Application of the crypt-isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms

Nakamura, Shinichi; Goto, Junko; Kitayama, Masahiko; Kino, Isamu

doi:10.1016/s0016-5085(94)94651-5

Cited by 48 publications

(83 citation statements)

References 32 publications

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“…Normal crypts were clearly distinguishable from tumor crypts by characteristic features as described elsewhere. 13 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.…”

Section: Crypt Isolation Methods and Dna Extractionmentioning

confidence: 99%

“…To analyze such tumors for specific genetic alterations accurately, the neoplastic glands must be separated from most of the non-neoplastic cells. 12,13 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.…”

Section: Patientsmentioning

confidence: 99%

“…An effective crypt isolation method was used to obtain pure tumor crypts. 9,12,13 The aim of the present study is to clarify the usefulness of this modified classification scheme that is based on an objective molecular analysis.…”

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confidence: 99%

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Molecular Validation of the Modified Vienna Classification of Colorectal Tumors

Sugai

Habano

Uesugi

et al. 2002

The Journal of Molecular Diagnostics

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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.

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Section: Crypt Isolation Methods and Dna Extractionmentioning

confidence: 99%

Section: Patientsmentioning

confidence: 99%

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Molecular Validation of the Modified Vienna Classification of Colorectal Tumors

Sugai

Habano

Uesugi

et al. 2002

The Journal of Molecular Diagnostics

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“…Crypt isolation from the tumor and normal mucosa was performed in accordance with a previously reported method. 18,19 Briefly, fresh mucosa and tumor were minced with a razor into minute pieces, then incubated at 37°C for 30 min in calcium-and magnesium-free Hanks' balanced salt solution (CMF) containing 30 mmol/L ethylene-diaminetetraacetic acid (EDTA). Following this procedure, the tissue was then stirred in CMF for 30 -40 min.…”

Section: Crypt Isolation Techniquementioning

confidence: 99%

Analysis of allelic imbalances at multiple cancer‐related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Sugai

Habano

Jiao

et al. 2004

Intl Journal of Cancer

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Genetic changes related to colorectal carcinomas are accumulated in individual tumor glands during disease progression. Microsatellite allelic analysis of individual tumor glands from 30 colorectal carcinomas using a polymerase chain reaction (PCR) assay coupled with crypt isolation was used to detect intratumoral genetic heterogeneity, the sequence of allelic imbalances (AIs) and the microsatellite instability status of single tumor glands during neoplastic progression. In addition, the CpG islands methylated phenotype (CIMP) status was examined using a methylation-specific PCR method. The specimens were divided into 2 groups: a pooled gland sample, which was composed of more than 50 tumor glands, and a single tumor gland sample. The latter consisted of 10 single tumor glands, which were obtained from the same tumor separately. Most colorectal carcinomas (27 of 30 tumors) examined were heterogeneous for at least one genetic alteration, with from 2 to 7 genotypically different subclones detected per tumor. In 12 of the 27 heterogeneous tumors, it was possible to define the order of genetic alterations during the tumor progression. By analyzing multiple single tumor glands within the same tumor, we found that various subclonal expansions were seen within the same tumors.

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“…Forty-®ve sporadic colorectal carcinomas were examined using a crypt isolation technique that allows the complete separation of neoplastic crypts from nonneoplastic stromal cells (Nakamura et al, 1993(Nakamura et al, , 1994Yoshida et al, 1996). This technique has been applied to both ow cytometric (Nakamura et al, 1994) and genetic analysis (Habano et al, 1996) of colorectal carcinomas and o ers advantages over conventional methods. In particular, the technique improves the sensitivity of LOH analysis for tumor suppressor genes (Habano et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Mismatch repair deficiency leads to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity

1998

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In order to determine the e ects of mismatch repair (MMR) de®ciencies in sporadic colorectal carcinomas, 45 such cancers were examined using a sensitive method called crypt isolation technique. Loss of heterozygosity (LOH) in the MSH2 or MLH1 gene was more frequently observed in replication error (RER) (+) carcinomas than in RER (7) carcinomas, which implied that loss of one normal allele could partly a ect repair capacity. MSH2 gene defects at both alleles were observed in two carcinomas, which showed severe repair de®ciencies. Interestingly, unlike the situation observed in the p53 gene, the MSH2 and MLH1 genes did not show complete LOH. Novel crypt isolation-based subpopulation (CISP) analysis demonstrated that at least two distinct carcinoma subpopulations existed in most carcinomas that showed incomplete LOH; one with and one without LOH. In one carcinoma that had germline mutation and somatic incomplete LOH of the MSH2 gene, the mutator phenotype was only observed in populations a ected in both alleles. Thus, the MSH2 gene appears to possess the two hits mechanism of tumor suppressor genes. However, unlike the tumor suppressor genes, MMR gene defects lead to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.

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Application of the crypt-isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms

Cited by 48 publications

References 32 publications

Molecular Validation of the Modified Vienna Classification of Colorectal Tumors

Molecular Validation of the Modified Vienna Classification of Colorectal Tumors

Analysis of allelic imbalances at multiple cancer‐related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Mismatch repair deficiency leads to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity

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