Molecular Validation of the Modified Vienna Classification of Colorectal Tumors
2002; Elsevier BV; Volume: 4; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60703-9
ISSN1943-7811
AutoresTamotsu Sugai, Wataru Habano, Noriyuki Uesugi, Yufei Jiao, Shinichi Nakamura, Kimihiko Sato, Toshimi Chiba, Motohiro Ishii,
Tópico(s)Colorectal Cancer Treatments and Studies
ResumoAlthough the Vienna classification has been introduced to resolve discrepancies in histological diagnoses of colorectal tumors between Western and Japanese pathologists, practical applications of this classification scheme have been problematic because invasion of the lamina propria of tumor cells is often difficult to recognize. Therefore, the following refinements of the classification criteria are needed: category 3, low-grade adenoma/dysplasia; category 4, intramucosal borderline neoplasia; 4-a, high-grade adenoma/dysplasia; 4-b, well-differentiated adenocarcinoma; category 5, definite carcinoma; 5-a, intramucosal moderately-differentiated adenocarcinoma; and 5-b, submucosal carcinoma. We attempted to test whether molecular genetic alterations are related to the modified classification scheme and whether they may help to further categorize the various intramucosal neoplasia grades of colorectal tumors. Two-hundred-thirty-two colorectal tumors were examined using flow cytometric analysis of DNA content, polymerase chain reaction microsatellite assays, and single-strand conformational polymorphism assays to detect abnormalities of DNA content, chromosomal allelic loss, and Ki-ras and p53 gene mutations. Microsatellite instability (MSI) was also examined. Frequencies of genetic alterations and DNA aneuploid states increased with an increase in the grade assigned according to the modified Vienna classification. MSI was a rare event in colorectal adenomas and their frequency of MSI did not correlate with tumor grade. The combined genetic and DNA ploidy data support the conclusion that analysis of genetic alterations and DNA aneuploid states may help in appropriate categorization of colorectal tumors according to the modified Vienna scheme. In addition, MSI-positive tumors may represent a specific subtype of colorectal adenomas. Although the Vienna classification has been introduced to resolve discrepancies in histological diagnoses of colorectal tumors between Western and Japanese pathologists, practical applications of this classification scheme have been problematic because invasion of the lamina propria of tumor cells is often difficult to recognize. Therefore, the following refinements of the classification criteria are needed: category 3, low-grade adenoma/dysplasia; category 4, intramucosal borderline neoplasia; 4-a, high-grade adenoma/dysplasia; 4-b, well-differentiated adenocarcinoma; category 5, definite carcinoma; 5-a, intramucosal moderately-differentiated adenocarcinoma; and 5-b, submucosal carcinoma. We attempted to test whether molecular genetic alterations are related to the modified classification scheme and whether they may help to further categorize the various intramucosal neoplasia grades of colorectal tumors. Two-hundred-thirty-two colorectal tumors were examined using flow cytometric analysis of DNA content, polymerase chain reaction microsatellite assays, and single-strand conformational polymorphism assays to detect abnormalities of DNA content, chromosomal allelic loss, and Ki-ras and p53 gene mutations. Microsatellite instability (MSI) was also examined. Frequencies of genetic alterations and DNA aneuploid states increased with an increase in the grade assigned according to the modified Vienna classification. MSI was a rare event in colorectal adenomas and their frequency of MSI did not correlate with tumor grade. The combined genetic and DNA ploidy data support the conclusion that analysis of genetic alterations and DNA aneuploid states may help in appropriate categorization of colorectal tumors according to the modified Vienna scheme. In addition, MSI-positive tumors may represent a specific subtype of colorectal adenomas. It is well known that there have been large discrepancies in the diagnosis of colorectal neoplasia by Western and Japanese pathologists.1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar These discrepancies are caused by differences in the histological criteria applied to colorectal intramucosal tumors according to accepted Western or Japanese protocols.1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar, 2Schlemper RJ Itabashi M Kato Y Lewin KJ Riddell RH Shimoda T Sipponen P Stolte M Watanabe H Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.Cancer. 1998; 82: 60-69Crossref PubMed Scopus (155) Google Scholar The presence of submucosal invasion is the most important diagnostic criterion for most Western pathologists, whereas in Japan, nuclear features and glandular structures are more important to the diagnosis.2Schlemper RJ Itabashi M Kato Y Lewin KJ Riddell RH Shimoda T Sipponen P Stolte M Watanabe H Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.Cancer. 1998; 82: 60-69Crossref PubMed Scopus (155) Google Scholar In fact, the actual degree of histological concordance between Western and Japanese pathologists is reported to be poor.1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar, 2Schlemper RJ Itabashi M Kato Y Lewin KJ Riddell RH Shimoda T Sipponen P Stolte M Watanabe H Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.Cancer. 1998; 82: 60-69Crossref PubMed Scopus (155) Google Scholar Schlemper et al2Schlemper RJ Itabashi M Kato Y Lewin KJ Riddell RH Shimoda T Sipponen P Stolte M Watanabe H Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.Cancer. 1998; 82: 60-69Crossref PubMed Scopus (155) Google Scholar indicated that the actual rate of concordance between Western and Japanese pathologists is less than 20% (3 of 20 cases). Recently, to resolve these discrepancies, an international panel of experts in gastrointestinal pathology proposed a new classification of these lesions on the basis of common histological criteria. The lesions were divided into five categories of colorectal neoplasia (Vienna classification of gastrointestinal neoplasia).1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar In brief, category 1 and 2 lesions are negative for neoplasia/dysplasia or indefinite for neoplasia/dysplasia, respectively. Category 3 means low-grade adenoma/dysplasia, in which the risk of developing invasive carcinoma is low. Clinicians may consider local treatment of the lesion or may choose to follow up. Category 4 includes lesions of high-grade adenoma/dysplasia that are either non-invasive or that carry a suspicion of invasive carcinoma; the risk of invasion is increased in this category. Local treatment such as endoscopic mucosal resection would be indicated here. Finally, category 5 includes invasive lesions carrying the risk of subsequent deeper invasion; the rate of metastases is so high that treatment is urgently needed. Indeed, it appears to be easy for the experienced general pathologist to categorize colorectal tumors into the appropriate group. However, there are some diagnostic problems associated with this classification. In particular, judgment of invasion beyond the basement membrane is very difficult to define.1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar Therefore, in many cases, neither Western nor Japanese pathologists were able to distinguish between high-grade adenoma/dysplasias versus most intramucosal carcinomas in a reproducible manner.1Schlemper RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preoser CM Flejou J-F Geboes K Hattori T Hirota T Itabashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Yamabe H The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar, 2Schlemper RJ Itabashi M Kato Y Lewin KJ Riddell RH Shimoda T Sipponen P Stolte M Watanabe H Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.Cancer. 1998; 82: 60-69Crossref PubMed Scopus (155) Google Scholar These results suggested that further refinement of the Vienna classification scheme was needed, and particular emphasis was placed on a better definition of the somewhat problematic category 4 and 5 lesions. In addition, Western and Japanese pathologists may not be in agreement even with the diagnosis of low-grade adenoma versus high-grade adenoma. When a pathologist differentiates a tumor from a low- or a high- grade adenoma, the criteria used to differentiate these adenomas should be derived from objective data, such as genetic and DNA ploidy data. Colorectal tumors are thought to follow a model of multi-step tumorigenesis, in which abnormalities in dominant oncogenes and tumor suppressor genes have been identified as frequent events.3Vogelstein B Fearon ER Hamilton SR Kern SE Preisinger AC Leppert M Nakamura Y White R Smits AMM Bos JL Genetic alterations during colorectal tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5816) Google Scholar, 4Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar Although many genetic alterations in colorectal tumors have been studied,3Vogelstein B Fearon ER Hamilton SR Kern SE Preisinger AC Leppert M Nakamura Y White R Smits AMM Bos JL Genetic alterations during colorectal tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5816) Google Scholar, 4Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar, 5Miyaki M Seki M Okamoto M Yamanaka A Maeda Y Tanaka K Kikuchi R Iwama T Ikeuchi T Tonomura A Nakamura Y White R Miki Y Utsunomiya J Koike M Genetic changes and histolopathological types in colorectal tumors from patients with familial adenomatous polyposis.Cancer Res. 1990; 50: 7166-7173PubMed Google Scholar mutations of APC (adenomatous polyposis coli), p53 and Ki-ras genes, as well as allelic losses of chromosome 5q, 17p, 18q, 1p, 8p, or 22q, are most frequently associated with the colorectal tumorigenesis.3Vogelstein B Fearon ER Hamilton SR Kern SE Preisinger AC Leppert M Nakamura Y White R Smits AMM Bos JL Genetic alterations during colorectal tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5816) Google Scholar, 4Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar, 5Miyaki M Seki M Okamoto M Yamanaka A Maeda Y Tanaka K Kikuchi R Iwama T Ikeuchi T Tonomura A Nakamura Y White R Miki Y Utsunomiya J Koike M Genetic changes and histolopathological types in colorectal tumors from patients with familial adenomatous polyposis.Cancer Res. 1990; 50: 7166-7173PubMed Google Scholar, 6Laurent-Puig Blons H Cugnenc P-H Sequence of molecular genetic events in colorectal tumorigenesis.Eur J Cancer Prev. 1999; 8: 539-547Crossref PubMed Scopus (69) Google Scholar On the other hand, recent molecular analysis has shown that there are two distinct genetic pathways in colorectal tumorigenesis that are characterized by genetic instability: chromosomal instability and microsatellite instability (MSI), the latter of which represents mutations in the short tandem repeat sequences distributed within the genome.7Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar Chromosomal instability is frequently found in association with a loss of heterozygosity (LOH) of cancer-related chromosomal loci, or with p53 and Ki-ras gene mutations. These changes characterize the chromosomal instability (CIN) or LOH genotype, which is often associated with DNA aneuploidy.7Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar, 8Lengauer C Kinzler KW Vogelstein B Genetic instability in colorectal cancers.Nature. 1997; 386: 623-627Crossref PubMed Scopus (1626) Google Scholar, 9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar Aneuploidy is well known as an indicator used to predict the malignant potential of colorectal adenomas. Most sporadic colorectal carcinomas arising in Western countries are believed to arise from adenoma10Muto T Bussey HJ Morson BC The evolution of cancer of the colon and rectum.Cancer. 1975; 36: 2251-2270Crossref PubMed Scopus (1871) Google Scholar and belong genetically to the LOH genotype.9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar, 11Watanabe T Muto T Colorectal carcinogenesis based on molecular biology of early colorectal cancer, with special reference to nonpolypoid (superficial) lesions.World J Surg. 2000; 24: 1091-1097Crossref PubMed Scopus (27) Google Scholar On the other hand, a few sporadic colorectal adenomas that occur with MSI do occur. Although MSI characterizes one of the two major genetic pathways, its role during development of colorectal tumors is not fully understood. Genetic data on lesions classified according to the various modified Vienna categories could conceivably provide the necessary means to further refine the histopathological Vienna classification criteria, and may also be useful for early detection of high-risk lesions. We attempted to identify such criteria through an extensive analysis of allelic loss of cancer-related chromosomal loci, mutations of p53 and Ki-ras genes, DNA ploidy, and MSI. An effective crypt isolation method was used to obtain pure tumor crypts.9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar, 12Arai T Kino I Morphometrical and cell kinetic studies of normal human colorectal mucosa: comparison between the proximal and the distal large intestine.Acta Pathol Jpn. 1989; 39: 725-730PubMed Google Scholar, 13Nakamura S Goto J Kitayama M Kino I Application of the crypt isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms.Gastroenterology. 1994; 106: 100-107PubMed Google Scholar The aim of the present study is to clarify the usefulness of this modified classification scheme that is based on an objective molecular analysis. In the present study, category 1 and 2 cases were excluded, given the facts that category 1 cases are thought to involve no genetic alterations, and category 2 lesions are rarely encountered during routine pathological examination. The objective of this study is to improve classification of lesions in categories 3 to 5. We excluded colorectal tumors that occurred in patients with a family history of colorectal tumors. All colorectal tumors are composed of a mixture of neoplastic and non-neoplastic cells. To analyze such tumors for specific genetic alterations accurately, the neoplastic glands must be separated from most of the non-neoplastic cells.12Arai T Kino I Morphometrical and cell kinetic studies of normal human colorectal mucosa: comparison between the proximal and the distal large intestine.Acta Pathol Jpn. 1989; 39: 725-730PubMed Google Scholar, 13Nakamura S Goto J Kitayama M Kino I Application of the crypt isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms.Gastroenterology. 1994; 106: 100-107PubMed Google Scholar Genetic alterations of tumor cells were analyzed after using a crypt isolation technique. This technique allowed for an accurate genetic analysis because pure tumor crypts were thus obtained. A total of 155 colorectal adenomas (from 102 men and 43 women ranging in age from 39 to 85 years; mean, 62.7) were obtained from Iwate Medical University and related hospitals (low-grade adenoma, n = 118; high-grade adenoma, n = 37). Thirty-two early carcinomas (characterized by invasion of malignant cells that were limited to the mucosa or submucosa) were also examined to investigate relationships between genetic alterations and high- grade dysplastic adenomas and early carcinomas. Furthermore, two groups of early carcinomas were sub-classified according to their depth of invasion, as follows: intramucosal (n = 22), and submucosal carcinoma (n = 10). The term “intramucosal” carcinoma was defined as a cancer limited to the lamina propria. The patients consisted of 22 men and 10 women (age 41 to 88 years; mean, 62.1). Histological diagnoses of the adenomas and intramucosal carcinomas were performed according to our hospital criteria, which were modified from those of the Japanese Research Society for cancer of the colon and rectum.14Japanese Society for Cancer of the Colon and Rectum Japanese Classification of Colorectal Carcinoma, first English edition. Kanehara Co., Tokyo1997: 30-63Google Scholar In brief, low-grade dysplasia was diagnosed by the presence of cells with enlarged, hyperchromatic nuclei that were largely confined to the basal portions of the cells, but that had some loss of polarity. Lesions containing cells with round-to-ovoid crypts and an obvious loss of polarity, but no architectural or glandular distortion, were classified as high-grade dysplastic adenomas. The intramucosal carcinomas were subdivided into two groups: well-differentiated and moderately-differentiated. A well-differentiated intramucosal carcinoma was defined by an obvious derangement of glandular architecture, with irregular branches of glands or “back to back” formations. Moderately-differentiated adenocarcinomas were defined by “gland in gland” or cribriform patterns of glands. Differentiation of the former from the latter is histologically easy for general pathologists. In the present study, moderately-differentiated adenocarcinomas often showed a higher cellular atypia than was seen in the intramucosal well-differentiated intramucosal carcinomas. However, it was often difficult to accurately distinguish the high dysplastic adenomas from the well-differentiated intramucosal carcinomas. A submucosal carcinoma was defined as a cancer characterized by submucosal invasion. In addition, an advanced carcinoma was defined as a cancer that has invaded beyond submucosa. Forty-five advanced colorectal carcinomas were analyzed as a positive control. The tumors were classified according to the Japanese Research Society for cancer of the colon and rectum14Japanese Society for Cancer of the Colon and Rectum Japanese Classification of Colorectal Carcinoma, first English edition. Kanehara Co., Tokyo1997: 30-63Google Scholar and modified Dukes' stage.15Turnbull RB Kyle K Watson FR Spratt J Cancer of the colon: the influence of the no-touch isolation technique on survival rates.Ann Surg. 1967; 166: 420-427Crossref PubMed Scopus (757) Google Scholar Histological features of adenomas and carcinomas are illustrated in Figure 1. Clinicopathological features of colorectal adenomas and carcinomas are listed in Table 1. Each tumor was classified into either category of modified Vienna classification by two experienced pathologists (S. T. and N. S.) according to our histological criteria. The Vienna and modified Vienna classification schemes are shown in Table 2.Table 1Clinicopathological Features of Colorectal NeoplasmsAdenomaEarly cancerAdvanced cancerTotal1553245Age (mean)62.762.165.3Gender (man/woman)102/4322/1033/12Location of tumor (right/left)51/1047/2513/32Size of tumor (mean, mm)5–28 (13.2)9–37 (18.4)20–87 (48.5)Grade of dysplasia Low dysplastic118 High dysplastic54Differentiation Well2015 Moderately1230 Open table in a new tab Table 2Vienna or Modified Vienna Classification of Gastrointestinal NeoplasiaVienna classificationModified Vienna classificationCategory 1Negative for neoplasia/dysplasiaNegative for neoplasia/dysplasiaCategory 2Indefinite for neoplasia/dysplasiaIndefinite for neoplasia/dysplasiaCategory 3Non-invasive low grade neoplasia (low grade adenoma/dysplasia)Low grade neoplasia (low grade adenoma/dysplasia)Category 4Non-invasive high grade neoplasiaIntramucosal borderline neoplasia 4.1 High grade adenoma/dysplasia 4.1 High grade adenoma/dysplasia 4.2 Non-invasive carcinoma (carcinoma in situ) 4.2 Intramucosal carcinoma, well differentiated 4.3 Suspicion of invasive carcinomaCategory 5Invasive neoplasiaDefinite carcinoma 5.1 Intramucosal carcinoma 5.1 Intramucosal carcinoma, moderately or poorly differentiated 5.2 Submucosal carcinoma or beyond 5.2 Submucosal carcinoma or beyond Open table in a new tab Tumor DNAs from adenomatous and cancer tissues were extracted from isolated adenomatous and cancer crypts. The tumor crypts were isolated from mucosa propria using a crypt isolation technique, as described elsewhere.16Habano W Sugai T Nakamura S Yoshida T A novel method for gene analysis of colorectal carcinomas using a crypt isolation technique.Lab Invest. 1996; 74: 933-940PubMed Google Scholar, 17Sugai T Habano W Nakamura S Yoshida T Uesugi N Sasou S Itoh C Katoh R Use of crypt isolation to determine loss of heterozygosity of multiple tumor suppressor genes in colorectal carcinoma.Pathol Res Pract. 2000; 196: 145-150Crossref PubMed Scopus (15) Google Scholar, 18Sugai T Habano W Nakamura S Uesugi N Sasou S Itoh C A unique method for mutation analysis of tumor suppressor genes in colorectal carcinomas using a crypt isolation technique.Arch Pathol Lab Med. 2000; 124: 382-386PubMed Google Scholar Briefly, fresh tumor samples and normal colonic mucosa were minced into small pieces using a razor, then incubated at 37°C for 50 minutes in Ca2+/Mg2+-free Hanks' balanced salt solution containing 30 mmol/L EDTA. The tissue was stirred in the salt solution for 20 minutes × 2. Crypts were separated from the lamina propria mucosa or fibrous stroma. These isolated tumor crypts were identified using a dissecting microscope (SZ60, Olympus, Tokyo, Japan). Normal crypts were clearly distinguishable from tumor crypts by characteristic features as described elsewhere.13Nakamura S Goto J Kitayama M Kino I Application of the crypt isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms.Gastroenterology. 1994; 106: 100-107PubMed Google Scholar A proportion of the isolated crypts were fixed in 70% ethanol and stored at 4°C until they were used for DNA extraction. The remaining isolated crypts were fixed with paraffin and examined by light microscopy to confirm the histopathological findings. There was no evidence of contamination by interstitial cells using this technique. However, it should be appreciated that this method cannot reliably differentiate between the crypts of adenomas with high-grade dysplasia and those with low-grade dysplasia. Accordingly, we confirmed the histological diagnosis by light microscopy of paraffin-embedded sections of these crypts. In general, colorectal adenomas tend to have a heterogeneous appearance, and in such cases the tumor grade was determined by the ratio of high-grade to low-grade dysplasia. DNAs from tumors and from some corresponding normal crypts were extracted by standard SDS proteinase K treatment. Samples were resuspended in TE buffer [10 mmol/L Tris-HCl, 1 mmol/L EDTA (pH 8.0)] to the equivalent of 1000 cells/μl. In adenomatous lesions and most early carcinomas, normal genomic DNA, obtained from blood samples from the corresponding patients, was isolated using DrGTL (TAKARA, Tokyo, Japan). A portion of each sample was prepared for flow cytometric analysis (flow cytometer; EPICS XL, Coulter, CA) as previously described.9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar, 19Sugai T Habano W Nakamura S Jiao Y-F Higuchi T Inomata M Chiba T Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.Cytometry. 2001; 46: 345-350Crossref PubMed Scopus (9) Google Scholar A total of 50 to 300 tumor crypts were isolated and examined in the present study. Briefly, normal and tumor crypts were incubated with 0.0125% pepsin (pH 2.0, Sigma, St. Louis, MO) for 5 minutes at 37°C, then washed twice with 0.2 mol/L Tris chloride-buffered saline and passed through a 27-gauge needle. The nuclei (from normal and tumor cells) were stained with propidium iodide (50 μg/ml, Sigma) containing ribonuclease (0.25 mg/ml, Sigma) in 0.2 mol/L Tris chloride-buffered saline at room temperature in the dark for 30 minutes. After filtration through a 37-μm nylon mesh (Tokyo Screen, Tokyo, Japan), samples were analyzed using a flow cytometer. At least 10,000 nuclei from each sample were examined. These samples were classified as diploid (D), multiploid (M), or aneuploid (A) carcinomas as determined by previous criteria.9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar, 13Nakamura S Goto J Kitayama M Kino I Application of the crypt isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms.Gastroenterology. 1994; 106: 100-107PubMed Google Scholar, 19Sugai T Habano W Nakamura S Jiao Y-F Higuchi T Inomata M Chiba T Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.Cytometry. 2001; 46: 345-350Crossref PubMed Scopus (9) Google Scholar Diploid and aneuploid carcinomas were defined as either a diploid tumor population alone or an aneuploid tumor population alone within a tumor. Multiploid carcinomas consisted of both diploid and aneuploid tumor populations. The DNA index (DI) was also calculated as defined previously.9Sugai T Habano W Nakamura S Sato H Uesugi N Takahashi H Jiao Y-F Yoshida T Itoh C Genetic alterations in DNA diploid, aneuploid, and multiploid colorectal carcinomas identified by the crypt isolation technique.Int J Cancer. 2000; 88: 614-619Crossref PubMed Scopus (33) Google Scholar, 19Sugai T Habano W Nakamura S Jiao Y-F Higuchi T Inomata M Chiba T Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.Cytometry. 2001; 46: 345-350Crossref PubMed Scopus (9) Google Scholar The DNA ploidy pattern of each of these samples was reconfirmed using cells remaining after extraction of the DNA used for genetic analysis. Normal and tumor DNA were analyzed for allelic loss by polymerase chain reaction (PCR) amplification of polymorphic dinucleotide repeat sequences. Seventeen markers on chromosomes 17p (TP53), 5q (D5S107; D5S346; D5S82; D5S299), 18q (D18S487; D18S34; DCC), 1p (D1S228; D1S548; D1S507), 8q (D8S201; D8S513; D8S532) and 22q (D22S274; D22S1140; D22S1168) were used. The sequences of these primers were obtained from “The Genome Database” (htt
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