Detection of colorectal carcinoma cells in circulating peripheral blood by reverse transcription-polymerase chain reaction targeting cytokeratin-20 mRNA

Funaki, Naomi; Tanaka, Junji; Itami, Atsushi; Kasamatsu, Takayuki; Ohshio, Gakuji; Onodera, Hisashi; Monden, Kazunobu; Okino, Takashi; Imamura, Masayuki

doi:10.1016/s0024-3205(96)00700-x

Cited by 44 publications

(19 citation statements)

References 14 publications

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“…Sense primer 1 (5'-CAGACACACGGTGAACTATGG-3') within exon 1 and antisense primer 2 (5'-GATCAGCTTCCACTGTTAGACG-3') within exon 3 are both identical to the published sequences of Burchill et al (1995). For the nested PCR, we used an original sense primer 3 (5'-CTGTTTGTTGGCAATGAGAAAATGG-3') within exon 1 and an original antisense primer 4 (5'-GTATTC-CTCTCTCAGTCTCATACT-3') covering both exon 2 (the 3' two bases, C and T) and exon 3; therefore, primer 4 could not anneal with the genomic DNA (Funaki et al, 1997b).…”

Section: Pcr Primersmentioning

confidence: 99%

“…First, PCR was performed by using primers 1 and 2, which amplify a 370-bp fragment as reported by Burchill et al (1995). We previously reported using a 100-g1 mixture for this step (Funaki et al, 1997b (Funaki et al, 1997b). The 50 p1 of the second-step PCR mixture was basically the same as that of the first-step PCR except for the different primers and the addition of 24 pl of DEPC-treated water.…”

Section: Pcr Protocolmentioning

confidence: 99%

“…After 35 cycles, 1 pl of this second-step product was used as the template for the third-step PCR. The third set of primers consisted of primers 3 and 4, which amplify a 303-bp fragment (Funaki et al, 1997b). The constituents of the third-step PCR were basically the same as those of the second PCR mixture except for the different template and primers.…”

Section: Pcr Protocolmentioning

confidence: 99%

“…However, we previously designed a system to detect the presence of hepatocellular carcinoma cells in circulation in the form of ax-fetoprotein mRNA (Funaki et al, 1995(Funaki et al, , 1997a and another to detect the presence of pancreatic and gastric carcinoma cells in circulation in the form of carcinoembryonic antigen mRNA (Funaki et al, 1996). By using these techniques as well as Burchill's primers as the first-step primers, we developed a highly sensitive reverse transcription (RT) and three-step nested polymerase chain reaction (PCR) method that was able to detect the presence of CK20 mRNA even in a healthy donor's blood containing only a single CK20-expressing cell (Funaki et al, 1997b). In addition, as our original 3' primer used in the second and third steps could not anneal with the genomic sequence, the specificity of the nested PCR product was always satisfactory on sequencing.…”

mentioning

confidence: 99%

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Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients

Funaki

Tanaka²,

Ohshio³

et al. 1998

Br J Cancer

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Summary A highly sensitive system was previously developed by us to detect the presence of colorectal carcinoma cells in blood in the form of cytokeratin 20 (CK20) mRNA. In the present study, we used an improved version of this system to analyse the peripheral blood of 28 patients with colorectal carcinoma, five patients with non-cancerous intestinal diseases and six normal controls for the presence or absence of CK20 mRNA and to investigate the relationship between the mRNA results and prognosis. All eight patients with recurrence were positive for CK20 mRNA, as were four patients in the Dukes' C stage with either distant metastasis or dissemination. Five of the nine patients in the Dukes' C stage with neither distant metastasis nor dissemination were positive, and three of these developed recurrence within 11 months after the analysis. Only one of the seven patients in the Dukes' A or B stage was positive, and none showed recurrence during the 1-19 months of observation. None of the five patients without carcinomas or of the six normal controls was positive. Although the follow-up period is limited and the recurrences were all local at present, these results suggest that the presence of CK20 mRNA in circulation may be a useful indicator for the screening of advanced colorectal carcinoma patients with a high risk of recurrence.

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Section: Pcr Primersmentioning

confidence: 99%

Section: Pcr Protocolmentioning

confidence: 99%

Section: Pcr Protocolmentioning

confidence: 99%

mentioning

confidence: 99%

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Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients

Funaki

Tanaka²,

Ohshio³

et al. 1998

Br J Cancer

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“…Expression of cytokeratin 19 (ck19; Genbank accession no Y00503) has been reported in a wide range of tissues (Quinlan et al, 1985) including normal lymph nodes, bone marrow and peripheral blood Gunn et al, 1996). Expression of cytokeratin 20 (ck20; Genbank accession no X73501), detected in most colorectal cancers and their metastases (Moll et al, 1992), initially appeared to be more limited (Moll et al, 1993) and its detection by RT-PCR has been used to identify occult metastases in colorectal cancer patients (Denis et al, 1997;Funaki et al, 1997;Dorudi et al, 1998). However, ck20 mRNA transcription (Denis et al, 1997) as well as protein expression (Litle et al, 1997) has been described recently in the blood of healthy controls, making its use in the detection of micrometastatic cells questionable (Wyld et al, 1998).…”

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confidence: 99%

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients

Bustin¹,

Gyselman²,

Williams³

et al. 1999

Br J Cancer

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SummaryThe clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5′-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.

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Comparative Analysis of Tumor Cell Dissemination in Mesenteric, Central, and Peripheral Venous Blood in Patients With Colorectal Cancer

Koch¹

2001

Arch Surg

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Background: Metastatic disease in colorectal cancer results from hematogenic dissemination of tumor cells. This dissemination can be explained by 2 concepts: (1) regional spread of tumor cells via portal venous drainage into the liver as the first site of metastasis and (2) early spread of tumor cells into central and peripheral venous blood as evidence of systemic hematogenic tumor cell dissemination. Hypothesis: Tumor cell detection in different blood compartments could help to understand the predominant pattern of hematogenic tumor cell dissemination in colorectal cancer.

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Detection of colorectal carcinoma cells in circulating peripheral blood by reverse transcription-polymerase chain reaction targeting cytokeratin-20 mRNA

Cited by 44 publications

References 14 publications

Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients

Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients

Comparative Analysis of Tumor Cell Dissemination in Mesenteric, Central, and Peripheral Venous Blood in Patients With Colorectal Cancer

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