (BMT) patients, and 6.0% in childhood diarrhea patients. Except for the GI inflammation patients, the patients’ positive rates for FOBT were all higher than the controls (p <0.05). The positive rate of the fecal CK-19 test was 8.2% in controls, 42.1% in GI cancer patients, 66.0% in GI inflammation patients, 84.8% in BMT patients, and 19.9% in childhood diarrhea patients. In all of the patient groups, positive rates for the CK-19 test were higher than in the controls (p <0.05). The fecal CK-19 test was more frequently positive (42.1%) in GI cancer patients than the FOBT; if both tests were used, the sensitivity was 49.1%. The fecal CK-19 test (but not the FOBT) gave a higher positive rate in GI inflammation patients than the controls, suggesting that the CK-19 test could serve as a screening test for GI inflammation. The highest positive rate of the fecal CK-19 test was found in the BMT group, indicating that significant GI epithelial desquamation had occurred. Although the positive rate of the fecal CK-19 test in childhood diarrhea patients was higher than in the controls, it was much lower than in adults with GI inflammatory disease. Evidently, children with GI inflammation do not desquamate as much intestinal epithelium as adult patients with GI inflammation. This study shows that the rapid fecal GK-19 test, used in conjunction with the FOBT, may be a valuable screening technique for GI diseases and can assist physicians in the differential diagnosis of GI diseases.
Keywords: fecal cytokeratin-19, fecal occult blood, graft vs host disease, bone marrow transplantation, GI cancer, GI inflammation, childhood diarrhea
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Introduction
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Gastrointestinal tract (GI) cancers account for the highest incidence and mortality of cancer worldwide [1]. The general survival rate of colorectal cancer patients is low [2,3], even though this cancer can be surgically cured if an early diagnosis is made. Therefore, there is an urgent need to detect and diagnose colorectal cancer at an early stage before the patient has developed an inoperable tumor or metastases. The fecal occult blood test (FOBT) is presently the only laboratory test suitable for screening a large population for colorectal cancer [4,5]. However, the efficacy of FOBT in reducing mortality from colorectal cancer is still controversial [4,6] because of the test’s high incidence of false positive and false negative results. Colonoscopy and tomography are effective in detecting colorectal lesions, but are too expensive and invasive for use as screening tools [7–9]. There is no simple laboratory test for screening for gastric cancer, either. Serum tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9), and anti-P53 antibody are not sufficient for making an early diagnosis of GI cancer [10–13].
Most diseases of the GI tract involve epithelial lesions. The number of patients with graft vs host disease (GVHD) and thrombotic microangiopathy (TMA) has recently increased because of the increased frequency of bone marrow transplantation (BMT) and high dose chemotherapy. GVHD is the most important problem following BMT and it frequently involves the colon. Apoptosis of the colonic mucosa is required for diagnosis of GVHD [14]. Patients with TMA show severe mucosal desquamation in the small intestine on autopsy, but there has been no laboratory test to estimate the severity of intestinal mucosal desquamation in vivo.
Cytokeratins (CKs) are a family of intracellular fibrous proteins that are present in almost all epithelia [15]. At least 20 subclasses of keratins have been identified on the basis of their molecular weights and isoelectric pH values. Cytokeratins are expressed in normal and tumor cell epithelium and constitute an excellent marker for epithelial differentiation [16]. Since malignant cells generally retain the intermediate filaments of their progenitor cell type, CKs can be used to characterize neoplastic cells of epithelial origin [17,18]. GI mucosal epithelial cells show strong expression of CK-19 [19]. Stool is an excretory product that is a rich source of epithelial cells derived from the GI tract [19]. Fecal CK-19 elimination has not hitherto been evaluated as a marker in screening for GI diseases, including cancer. To evaluate the screening power of fecal CK-19, we have developed a rapid fecal CK-19 test kit and we have conducted this study on 1,329 stool samples from normal persons and from patients with various GI diseases.
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Materials and Methods
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Patients.
From February 2004 to April 2005, we studied stool specimens that were submitted to the clinical laboratory of St. Mary’s Hospital for FOBT. A total of 1,329 stool samples was analyzed. The patients’ diagnoses were obtained from the results of histopathological and/or radiological examinations and by review of the medical records. The patients were classified as having GI cancer (N = 57), GI inflammation (N = 224), GVHD after BMT (N = 33), and childhood diarrhea (N = 201). The controls included patients without any GI diseases, as indicated by histopathological or radiological examinations (N = 814). Stool specimens from 110 healthy persons who were receiving a medical checkup were included in the controls; they were adults (age 22 to 60 yr) and showed no abnormality in the medical examination, which included gastric endoscopy and tests for serum alpha fetoprotein, CEA, CK-19 fragments, CA 19-9, and squamous cell carcinoma-related antigen. All of the patients gave informed written consent to the Catholic Human Sample Bank, Seoul, Korea, for research use of any specimens that remained after routine laboratory tests.
Fecal occult blood test.
The FOBT was performed <2 hr after receipt of the specimen, using a latex agglutination inhibition test for human hemoglobin in stool (Iatron Lab, Tokyo, Japan). After the stool specimen was thoroughly mixed with buffer, one drop of the specimen was mixed with a drop of rabbit antihuman hemoglobin antibody solution. One drop of latex sensitized with human hemoglobin was then added and mixed gently. The results were reported as positive when agglutination was not evident and as negative when agglutination was observed.
Rapid fecal CK-19 test.
The CK-19 test was developed by DiNonA Research Institute (Seoul, Korea) and is owned by DiNonA, Inc.; it has not yet been marketed. The principle of the test is based on gold immunochromatography. The sensitivity is 1 ng/ml (Fig. 1
), and the test gave positive results with the lysates of CK19 positive cell lines (SNU638: 56 ng/ ml; MKN74: 88 ng/ml; MKN28: 72 ng/ml) and negative result with the lysate of a CK19 negative cell line (Jurkat cells). Crossreactivity was not detected with <100 ng/ml of CK7. The anti-CK-19 monoclonal antibody was developed using recombinant GST-CK19 fusion protein as an immunogen; the coating antibody was 25B2(IgG1); the labeling antibody was 4B4(IgG1). Purity of the recombinant GST-CK19 protein standard was >95%. It was used to test the stability of the CK-19 kit. The fecal samples used for the rapid fecal CK-19 test were identical to those used for FOBT. After FOBT, the remaining stool sample was mixed with buffer solution and stored at –80°C until analysis. Two drops of sample were applied to the sample application area of the test cartridge. After 15 min, the results were read as positive when both the test and control lines were visible or as negative when only the control lines were visible (Fig. 2).
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Fig. 1. Senstitivity of the rapid fecal CK-19 test was 1 ng/ml. Strips C1 (10 ng/ml) and C2 (1 ng/ml) show both control and test bands; strip C3 (0.1 ng/ml) shows a very faint test band; strips C4 (0.01 ng/ml) and C5 (0.001 ng/ml) do not show visible test bands, and show only the control bands.
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Fig. 2. Results of the rapid fecal CK-19 test at 15 min after adding the specimen (stool mixed in buffer) showing positive results. The test and control bands are both seen and the intensities of the test bands are different according to samples (A: strong; B: intermediate; C: weak).
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Statistics.
All data were compared using Chi-square tests; p values <0.05 were considered statistically significant.
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Results
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Controls.
Among 814 stool samples from control subjects, 17 samples (2.1%) were FOBT+ and 67 (8.2%) were CK-19+. Eighty samples (9.5%) gave a positive result on either the FOBT or CK-19 test (Table 1).
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Table 1. Results of the rapid fecal CK-19 test and FOBT in control subjects and patients with various gastrointestinal diseases.*
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GI cancer.
Among 57 stool samples from patients with GI cancer, 8 samples (14.0%) were FOBT+ and 24 (42.1%) were CK-19+. Twenty eight samples (49.1%) gave a positive result on either the FOBT or CK-19 test. The FOBT and CK-19 tests both gave higher positive rates for samples from patients with GI cancer than from controls (p <0.05).
GI inflammation.
Among 224 stool samples from patients with GI inflammation, 8 (3.5%) samples were FOBT+, which did significantly differ from the positivity rate of controls (p = 0.1998), and 148 (66.0%) samples were CK-19+, which was much higher than the controls (p <0.0001) or the patients with GI cancer (p = 0.0009). One hundred fifty-six samples (69.7%) gave positive results on either the FOBT or CK-19 test.
Bone marrow transplantation.
Among 33 stool samples from patients with BMT, 19 samples (11.7%) were FOBT+ and 28 (84.8%) were CK-19+. Thirty-one samples (87.7%) gave positive results on either the FOBT or CK-19 test. All of these results showed higher positive rates for the BMT patients than the controls (p <0.05). The fecal CK-19 positivity rate of the BMT patients was similar to the GI inflammation patients (p = 0.3131), but the FOBT positivity rate for the BMT patients was much higher than the GI inflammation patients (p <0.0001).
Childhood diarrhea.
Among 201 stool samples from childhood diarrhea patients, 12 samples (6.0%) were FOBT+ and 40 (19.9%) were CK-19+. Forty-six samples (22.9%) revealed positive results on either the FOBT or CK-19 test. All of these results showed higher positivity rates for the childhood diarrhea patients than the controls (p <0.05). The FOBT positivity rate of the childhood diarrhea patients was similar to the GI inflammation patients (p = 0.2437), but the fecal CK-19 positivity rate of childhood diarrhea patients was lower than the GI inflammation patients (p <0.0001).
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Discussion
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In two controlled trials, the positive predictive value of FOBT as an initial screening test was 10% for carcinoma and 30% for adenomas, indicating that FOBT is a poor screening procedure for colorectal neoplasia [20]. In the present study, only 8 of 57 stool samples (14%) from patients suffering with GI cancer gave positive results for the FOBT. Although this was higher than that for healthy persons, the sensitivity was still very low. The fecal CK-19 test gave a much higher rate of positivity (42.1%) for GI cancer patients; if both tests were used, the sensitivity was 49.1%. Only 9.5% of samples from persons without GI diseases revealed positive results on either the FOBT or CK-19 test. These results were similar to a previous report [21].
Among the 224 stool samples from patients with GI inflammation, only the fecal CK-19 test gave a higher positivity rate than the controls (p <0.0001), and the patients’ FOBT results were not significantly different from the controls. These results are important because the fecal CK-19 test could be used as a screening test for GI inflammation. There is currently no adequate screening method for GI inflammation.
The incidence of clinically significant GVHD after BMT is 20–50% [22–24]. Currently, the screening and diagnosis of gastrointestinal GVHD is based upon endoscopic findings and histologic examination of biopsy specimens, although endoscopy and mucosal biopsies in BMT patients have procedural risks. These patients frequently have neutropenia and thrombocytopenia, which increase the risk of bleeding and infection. Some centers do not perform biopsy until the platelet counts are >50,000/mm3 [25]. Endoscopic procedural mortality in BMT patients is reported to be as high as 1.8% [26]. There is no simple, noninvasive method to diagnose GVHD after BMT. In the present study, among the 33 stool samples from patients with BMT, 28 samples (84.8%) were CK-19+. This was the highest positivity rate for fecal CK-19, and it indicates that much GI epithelial desquamation has occurred. If a semiquantitative estimation of the fecal CK-19 is performed, screening for GVHD using the rapid fecal CK-19 test might be possible.
Among the 201 stool samples from the children with diarrhea, 12 samples (6.0%) were FOBT+ and 40 (19.9%) were CK-19+. Although the positivity rate of the fecal CK-19 test was higher than the controls, it was lower than other GI inflammatory diseases (p <0.0001). This suggests that children with GI inflammation do not desquamate as much as adults with GI inflammation.
In conclusion, the rapid fecal CK-19 test, used in conjunction with FOBT, may be a valuable screening method for GI diseases, and the results of these tests may aid physicians in the differential diagnosis of GI diseases.
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Acknowledgement
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This study was supported by a grant (02-PJ1-PG11-VN01-SV01-0029) of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. The CK-19 test kits used in this study were donated by DiNonA Research Institute.
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Abstract
Introduction
