Introduction 5 To eliminate the risk of colorectal cancer, a restorative proctocolectomy with ileal pouch anal anastomosis (IPAA) is accepted as one of the surgical treatments of choice in these patients. 4 14 14 23 34 14 26 33 38 14 23 34 2 3 7 9 13 24 25 29 35 37 10 20 21 17 Min/+ 22 30 The aim of this study is to investigate changes in apoptosis and cell proliferation rates, occurring in the mucosa of the pouch of patients with FAP, in comparison with the ileum of the afferent loop. The results may contribute to a better understanding of the enhanced adenoma formation in the pouch compared to normal ileum. Materials and methods Patients and tissues The study was approved by the regional medical ethical commission, and informed consent was obtained from all patients. Patients with FAP and an IPAA, who were under surveillance in the Radboud University Nijmegen Medical Centre or regional affiliated hospitals, were invited to participate in this study. Thirty-two patients with FAP were included. The diagnosis FAP was based on either a clinical presentation of at least 100 colonic adenomas or a mutation in the APC gene. Data concerning the surgical procedures were obtained from medical records. From each patient, mucosal biopsy specimens of both the pouch and the afferent ileal loop were obtained during a regular surveillance endoscopy, in the period January 2002 until April 2004. Patients were fasted overnight. On the day of examination, patients were encouraged to drink liberally. No laxatives or cathartic enemas were given. To clear the pouch of fecal ruminants, two 250-ml water enemas were given before the endoscopy. The endoscopy was performed with an Olympus GIF-1T140 video endoscope. From January 2002 until August 2003, a 2.8-mm diameter biopsy forceps (FB 13K-1 Olympus, Tokyo, Japan) was used, and from September 2003 until April 2004, a 3.0-mm diameter biopsy forceps (B102-C1-30.160 MedWork/Treier Endoscopie GA, Beromünster, Switzerland) was used. The afferent loop was introduced up to 20 cm proximal of the pouch. The mucosa was sprayed with 1% indigo carmine dye (Laboratoires SERB, Paris) at 1:1 dilution with water, where after photographs were taken to evaluate number and size of adenomas present. For pathological examination, at least four biopsies were taken at random locations from the afferent ileal loop (10 to 20 cm proximal from the pouch), four biopsies from the pouch mucosa (at least 5 cm proximal from the anal verge), and four biopsies from adenomas if present. The biopsies from adenomas were used only for pathological evaluation, i.e., to exclude serious dysplasia in pouch adenomas, and were not used for research purposes. The biopsies were stretched on filter paper to maintain correct orientation of crypts, fixated in formalin, and embedded in paraffin. Immunohistochemistry 8 18 Tissue sections of 4-μm thickness were cut from paraffin blocks, mounted on electrostatic slides (Super Frost Plus, Menzel-Gläser, Germany), and dried overnight, followed by drying in a stove at 50°C for 15 min. Sections were put in xylol for 10 min and taken from xylol through 100% alcohol to water. After deparaffinization, endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min. Pretreatment was performed by heating the tissue sections in citrate buffer (10 mmol/l, pH 6.0) at 180-W power in a microwave oven for 10 min. After cooling at room temperature for 1.5 h, sections were rinsed with PBS. Then, 20% normal horse serum (Vector Laboratories, Burlingame, CA, USA) was applied for 10 min. The sections were then incubated overnight at 4°C with either the mouse monoclonal antibodies MIB-1 at 1:1,000 dilution or M30 at 1:100 dilution. 4 Tissue sections of rectal carcinoma were used as positive controls. Evaluation of immunostaining results Investigators were blinded for the origin of the tissue sections, regarding patient and biopsy location. For evaluation of M30 staining, tissue sections were examined by light microscopy. M30 positivity was identified as brown cytoplasmic staining. M30-positive cells were marked by a first investigator (BvH) and reevaluated by an expert pathologist (IN). In all cases, the complete section was evaluated, and all M30-positive epithelial cells were counted. The apoptotic index was expressed as the number of M30-positive cells per tissue area in square millimeters. Tissue area was assessed by using a Zeiss KS400 computer-aided system. In each MIB-1-immunostained tissue section, crypts whose entire length could be visualized were photographed under ×400 magnification using a Zeiss KS400 computer-aided system. Crypts were excluded when they did not reach the muscularis mucosae or had multilayered bases. MIB-1 positivity was identified as brown nuclear staining. The number of MIB-1-positive epithelial cells and the total number of epithelial cells in up to five crypts per tissue section were counted from screen. The labeling index for each crypt was given by the ratio of MIB-1-positive cells and the total number of crypt epithelial cells and is expressed as percentage of total. For each patient, the labeling indices of pouch and afferent ileal loop were expressed as means of three to five counted crypts. If less than three crypts could be photographed for either pouch or ileal loop, the patient was excluded from analysis. The photographed crypts of five randomly selected patients were counted twice by one investigator (BvH) to determine intraobserver variability. Although great effort was made to obtain well-orientated mucosal crypts when using the quantitative method, however, not all biopsies reached the criteria mentioned above, and therefore, could not be examined. This problem mainly occurred in the biopsies taken from the pouch and might be due to friability of the pouch mucosa. We therefore developed a new semiquantitative scoring system. A representative part of the biopsies showing several complete crypt/villous axes was photographed under ×100 magnification. The photographs were judged pair-wise (pouch vs afferent loop) during which the investigators had to choose from four possible outcomes; one of the two locations showed most MIB-1 positivity. MIB-1 positivity did not differ or no judgement could be made. Judgement was based on relative length of the area of positive cells and the relative size of the stem cell compartment. Five investigators, two pathologists (IN, HvK), two gastroenterologists (PF, FN), and one junior investigator (BvH) independently compared the paired photographs of biopsies of pouch and afferent ileal loop of all patients. When three or more observers agreed in their judgement, this judgement was denoted as consensus judgement. If this criterion was not met, no consensus was reached. If the quality of the tissue sections was poor, no judgment was made. One investigator (BvH) judged the whole series twice for evaluation of intraobserver reliability. Statistical analyses Values for apoptosis and cell proliferation in the quantitative study were not expected to be normally distributed; therefore, they were presented as median and range. The Wilcoxon matched-pairs signed-ranks test was used to compare the paired observations in the apoptosis staining and the paired mean labeling indices in the cell proliferation study. Consensus judgements on the semiquantitative assessment of cell proliferation, favoring either pouch or ileal afferent loop, were compared with a Sign test. To evaluate the reliability of this semiquantitative method to assess cell proliferation, Cohen’s kappa was calculated for the first and second series of judgements by the prime investigator to determine intraobserver reliability. Also, for each pair of investigators, a Cohen’s kappa was calculated. The mean Cohen’s kappa was taken as value for interobserver reliability. Consensus judgments were compared to the difference in mean labeling index between pouch and ileal afferent loop for each evaluable patient. p Results Patient characteristics 1 Table 1 Patient characteristics   Apoptosis Cell proliferation Number of patients studied 32 20 Male/Female 19/13 12/8 Median age in years (range) 32 (16–72) 29 (16–62) Median age at surgery in years (range) 24 (10–55) 20 (10–52) Median age pouch in months (range) 96 (9–216) 105 (9–216) IPAA: hand-sewn/double-stapled/ unknown 9/21/2 5/14/1 Carcinoma at surgery 4 0 Patients with adenomas at biopsy:yes/no 24/8 15/5 Materials and methods Twenty-three patients were operated in the Radboud University Nijmegen Medical Centre. The median age at the time of reconstructive colectomy was 24 (range 10–55) years. A mucosectomy with hand-sewn IPAA was performed in 9 patients, and a double-stapled IPAA was performed in 21 patients. For two patients, the information about the performed technical procedure could not be retrieved. At the moment of colectomy, four patients had a colorectal adenocarcinoma localized in the rectum, sigmoid, hepatic flexure, or appendix, respectively. At the time of endoscopy, the median age of the pouch was 96 (range 9–216) months. The medication used was loperamide by 18 patients, psyllium fibres by 3 patients, and iron, metoclopramide, colestyramine, omeprazole, sulindac, tramadol, tamoxifen, gosereline, nifedipine, metoprolol, furosemide, or losartan each by 1 patient. Thirteen patients were not on medication 3 months before endoscopy. 15 27 Histological examination revealed pouch adenomas in 24 patients (75%). Apoptosis 1 Fig. 1 encircled left right  2 2 2 Fig. 2 2 fat line green box error bars  Cell proliferation Quantitative comparison 3 r s p Fig. 3 Brown stained nuclei  p 4 Fig. 4 p fat line red box error bars  Semiquantitative comparison 5 6 p Fig. 5 left right  Fig. 6 0 1 2 3 4  κ 6 7 n p Fig. 7 0 1 2  Discussion 14 26 33 38 16 To overcome the problem of the relative low number of assessable crypts, another quicker but less quantitative method was applied in which five investigators compared photographs of tissue sections of pouch and ileal mucosae. In accordance with the results of the first method, we found significantly higher cell proliferation in the mucosa of the pouch compared to that of the ileal mucosa. Although no significant correlation could be found in a case-by-case comparison between both methods, in all cases in which the semiquantitative analyses showed more proliferation in the pouch, this was confirmed by the quantitative analyses of crypts. Furthermore, the inter- and intraobserver variability was good. In addition, the semiquantitative method is far less time-consuming and can therefore give a relatively fast and easy impression of eventual differences in cell proliferation. Evaluation of this method in a larger study may further demonstrate its value. 11 12 1 28 30 32 19 31 The higher proliferation found in the pouch mucosa in comparison to mucosa of the afferent ileal loop can only be explained by intraluminal changes that occur after construction of the pouch. Whether changes in bacterial flora, bile acid composition, short-chain fatty acids, or other compounds are responsible for this finding remains unclear, but a better understanding of this process is necessary to find a possible treatment for this group of patients. In conclusion, the increased cell proliferation in the ileal pouch mucosa compared to the mucosa of the afferent ileal loop may contribute to the enhanced risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopical surveillance of the pouch in these patients. In addition, cell proliferation can be used as an early endpoint marker in chemopreventive studies in these patients. The applied new method for semiquantitative evaluation of cell proliferation in immunohistochemically stained tissue sections seems promising, as it offers a relatively fast and easy means of assessment.