Detection of colorectal neoplasms by the highly sensitive hemoglobin-haptoglobin complex in feces

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1 Int J Colorectal Dis (1999) 14: Springer-Verlag 1999 ORIGINAL ARTICLE Andreas Sieg Christine Thoms Kai Lüthgens Markus R. John Heinrich Schmidt-Gayk Detection of colorectal neoplasms by the highly sensitive hemoglobin-haptoglobin complex in feces Accepted: 25 October 1999 Abstract Screening for fecal occult blood by means of guaiac tests has an unsatisfactory sensitivity for the detection of colorectal neoplasms. The immunological determination of human hemoglobin in feces has a higher sensitivity and specificity, but hemoglobin is degraded during its transport through the gastrointestinal tract. We compared the hemoglobin test to a newly developed immuno-chemiluminometric (ILMA) assay for quantifying the hemoglobin-haptoglobin complex in feces which shows high stability against degradation. From each of 621 patients with gastrointestinal complaints before scheduled colonoscopy we collected two 1-ml samples from a single stool; there were no dietary restrictions. The sensitivity for detecting colorectal carcinomas proved 87% with hemoglobin. With the hemoglobinhaptoglobin complex it was 87% at a cutoff level of 1.5 µg/g feces, 83% at 2. µg/g feces, and 78% at 2.5 and 3. µg/g feces. The sensitivity for detecting large adenomatous polyps was 54% with hemoglobin, 76% with the hemoglobin-haptoglobin complex at a cutoff point of 1.5 µg/g feces, 73% with the hemoglobin-haptoglobin complex at 2. and 2.5 µg/g feces, and 65% with the hemoglobin-haptoglobin complex at 3. µg/g feces. The optimal cutoff point for the hemoglobin-haptoglobin complex was estimated to be 2. µg/g stool. The specificity for hemoglobin (99%) was significantly higher than that for the hemoglobin-haptoglobin complex at 2. µg/g feces (96%). Immunological determination of the hemoglobin-haptoglobin complex in feces has a comparable sensitivity as the fecal hemoglobin assay for A. Sieg ( ) Practice of Gastroenterology, Hauptstrasse 45, D Östringen, Germany dr.andreas.sieg@t-online.de, Tel.: , Fax: K. Lüthgens M.R. John H. Schmidt-Gayk Laboratory Group, University of Heidelberg, Heidelberg, Germany A. Sieg C. Thoms M.R. John H. Schmidt-Gayk Department of Medicine, University of Heidelberg, Heidelberg, Germany colorectal carcinomas and a significantly higher sensitivity for adenomatous polyps but a significantly lower specificity. Its use for colorectal cancer prevention is currently being evaluated in a screening study. Key words Colorectal cancer Colonic polyps -haptoglobin complex Occult blood Introduction Guaiac tests are widely used in colorectal cancer screening to detect fecal occult blood (FOB), although they have an unsatisfactory sensitivity for detecting colorectal neoplasms in asymptomatic patients [1 4]. In a group of 248 patients with positive fecal guaiac tests who underwent colonoscopy followed by esophago-gastro-duodenoscopy upper gastrointestinal lesions were identified more frequently than colonic lesions [5]. FOB screening with guaiac tests reveal significantly fewer colorectal neoplasms than sigmoidoscopy [6]. A major disadvantage of guaiac tests is the fact that they are not specific for human blood and can produce positive results when meat, fruit, or vegetables containing peroxidase have been ingested [7]. The rate of false-negative results is increased by large amounts of vitamin C [8]. Nevertheless, the mortality rate from colorectal cancer could reportedly be reduced by 15 18% with biennial screening using nonrehydrated guaiac tests [9, 1] and by 33% with annual screening using rehydrated guaiac tests [11]. A meta-analysis with four randomized and two nonrandomized trials of Hemoccult screening showed a 16% reduction in mortality from colorectal cancer [12]. When adjusted for attendance at screening, this reduction was 23% for persons actually screened [12]. Given the more sophisticated immunological screening techniques to detect constituents of human blood in feces, it should be possible to reduce mortality from colorectal cancer even further. A higher sensitivity and specificity of immunological tests for hemoglobin (Hb) in the detection of co-

2 268 lorectal neoplasms in comparison to guaiac tests has been reported in screening [1, 4, 13] and evaluation studies [14, 15], and Hb has proven more sensitive than albumin in detecting colorectal neoplasms [16]. The Hb molecule, however, is partly degraded during passage through the gastrointestinal tract and may become unrecognizable to the antibody in the assay. This is supported by the finding in our previous study that 2 of 12 right-sided cancers were not detected by the Hb assay, in contrast to 31 left-sided cancers which all were detected [16]. This problem can be overcome by employing an immunoassay for α 1 -antitrypsin [17] which degrades at a much lower rate [18], or an immunological test of the Hbhaptoglobin (Hp) complex which shows high stability in gastric juice and fecal extracts [19]. The aim of this study was to compare a newly developed immuno-chemiluminometric assay (ILMA) of the HbHp complex [2] with our standard luminescence immunoassay of Hb in feces in an evaluation study for the detection of colorectal neoplasms. Methods Human Hb and the HbHp complex in feces were determined immunologically in 621 patients (28 men, 341 women; aged years, median 59) scheduled for colonoscopy. The patients had been referred to a practice for gastroenterology by their family physicians for the investigation of gastrointestinal symptoms. The patients collected 1-ml samples into 1-ml air-tight stool specimen tubes (Sarstedt, Nümbrecht-Rommelsdorf, Germany) from two different sites of a single stool at least 2 days prior to examination. Collection into vials provides a more representative amount of feces than paper smears and allows a quantitative determination of the constituents. No dietary restrictions were made. The samples were stored in the deep-freeze for 1 week before examination. Those handling the laboratory results were blinded to the colonoscopic results, and vice versa. The laboratory samples were thawed, weighed, and diluted tenfold with a phosphate buffer ph 7.4 containing 2 g gelatin, 1 g sodium azide, and.4 g EDTA per liter. The diluted samples were mixed thoroughly and centrifuged for 15 min at 3 g, and the supernatants were measured in luminescence immunoassays specific for human Hb or the HbHp complex. The luminescence immunoassay for Hb was set up according to Gao et al. [21] and that for the HbHp complex according to Lüthgens et al. [2]. The upper limit of normal for human hemoglobin was set at 1 µg/g feces, according to our earlier findings [16]. For the HbHp complex the optimal threshold value was to be found. The detection limit was about 1 µg/g feces for human Hb and.3 µg/g feces for the HbHp complex. Diagnosis was based on the endoscopic and histopathological findings. Carcinomas were classified according to the UICC stages I IV of the TNM system, and location. Adenomatous polyps were classified as to histopathological characteristics, size (large polyps: >1 cm; small polyps <1 cm) and location. We defined the left colon as consisting of the rectum, sigmoid, descending colon, and the splenic flexure; and the right colon as consisting of the cecum, ascending colon, hepatic flexure, and transverse colon. The results were classified as true-positive if a neoplasm (carcinoma or large adenomatous polyp) was found, and as false-positive if a normal colonic mucosa and no cause of extracolonic gastrointestinal bleeding was found. In patients with upper abdominal complaints and normal colonic mucosa the upper gastrointestinal tract was examined by esophago-gastro-duodenoscopy. Sensitivity and specificity are expressed below as percentages defined in the standard manner [22 24]. Fecal Hb and the HbHp complex were compared by McNemar s χ 2 test [25]; P<.5 was considered as statistically significant. Results In 23 patients with colorectal carcinoma the sensitivity of fecal Hb was 87% (Table 1). The sensitivity of the HbHp complex depended on the cutoff value and was 87% at 1.5 µg/g stool, 83% at 2. µg/g stool, and 78% at 2.5 and 3. µg/g stool. The sensitivity of fecal Hb in 37 patients with large colorectal adenomas was 54% and that of the HbHp complex was 76% at 1.5 µg/g stool, 73% at 2. and 2.5 µg/g stool, and 65% at 3. µg/g stool. The number patients with early carcinoma (UICC stage Table 1 Sensitivity of immunologically determined fecal human Hb and the HbHp complex (at various cutoff values: µg/g stool) in patients with carcinomas and large adenomatous polyps: percentages of positive test results Findings n Hb HbHp 1.5 HbHp 2. HbHp 2.5 HbHp 3. Carcinomas (all cases) Left-sided Right-sided Large adenomas (all cases) Left-sided Right-sided Early cancers (UICC stage I) Table 2 Specificity of fecal human Hb and the HbHp complex (at various cutoff values: µg/g stool) in 357 patients with normal colonic mucosa Hb HbHp 1.5 HbHp 2. HbHp 2.5 HbHp 3. n % n % n % n % n % Positive test results Positive test results without reason for GI bleeding Specificity

3 Sensitivity Sensitivity a haptoglobin Specificity -haptoglobin 269 I; n=5) was too small for statistical evaluation, as was the number of right-sided carcinomas and adenomas. Of 357 patients with normal colonic mucosa 19 had positive hemoglobin (5%), 47 (13%) positive HbHp complex at 1.5 µg/g stool, 36 (1%) at 2. µg/g stool, 31 (9%) at 2.5 µg/g stool and 25 (7%) at 3. µg/g stool (Table 2). In 26 of these 47 patients we found the following causes of gastrointestinal bleeding: gastric ulcer, 1; erosive gastritis, 2; duodenal ulcer, 3; enlarged hemorrhoids, 18; coumarine therapy, 1; nonsteroidal antiinflammatory drugs, 2; Oslers disease with epistaxis, 1. Excluding these patients from the group with normal colonic mucosa and positive FOB tests, only 21 patients remained with false-positive findings. Thus the specificity of the test, defined by false-positive results if a normal colonic mucosa and no other cause of gastrointestinal bleeding was found, was 99% for Hb (Table 2). For the HbHp complex the specificity was 93% at 1.5 µg/g stool, 96% at 2. µg/g stool, 97% at 2.5 µg/g stool, and 98% at 3. µg/g stool. The receiver operating characteristic curves of Hb and the HbHp complex for the detection of carcinomas and adenomatous polyps are shown in Fig. 1. The optimal cutoff point for the HbHp complex was calculated at 2. µg/g stool. With this value we compared the sensitivity and specificity for Hb, the HbHp complex, and the combined test (Table 3). The positive predictive value of the combined test was 14% for carcinomas, 19% for large adenomas, and 33% for colorectal neoplasms (carcinomas and large adenomas). The results for miscellaneous diagnoses other than colorectal cancer and large adenomatous polyps are shown in Table 4..1 b Specificity Fig. 1a,b Receiver operating characteristic curves for fecal human Hb and the HbHp complex. a In 23 patients with colorectal carcinoma. b In 37 patients with large adenomatous polyps Discussion We found the sensitivity of the HbHp complex in detecting colorectal cancers to be comparable or, at higher cutoff levels, slightly below that of the Hb assay. However, the HbHp complex and the combined test were signifi- Table 3 Sensitivity and specificity of fecal human Hb (normal value <1 µg/g stool), the HbHp complex (normal value <2 µg/g stool), the combined test, and the significance of the χ 2 test for Hb vs. HbHp at a cutoff of 2. µg/g feces Hb (%) HbHp (%) P Hb+HbHp (%) Sensitivity for colorectal carcinoma (n=23) NS 87 Sensitivity for large adenomas (n=37) <.5 73 Specificity <.5 96 Table 4 Sensitivity of fecal Hb (normal value <1 µg/g stool), the HbHp complex (normal value <2 µg/g stool), and the combined test in patients with miscellaneous diagnoses: positive test results Diagnoses n Hb HbHp Hb+HbHp n % n % n % Small adenomas (<1 mm) Hyperplastic polyps Diverticulosis Diverticulitis Ulcerative colitis Crohn s disease Rectal ulcer Unspecific colitis

4 27 cantly superior to Hb in detecting large adenomatous polyps. This offers important progress in screening since adenomas are precursors of most of the carcinomas and systematic removal of adenomas may reduce the incidence of colorectal cancers by up to 9% [26]. The optimal cutoff point for the HbHp complex was determined to be 2 µg/g feces. The sensitivity in the present study was in the same range or even better than that reported by other authors for hemoglobin tests [1, 4, 13 15], and far higher than the values reported for guaiac tests without rehydration [1 4, 13 15], although we examined only two samples from a single stool. This is more convenient for the participants in a screening program and, according to a previous study [27], results in better compliance of 83% compared to 3 67% reported for guaiac tests [4, 9 11]. However, our study was not performed under screening conditions, under which the values for sensitivity would possibly be lower due to the small proportion of participants with symptoms. In the specific setting of a gastroenterological practice the positive predictive value of the combined test, describing the possibility of having the disease if the test is positive, was 14% for colorectal carcinomas, 19% for large adenomas, and 33% for colorectal neoplasms (carcinomas and large adenomas). These values are comparable to those obtained in screening studies with Hb, in which the respective positive predictive values were 7.6% [27] and 5.% [4] for colorectal cancer and 49.6% [27] and 2.5% [4] for all neoplasias, and comparable to nonrehydrated Hemoccult tests with positive predictive values of 6.6% [4] and 12% [9] for colorectal cancer and 23.2% [4] and 46% [9] for all neoplasias, but superior to studies using the rehydrated Hemoccult test, with a positive predictive value of only 2.2% for colorectal cancer [11]. The low probability of detecting cancer after a positive FOB test is due to the wide variety of diseases that may produce a positive FOB test. Especially with the immunological tests a positive result may, on the one hand, be false-positive for cancer but, on the other, true-positive for another disease of the gastrointestinal tract. This is not atypical for screening tests. Specificity is an important determinant of the cost of FOB screening. Even small changes in test specificity result in large effects on the number of false-positives in a screening study, which are responsible for unnecessary colonic examinations [28]. A 33% annual reduction in colorectal cancer mortality has been reported possible for patients screened by a rehydrated Hemoccult II test [11]. Rehydration, however, decreases the specificity of the test to 9.4% resulting in a high false-positive rate; colonoscopy has been reported as necessary in 38% of screened individuals [11]. In our study the specificity defined by false-positive results if a normal colonic mucosa and no other cause of gastrointestinal bleeding was found was 99% for hemoglobin. This was significantly higher than the values for the HbHp complex and the combined test (96%, P<.5). This rather high specificity of immunological FOB tests was obtained under conditions of an evaluation study in patients with symptoms. The specificity in a screening study is expected to be higher. For the Hb test the specificity under screening conditions was 99.5% [27]. Whether Hb alone, the HbHp complex, or the combined test should be used for colorectal cancer screening depends on the decision of public health authorities. In general, compliance and sensitivity of FOB tests are important determinants of the cost-effectiveness of colon screening programs [29 31]. A major advantage of immunological FOB tests is the possibility of varying the normal range and thereby affecting sensitivity and specificity according to the risk of the population screened and according to the financial resources of the country. Fecal hemoglobin can be examined in every laboratory with standard equipment, although it is not a bedside test. According to our experience, a bedside test is not required for a mass screening of healthy adults. The time required to obtain a test result is similar for the Hemoccult and for our immunological test. The Hemoccult test has a sample collection period of at least 3 days. The immunological test has a sample collection period of only 1 day, and analysis can be carried out in 1 day. The costs of the immunological hemoglobin test or the HbHp complex in Germany are about four times those of the Hemoccult test. Previous data have shown that cost of screening by immunological methods are less than the savings from avoiding carcinoma costs by endoscopic polypectomy of Dukes A carcinomas [27, 32]. Immunological determination of the fecal HbHp complex exhibits a high sensitivity for colorectal neoplasms, especially for adenomatous polyps, at the expense of decreased specificity. Whether immunological assays of fecal Hb, the HbHp complex, or a combined test should be used in colorectal cancer screening can be decided after evaluation of an ongoing screening study comparing fecal Hb and the HbHp complex. Acknowledgements We are indebted to W. Bersch, M.D., and to J. Bertling, M.D., Speyer, Germany for the histopathological evaluation. References 1. Frommer DJ, Kapparis A, Brown MK (1988) Improved screening for colorectal cancer by immunological detection of occult blood. BMJ 96: Allison JE, Feldman R, Tekawa IS (199) Hemoccult screening in detecting colorectal neoplasm: sensitivity, specificity, and predictive value: long-term follow-up in a large group practice setting. Ann Intern Med 112: Ahlquist DA, Wieand HS, Moertel CG, McGill DB, Loprinzi CL, O Connell MJ, Maillard JA, Gerstner JB, Pandya K, Ellefson RD (1993) Accuracy of fecal occult blood screening for colorectal neoplasia: a prospective study using Hemoccult and Hemoquant tests. JAMA 269: Allison JE, Tekawa IS, Ransom LR, Adrain AL (1996) Comparison of fecal occult-blood tests for colorectal cancer screening. N Engl J Med 334: Rockey DC, Koch J, Cello JP, Sanders LL, McQuaid K (1998) Relative frequency of upper gastrointestinal and colonic lesions in patients with positive fecal occult-blood tests. N Engl J Med 339:

5 Verne JECW, Aubrey R, Love SB, Talbot IC, Northover JMA (1998) Population based randomised study of uptake and yield of screening by flexible sigmoidoscopy compared with screening by faecal occult blood testing. BMJ 317: Macrae FA, St John DJB, Calagiore P, Taylor LS, Legge WJ (1982) Optimal dietary conditions for Hemoccult testing. Gastroenterology 82: Jaffe RM, Kasten B, Young DS, MacLowry JD (1975) Falsenegative stool occult blood tests caused by ingestion of ascorbic acid (vitamin C). Ann Intern Med 83: Hardcastle JD, Chamberlain JO, Robinson MHE, Moss SM, Satya SA, Balfour TW, James PD, Mangham CM (1996) Randomised controlled trial of faecal-occult-blood screening for colorectal cancer. Lancet 348: Kronborg O, Fenger C, Olson J, Jörgensen OD, Söndergaard O (1996) Randomised study of screening for colorectal cancer with faecal-occult-blood test. Lancet 348: Mandel JS, Bond JH, Church TR, Snover DC, Bradley MG, Schumann LM, Ederer F (1993) Reducing mortality from colorectal cancer by screening for fecal occult blood. N Engl J Med 328: Towler B, Irwig L, Glasziou P, Kewenter J, Weller D, Silagy C (1998) A systematic review of the effects of screening for colorectal cancer using the faecal occult blood test, Hemoccult. BMJ 317: Robinson MHE, Marks CG, Farrands PA, Whynes DK, Bostok K, Hardcastle JD (1995) Is an immunological faecal occult blood test better than Haemoccult? A cost-benefit study. Eur J Surg Oncol 21: Hope RL, Chu G, Hope AH, Newcombe RG, Gillespie PE, Williams SJ (1996) Comparison of three faecal occult blood tests in the detection of colorectal neoplasia. Gut 39: St John DJB, Young GP, Alexeyeff MA, Deacon MC, Cuthbertson AM, Macrae FA, Penfold JC (1993) Evaluation of new occult blood tests for detection of colorectal neoplasia. Gastroenterology 14: Sieg A, Scheida M, John MR, Hertel A, Schröter M, Lüthgens K, Schmidt-Gayk H (1998) Validity of new immunological human hemoglobin and albumin tests in detecting colorectal neoplasms an endoscopy-controlled study. Z Gastroenterol 36: Morrow RJ, Lawson N, Hussaini SH, Asquith P (199) The usefulness of faecal haemoglobin, albumin and alpha-1-antitrypsin in the detection of gastrointestinal bleeding. Ann Clin Biochem 27: Florent C, L Hirondel C, Desmazures C, et al (1981) Intestinal clearance of alpha-1-antitrypsin. A sensitive method for the detection of protein losing enteropathy. Gastroenterology 81: Meguro T (1994) Measurement of fecal hemoglobin-haptoglobin complex as a new diagnostic tool of lower gastrointestinal tract diseases. Hokkaido Igaku Zasshi 69: Lüthgens K, Maier A, Kampert I, Sieg A, Schmidt-Gayk H (1998) -haptoglobin complex. A highly sensitive assay for the detection of fecal occult blood. Clin Lab 44: Gao P, John MR, Schmidt-Gayk H, Arndt B, Scheida M, Theuer D (1995) Solid-phase competitive luminescence immunoassay for lysozyme in faeces. Clin Chim Acta 239: Abel U (1993) Die Bewertung diagnostischer Tests. Hippokrates, Stuttgart 23. Royall RM (1976) Current advances in sampling theory: implications for human observational studies. Am J Epidemiol 14: Saah AJ, Hoover DR (1997) Sensitivity and specificity reconsidered: the meaning of these terms in analytical and diagnostic settings. Ann Intern Med 126: Zöfel P (1992) Statistik in der Praxis. Fischer, Stuttgart 26. Winaver SJ, Zauber AG, Ho MN, O Brien MJ, Gottlieb LS, Sternberg SS, Waye JD, Schapiro M, Bond JH, Panish JF, Ackroyd F, Shike M, Kurtz RC, Hornsby-Lewis L, Gerdes H, Stewart ET, and the National Polyp Study Workgroup (1993) Prevention of colorectal cancer by colonoscopic polypectomy. N Engl J Med 329: Sieg A Hertel A, John MR, Lüthgens K, Schmidt-Gayk H (1998) Screening for colorectal neoplasms with a new immunological human faecal haemoglobin and albumin test. Eur J Cancer Prev 7: Ransohoff DF, Lang CA (1997) Screening for colorectal cancer with the fecal occult blood test: a background paper. Ann Intern Med 126: Liebermann DA (1995) Cost-effectiveness model for colon cancer screening. Gastroenterology 19: Toribara NW (1995) Sleisinger MH. Screening for colorectal cancer. N Engl J Med 332: Winaver SJ, Fletcher RH, Miller L, Godlee F, Stolar MH, Mulrow CD, Woolf SH, Glick SN, Ganiaths TG, Bond JH, Rosen L, Zapka JG, Olsen SJ, Giardello FM Sisk JE, Van Antwerp R, Brown-Davis C, Marciniak DA, Mayer RJ (1997) Colorectal cancer screening: clinical guidelines and rationale. Gastroenterology 112: Sieg A, Schröter M, Hertel A, Schmidt-Gayk H (1996) Die Prävention des kolorektalen Karzinoms nach dem Östringer Modell ist kosteneffektiv. Z Gastroenterol 9:61(A)