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• W2109970382 abstract "Background & Aims: The early detection of colorectal cancer is desired because this cancer can be cured surgically if diagnosed early. The purpose of the present study was to determine the feasibility of a new methodology for isolating colonocytes from naturally evacuated feces, followed by cytology or molecular biology of the colonocytes to detect colorectal cancer originating from any part of the colorectum. Methods: Several simulation studies were conducted to establish the optimal methods for retrieving colonocytes from any portion of feces. Colonocytes exfoliated into feces, which had been retrieved from 116 patients with colorectal cancer and 83 healthy volunteers, were analyzed. Part of the exfoliated colonocytes was examined cytologically, whereas the remainder was subjected to DNA analysis. The extracted DNA was examined for mutations of the APC, K-ras, and p53 genes using direct sequence analysis and was also subjected to microsatellite instability (MSI) analysis. Results: In the DNA analysis, the overall sensitivity and specificity were 71% (82 of 116) of patients with colorectal cancer and 88% (73 of 83) of healthy volunteers. The sensitivity for Dukes A and B was 72% (44 of 61). Furthermore, the sensitivity for cancers on the right side of the colon was 57% (20 of 35). The detection rate for genetic alterations using our methodology was 86% (80 of 93) when the analysis was limited to cases in which genetic alterations were present in the cancer tissue. Conclusions: We have developed a new methodology for isolating colonocytes from feces. The present study describes a promising procedure for future clinical evaluations and the early detection of colorectal cancers, including right-side colon cancer. Background & Aims: The early detection of colorectal cancer is desired because this cancer can be cured surgically if diagnosed early. The purpose of the present study was to determine the feasibility of a new methodology for isolating colonocytes from naturally evacuated feces, followed by cytology or molecular biology of the colonocytes to detect colorectal cancer originating from any part of the colorectum. Methods: Several simulation studies were conducted to establish the optimal methods for retrieving colonocytes from any portion of feces. Colonocytes exfoliated into feces, which had been retrieved from 116 patients with colorectal cancer and 83 healthy volunteers, were analyzed. Part of the exfoliated colonocytes was examined cytologically, whereas the remainder was subjected to DNA analysis. The extracted DNA was examined for mutations of the APC, K-ras, and p53 genes using direct sequence analysis and was also subjected to microsatellite instability (MSI) analysis. Results: In the DNA analysis, the overall sensitivity and specificity were 71% (82 of 116) of patients with colorectal cancer and 88% (73 of 83) of healthy volunteers. The sensitivity for Dukes A and B was 72% (44 of 61). Furthermore, the sensitivity for cancers on the right side of the colon was 57% (20 of 35). The detection rate for genetic alterations using our methodology was 86% (80 of 93) when the analysis was limited to cases in which genetic alterations were present in the cancer tissue. Conclusions: We have developed a new methodology for isolating colonocytes from feces. The present study describes a promising procedure for future clinical evaluations and the early detection of colorectal cancers, including right-side colon cancer. Colorectal cancer is one of the most common malignancies worldwide. In Japan, colorectal cancer is the third and second leading cause of death from cancer in men and women, respectively.1The Editorial Board of the Cancer Statistics in Japan.Cancer statistics in Japan—. 2003Google Scholar However, colorectal cancer is curable by surgical resection if diagnosed at a sufficiently early stage. This incentive has prompted investigators to develop new methods enabling the early diagnosis of colorectal cancer and has led to the introduction of cancer screening programs in many countries. For mass cancer screenings, a simple, economic, and noninvasive method of cancer detection is desired. The Hemoccult test is currently used in many countries for this purpose.2Mandel J.S. Bond J.H. Church T.R. Snover D.C. Bradley G.M. Schuman L.M. Ederer F. Minnesota Colon Cancer Control StudyReducing mortality from colorectal cancer by screening for fecal occult blood.N Engl J Med. 1993; 328: 1365-1371Crossref PubMed Scopus (2898) Google Scholar, 3Hardcastle J.D. Chamberlain J.O. Robinson M.H. Moss S.M. Amar S.S. Balfour T.W. James P.D. Mangham C.M. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer.Lancet. 1996; 348: 1472-1477Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar, 4Kronborg O. Fenger C. Olsen J. Jorgensen O.D. Sondergaard O. Randomized study of screening for colorectal cancer with faecal-occult-blood test.Lancet. 1996; 348: 1467-1471Abstract Full Text Full Text PDF PubMed Scopus (2175) Google Scholar, 5Towler B. Irwig L. Glasziou P. Kewenter J. Weller D. Silagy C. A systematic review of the effects of screening for colorectal cancer using the faecal occult blood test, hemoccult.BMJ. 1998; 317: 559-565Crossref PubMed Scopus (427) Google Scholar, 6Winawer S. Fletcher R. Rex D. Bond J. Burt R. Ferrucci J. Ganiats T. Levin T. Woolf S. Johnson D. Kirk L. Litin S. Simmang C. Colorectal cancer screening and surveillance clinical guidelines and rationale—update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (1971) Google Scholar However, this test is nonspecific and is not sufficiently sensitive to detect early stage colorectal cancer, although a higher sensitivity has been reported for advanced-stage colorectal cancer.7Mandel J.S. Church T.R. Bond J.H. Ederer F. Geisser M.S. Mongin S.J. Snover D.C. Schuman L.M. The effect of fecal occult-blood screening on the incidence of colorectal cancer.N Engl J Med. 2000; 343: 1603-1607Crossref PubMed Scopus (1230) Google Scholar Radioimmunoassays using tumor markers, such as carcinoembryonic antigen, also are not suitable for the detection of early cancer, although such tests can be used to monitor patients for an increasing tumor burden or tumor recurrence. Diagnosis by barium enema study and fiberoptic colonoscopy is accurate but time-consuming, expensive, and invasive. Therefore, an urgent need exists to establish a sensitive, reliable, and noninvasive method for the detection of colorectal cancer at an early stage.To date, several screening methods for colorectal cancer based on the detection of mutated DNA in feces have been reported.8Sidransky D. Tokino T. Hamilton S.R. Kinzler K.W. Levin B. Frost P. Vogelstein B. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors.Science. 1992; 256: 102-105Crossref PubMed Scopus (677) Google Scholar, 9Hasegawa Y. Takeda S. Ichii S. Koizumi K. Maruyama M. Fujii A. Ohta H. Nakajima T. Okuda M. Baba S. et al.Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA).Oncogene. 1995; 10: 1441-1445PubMed Google Scholar, 10Smith-Ravin J. England J. Talbot I.C. Bodmer W. Detection of c-Ki-ras mutations in faecal samples from sporadic colorectal cancer patients.Gut. 1995; 36: 81-86Crossref PubMed Scopus (101) Google Scholar, 11Eguchi S. Kohara N. Komuta K. Kanematsu T. Mutations of the p53 gene in the stool of patients with resectable colorectal cancer.Cancer. 1996; 77: 1707-1710Crossref PubMed Scopus (84) Google Scholar, 12Nollau P. Moser C. Weinland G. Wagener C. Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR.Int J Cancer. 1996; 66: 332-336Crossref PubMed Scopus (104) Google Scholar, 13Ratto C. Flamini G. Sofo L. Nucera P. Ippoliti M. Curigliano G. Ferretti G. Sgambato A. Merico M. Doglietto G.B. Cittadini A. Crucitti F. Detection of oncogene mutation from neoplastic colonic cells exfoliated in feces.Dis Colon Rectum. 1996; 39: 1238-1244Crossref PubMed Scopus (49) Google Scholar, 14Deuter R. Muller O. Detection of APC mutations in stool DNA of patients with colorectal cancer by HD-PCR.Hum Mutat. 1998; 11: 84-89Crossref PubMed Scopus (28) Google Scholar, 15Ahlquist D.A. Skoletsky J.E. Boynton K.A. Harrington J.J. Mahoney D.W. Pierceall W.E. Thibodeau S.N. Shuber A.P. Colorectal cancer screening by detection of altered human DNA in stool feasibility of a multitarget assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 16Dong S.M. Traverso G. Johnson C. Geng L. Favis R. Boynton K. Hibi K. Goodman S.N. D’Allessio M. Paty P. Hamilton S.R. Sidransky D. Barany F. Levin B. Shuber A. Kinzler K.W. Vogelstein B. Jen J. Detecting colorectal cancer in stool with the use of multiple genetic targets.J Natl Cancer Inst. 2001; 93: 858-865Crossref PubMed Scopus (322) Google Scholar, 17Rengucci C. Maiolo P. Saragoni L. Zoli W. Amadori D. Calistri D. Multiple detection of genetic alterations in tumors and stool.Clin Cancer Res. 2001; 7: 590-593PubMed Google Scholar, 18Traverso G. Shuber A. Olsson L. Levin B. Johnson C. Hamilton S.R. Boynton K. Kinzler K.W. Vogelstein B. Detection of proximal colorectal cancers through analysis of faecal DNA.Lancet. 2002; 359: 403-404Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 19Traverso G. Shuber A. Levin B. Johnson C. Olsson L. Schoetz Jr, D.J. Hamilton S.R. Boynton K. Kinzler K.W. Vogelstein B. Detection of APC mutations in fecal DNA from patients with colorectal tumors.N Engl J Med. 2002; 346: 311-320Crossref PubMed Scopus (290) Google Scholar, 20Boynton K.A. Summerhayes I.C. Ahlquist D.A. Shuber A.P. DNA integrity as a potential marker for stool-based detection of colorectal cancer.Clin Chem. 2003; 49: 1058-1065Crossref PubMed Scopus (86) Google Scholar These methods, however, are time-consuming and are not sufficiently sensitive. The major reason for this inaccuracy is the fact that nucleic acids in feces are derived from an enormous number and variety of bacteria and normal cells. Accordingly, the proportion of genes derived from cancer cells in feces is as low as 1%, at most.9Hasegawa Y. Takeda S. Ichii S. Koizumi K. Maruyama M. Fujii A. Ohta H. Nakajima T. Okuda M. Baba S. et al.Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA).Oncogene. 1995; 10: 1441-1445PubMed Google Scholar This makes the application of gene-detecting methods difficult in clinical practice.We previously reported that the expression of CD44 variants in exfoliated colonocytes isolated from feces according to the Percoll centrifugation method could serve as a noninvasive diagnostic marker for early colorectal cancer.21Yamao T. Matsumura Y. Shimada Y. Moriya Y. Sugihara K. Akasu T. Fujita S. Kakizoe T. Abnormal expression of CD44 variants in the exfoliated cells in the feces of patients with colorectal cancer.Gastroenterology. 1998; 114: 1196-1205Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar However, the repetition of the Percoll centrifugation method was found to distort the morphology of the exfoliated colonocytes. Accordingly, the sensitivity of this method also appeared to be unsatisfactory because of the low retrieval rate of the exfoliated colonocytes. Another study described a processing method that involved scraping or washing the stool’s surface with a buffer to collect exfoliated colonocytes.22Davies R.J. Freeman A. Morris L.S. Bingham S. Dilworth S. Scott I. Laskey R.A. Miller R. Coleman N. Analysis of minichromosome maintenance proteins as a novel method for detection of colorectal cancer in stool.Lancet. 2002; 359: 1917-1919Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar In the ascending colon, however, the feces remains unformed. Therefore, most cancer cells exfoliated from the walls of the ascending colon would be incorporated into the inner core of the feces during the course of its formation. Thus, recovering cancer cells that originated from the ascending colon might be difficult using methods that involve scraping or washing solid feces.Under these circumstances, we succeeded in developing a new, very effective methodology that allows the simple isolation of exfoliated colonocytes from not only the surface but also the central portion of feces while maintaining the colonocytes’ initial morphology. Currently, we are attempting to apply a molecular biologic tool to purified colonocytes exfoliated into feces to detect cells from early colorectal cancers, including right-side colon cancer.Materials and MethodsStudy DesignThis was a prospective study conducted between December 2002 and August 2004. The study protocol was reviewed and approved by the Institutional Review Board of the National Cancer Center, Japan. Written informed consent was obtained from all patients and healthy volunteers. No modifications to the protocol procedures were made during the course of the study.Study PopulationA total of 116 patients with histologically confirmed colorectal cancer and 83 healthy volunteers were enrolled. The healthy volunteers consisted of 37 men and 46 women with no apparent abnormalities, such as adenoma or carcinoma (including hyperplastic polyps), found during a total colonoscopy performed at the National Cancer Center Research Center for Cancer Prevention and Screening. The median age of these volunteers was 58.4 years (range, 40–70 years). The characteristics of the patients and healthy volunteers are summarized in Table 1. All the patients with colorectal cancer had undergone surgical resection of their primary tumor at the National Cancer Center Hospital, Tsukiji, or at Hospital East, Kashiwa, Japan. The median age of the patients was 62.0 years (range, 32–82 years). There were 69 men and 47 women patients. The primary tumors were located in the following sites: rectum in 53 patients, sigmoid colon in 21 patients, descending colon in 7 patients, transverse colon in 6 patients, ascending colon in 23 patients, and cecum in 6 patients. The clinical stage of the patients according to Dukes’ classification was as follows: Dukes’ stage A in 30 patients, stage B in 31 patients, stage C in 53 patients, and stage D in 2 patients.Table 1Characteristics of Patients and Healthy VolunteersCharacteristicPatient (N = 116)Healthy volunteer (N = 83)Age, y Mean62.058.4 Range32–8240–70Sex, no (%) Male69 (59.5)37 (44.6) Female47 (40.5)46 (55.4)DNA, ng/gram of stool Mean570.8175.3 Range2.0–7462.80.2–1907.5Tumor location, no (%) Cecum6 (5.2) Ascending colon23 (19.8) Transverse colon6 (5.2) Descending colon7 (6.0) Sigmoid colon21 (18.1) Rectum53 (45.7)Size, mm Mean40.0 Range4.0–120.0Histology, no (%) W/D55 (47.4) M/D56 (48.3) P/D2 (1.7) Mucinous carcinoma2 (1.7) Carcinoid tumor1 (0.9)Depth, no (%) T110 (8.6) T232 (27.6) T371 (61.2) T43 (2.6)Dukes’ stage, no (%) A30 (25.9) B31 (26.7) C53 (45.7) D2 (1.7)W/D, Well-differentiated adenocarcinoma; M/D, moderately differentiated adenocarcinoma; P/D, poorly differentiated adenocarcinoma. Open table in a new tab Stool SamplesBefore surgical resection, stool samples were obtained from 116 patients with colorectal cancer. Stool samples were also obtained from 83 healthy volunteers a few weeks after they had undergone a total colonoscopy. Naturally evacuated feces from subjects who had not taken laxatives were used as stool samples. Each patient was instructed to evacuate into a polystyrene disposable tray (AS one, Osaka, Japan) measuring 5 × 10 cm in size at home and bring the sample to the reception counter at the outpatient clinic or the Cancer Prevention and Screening Center of the National Cancer Center. The samples were collected and transferred to a laboratory at which they were allowed to stand at room temperature. Preparation of the stool samples for examination was conducted within 1–6 hours after the evacuation.Magnetic BeadsDynabeads Epithelial Enrich are uniform, superparamagnetic, polystyrene beads (4.5-μm diameter) coated with a mouse IgG1 monoclonal antibody (mAb Ber-EP4) specific for the glycopolypeptide membrane antigen Ep-CAM, which is expressed on most normal and neoplastic human epithelial tissues (Dynal, Oslo, Norway). Ep-CAM is widely expressed in the highly proliferative cells of the intestinal epithelium, from the basal cells to cells throughout the crypts at the basolateral membranes, and only the apical membrane facing the lumen is negative. The development of adenomas has been reported to be associated with increased Ep-CAM expression, and Ep-CAM over expression (mAb GA733) has frequently been demonstrated in colorectal carcinomas.23Winter M.J. Nagtegaal I.D. van Krieken J.H. Litvinov S.V. The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology.Am J Pathol. 2003; 163: 2139-2148Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 24Balzar M. Prins F.A. Bakker H.A.M. Fleuren G.J. Warnaar S.O. Litvinov S.V. The structural analysis of adhesions mediated by Ep-CAM.Exp Cell Res. 1999; 246: 108-121Crossref PubMed Scopus (67) Google Scholar, 25Salem R.R. Wolf B.C. Sears H.F. Lavin P.T. Ravikumar T.S. DeCoste D. D’Emilia J.C. Herlyn M. Schlom J. Gottlieb L.S. Expression of colorectal carcinoma-associated antigens in colonic polyps.J Surg Res. 1993; 55: 249-255Abstract Full Text PDF PubMed Scopus (22) Google ScholarSimulation StudiesA series of simulation studies were conducted to establish the optimal conditions for retrieving HT-29 colorectal cancer cells from feces. Feces from healthy volunteers were divided into several portions, each of which was seeded with 100 μL HT-29 cells (1 × 106/approximately 5 g feces). The cells were retrieved under several different conditions as follows: use of a Hank’s solution and 25 mmol/L Hepes buffer (pH 7.35); processed feces of 5, 10, or 30 g volume; filter with a pore size of 48, 96, 512, or 1000 μm; incubation of homogenized solution with magnetic beads at 4°C or room temperature; application of 20, 40, 80, 200, or 400 μL magnetic beads; incubation of homogenized solution with magnetic beads under gentle rolling at 15 rounds/minute in a mixer for 10, 20, 30, or 40 minutes; and the reaction time between the cell-magnetic bead complexes and a magnet on a shaking platform for 0, 2, 10, 20, 30, 40, 50, or 60 minutes. Finally, the cell retrieval rate calculated for the magnetic beads method under the conditions determined to be the most suitable for this simulation study was compared with that calculated for the Percoll centrifugation method. The retrieval rate was calculated by dividing the number of cells that bound to the retrieved beads by the number of cells initially added to the feces. The cells were counted using a NucleoCounter (ChemoMetec A/S, Allerød, Denmark).Isolation of Exfoliated Cells From FecesThe procedure was conducted using the most suitable and optimal conditions determined by the simulation study (Figure 1). Approximately 5–10 g of naturally evacuated feces were used to isolate exfoliated cells. Feces were collected into Stomacher Lab Blender bags (Seward, Thetford, United Kingdom). The stool samples were homogenized with a buffer (200 mL) consisting of Hank’s solution, 10% fetal bovine serum (FBS), and 25 mmol/L Hepes buffer (pH 7.35) at 200 rpm for 1 minute using a Stomacher (Seward). The homogenates were then filtered through a nylon filter (pore size, 512 μm), followed by division into 5 portions (40 mL each). Subsequently, 40 μL of magnetic beads were added to each homogenized solution portion, and the mixtures were incubated for 30 minutes under gentle rolling in a mixer at room temperature. The samples on the magnet were then incubated on a shaking platform for 15 minutes at room temperature. Colonocytes isolated from 5 tubes were smeared onto slides and then stained using the Papanicolaou method. The remainder of the samples was centrifuged, and the sediments were stored at −80°C until DNA extraction.Extraction of DNAFresh tissue samples were obtained from the surgically resected specimens of 116 patients with colorectal cancer. The samples were snap frozen in liquid nitrogen within 20 minutes of their arrival at the pathologic specimen reception area and were stored in liquid nitrogen until analysis.Genomic DNA was extracted from each tumor tissue specimen using a DNeasy kit (QIAGEN, Valencia, CA). Genomic DNA was also extracted from colonocytes isolated from feces using the SepaGene kit (Sanko-Junyaku, Tokyo, Japan).Direct Sequence AnalysisDirect sequencing was conducted to identify mutations in the APC codon 1270–1594, in codons 12 and 13 of the K-ras gene, and in exons 5, 6, 7, and 8 of the p53 gene.The PCR primers used in this study were as follows: APC (5′-AAACACCTCAAGTTCCAACCAC-3′, 5′-GGTAATTTTGAAGCAGTCTGGGC-3′); K-ras (5′-CTGGTGGAGTATTTGATAGTG-3′, 5′-CCCAAGGAAAGTAAAGTTC-3′); p53 exon 5 (5′-GCCGTCTTCCAGTTGCTTTAT-3′, 5′-CCAAATACTCCACACGCAAAT-3′); p53 exon 6 (5′-CATGAGCGCTGCTCAGATAG-3′, 5′-TGCACATCTCATGGGGTTATAG-3′); p53 exon 7 (5′-CTTGGGCCTGTGTTATCTCCTA-3′, 5′-AAGAAAACTGAGTGGGAGCAGT-3′); and p53 exon 8 (5′-ACCTCTTAACCTGTGGCTTC-3′, 5′-TACAACCAGGAGCCATTGTC-3′).The sequence primers used in this study were as follows: APC (5′-CAAAAGGCTGCCACTTGCAAAG-3′, 5′-AAAATAAAGCACCTACTGCTG-3′, 5′-GAATCAGCCAGGCACAAAGC-3′); K-ras (5′-CTGGTGGAGTATTTGATAGTG-3′); p53 exon 5 (5′-CCAAATACTCCACACGCAAAT-3′); p53 exon 6 (5′-CATGAGCGCTGCTCAGATAG-3′); p53 exon 7 (5′-AAGAAAACTGAGTGGGAGCAGT-3′); and p53 exon 8 (5′-ACCTCTTAACCTGTGGCTTC-3′). Each fragment was sequenced by direct sequencing using the Big Dye Terminator v 3.1/1.1 cycle kit (Applied Biosystems, Forester City, CA).All obtained sequences were aligned with previously published sequences (National Center for Biotechnology Information [NCBI] Genbank accession No. M74088 [APC], M54968 [K-ras], and X54156 [p53]) for each of the target genes and were analyzed using Phred/Phrp/DNASIS pro (Hitachi Software Engineering, Tokyo, Japan). The presence and nature of each mutation were confirmed by repeated PCR and sequencing.BAT26The BAT26 gene, an indicator of microsatellite instability (MSI), was amplified by PCR. Each fragment was electrophoresed using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) and then analyzed by GeneScan v 3.7 (Applied Biosystems). The PCR primers used in this study were 5′-TGACTACTTTTGACTTCAGCC-3′ and 5′-AACCATTCAACATTTTTAACCC-3′.CytologyColonocytes isolated from feces were examined by 2 experienced cytotechnologists after Papanicolaou staining.Study BlindingWe followed the guidelines of our medical institution for preparing blinded samples. Technicians processed the stool samples and prepared the slides for cytology and the cell pellets for DNA extraction. The samples were blinded to prevent the identification of individuals and the samples’ origins. Two cytologists assessed the blinded samples, and the Life Science Group of Hitachi, Ltd, analyzed the DNA sequences.Statistical AnalysisA Fisher exact test was used to compare all proportions. All reported P values are 2-sided. A value of P < .05 was considered statistically significant.ResultsSimulation StudiesThe cell retrieval rate was found to decrease when Hank’s solution without FBS was used, thus indicating the effectiveness of adding serum to the homogenizing buffer (Figure 2A). The cell retrieval rate was found to decrease when more than 30 g of feces were processed (Figure 2B). The cell retrieval rates were similar when incubation was conducted at room temperature and at 4°C (Figure 2C). Filtering of the stool suspension with the 48- or 96-μm filter resulted in significant clogging and thus hampered cell retrieval. However, a lot of fecal residue remained after filtering with the 1000-μm filter, hindering the handling of the stool suspension thereafter. We therefore decided to use the 512-μm filter (Figure 2D). The dose of the magnetic beads applied was also examined. The cell retrieval rate increased in a dose-dependent manner up to 80 μL. In reality, a sufficient amount of genomic DNA derived from exfoliated colonocytes was obtained, even when 40 μL of magnetic beads were used (Figure 2E). Regarding the optimal incubation time of the magnetic beads for the complete binding of HT-29 cells to the beads, 30 minutes of incubation was found to be sufficient for the satisfactory binding of HT-29 cells to the beads (Figure 2F). For the retrieval of the cell-magnetic bead complexes on the magnet, a 10-minute reaction period was sufficient (Figure 2G).Figure 2Simulation study to establish the optimal conditions for retrieving HT-29 colorectal cancer cells from feces and to compare the cell retrieval rates for the magnetic beads methods and the Percoll centrifugation method. Feces from healthy volunteers were divided into several portions, each of which was seeded with 100 μL HT-29 colorectal cancer cells (1 × 106/approximately 5 grams of feces). The procedure for retrieving the HT-29 cells was conducted under various conditions as follows: (A) homogenizing buffer with or without FBS; (B) stool weight (5, 10, or 30 g); (C) temperature during the cell-yielding procedure (4°C or room temperature); (D) filter pore size (48, 96, 512, or 1000 μm); (E) volume of applied magnetic beads (20, 40, 80, 200, or 400 μL); (F) incubation time of the homogenized solution with the magnetic beads under gentle rolling in a mixer (10, 20, 30, or 40 minutes); and (G) reaction time for the cells-magnetic bead complexes and the magnet on the shaking platform (0, 2, 10, 20, 30, 40, 50, or 60 minutes). The cell retrieval ratio (%) was calculated using the following formula: 100 × number of HT-29 cells retrieved/number of applied HT-29 cells. (H) Comparison of cell retrieval rates for the magnetic beads methods (open column) and the Percoll centrifugation method (solid column).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The cell retrieval rates were 0.8% and 33.5% using the Percoll centrifugation method and the magnetic beads method, respectively, thus underscoring the advantage of the magnetic beads method (Figure 2H).CytologyAtypical cells were observed in colonocytes isolated from the feces of 32 of 116 patients with colorectal cancer, with a sensitivity rate of 28% (95% CI: 20–37; Table 2, Figure 3A and 3B). No atypical cells were observed in any of the 83 healthy volunteers, with a specificity rate of 100% (95% CI: 96–100). A significant difference (P < .0001) was found in the positivity rate between the patient group and the healthy volunteer group. The sensitivity rates for Dukes’ A, B, and C or D colorectal cancers were 23% (7 of 30; 95% CI: 10–42), 32% (10 of 31; 95% CI: 17–51), and 27% (15 of 55; 95% CI: 16–41), respectively. No significant differences in the positivity rates were found among any of the stages. Furthermore, the sensitivity rates for cancers on the right side of the colon, including the cecum, ascending colon, and transverse colon, and for those on the left side of the colon, including the descending colon, sigmoid colon, and rectum, were 9% (3 of 35; 95% CI: 2–23) and 36% (29 of 81; 95% CI: 25–47), respectively. Therefore, the positivity rate was significantly higher for cancers on the left side of the colon (P < .01).Table 2Incidences of Genetic Alterations of the APC, K-ras, p53, and MSI (BAT26) Genes as Well as Results From Cytology in all Patients and Healthy VolunteersPatientHealthy volunteerTumor tissueIsolated cellIsolated cellMarkerNo.Positivity (%) (95% CI)No.Sensitivity (%) (95% CI)No.Specificity (%) (95% CI)OverallCombined marker9380 (72–87)8271 (62–79)1088 (79–94)Patients (n = 116), healthy volunteers (n = 83)APC5144 (35–53)4741 (32–50)199 (93–100)K-ras3328 (20–38)3328 (20–38)199 (93–100)p536253 (44–63)4539 (30–48)693 (85–97)BAT2665 (2–11)43 (1–9)396 (90–99)Cytology3228 (20–37)0100 (96–100)Dukes’ stage A (n = 30)Combined marker2480 (61–92)1860 (41–77)APC1447 (28–66)1137 (20–56)K-ras620 (77–39)517 (6–35)p53620 (77–39)930 (15–49)BAT2613 (1–17)13 (1–17)Cytology723 (10–42)Dukes’ stage B (n = 31)Combined marker3097 (83–100)2684 (66–95)APC1755 (36–73)1755 (36–73)K-ras1032 (17–51)929 (14–48)p531858 (39–75)1342 (25–61)BAT2626 (1–21)13 (1–17)Cytology1032 (17–51)Dukes’ stages C and D (n = 55)Combined marker3971 (57–82)3869 (55–81)APC2036 (24–50)1935 (22–49)K-ras1731 (19–45)1935 (22–49)p532749 (35–63)2342 (29–56)BAT2635 (1–15)24 (0–13)Cytology1527 (16–41)Right-sided colon cancer (n = 35)Combined marker2777 (60–90)2057 (39–74)APC1131 (17–49)823 (10–40)K-ras1646 (29–63)1234 (19–52)p531749 (31–66)1131 (17–49)BAT2626 (1–19)13 (1–15)Cytology39 (2–23)Left-sided colon cancer (n = 81)Combined marker6681 (71–89)6277 (66–85)APC4049 (38–61)3948 (37–60)K-ras1721 (13–31)2126 (17–37)p534556 (44–67)3442 (31–53)BAT2645 (1–12)34 (1–10)Cytology2936 (25–47) Open table in a new tab Figure 3Cytology and DNA sequencing. Papanicolaou staining of colonocytes isolated from the feces of patients with colorectal cancer. (A) A patient with ascending colon cancer, Dukes’ stage A. (B) A patient with rectal cancer, Dukes’ stage C. Detection of mutations in tumor tissues and colonocytes isolated from the feces of patients with colorectal cancer. (C) A point mutation of the APC gene in a tumor tissue specimen obtained from a patient with rectal cancer, Dukes’ stage B. (D) An identical mutation was detected in colonocytes isolated from the feces of the patient. (E) A point mutation of the p53 gene i" @default.
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• W2109970382 date "2005-12-01" @default.
• W2109970382 modified "2023-10-17" @default.
• W2109970382 title "A New Method for Isolating Colonocytes From Naturally Evacuated Feces and Its Clinical Application to Colorectal Cancer Diagnosis" @default.
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