A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood

Helfrich, Wijnand; tenPoele, R; Meersma, Gj; Mulder, Nh; Devries, Ege; Deleij, L; Smit, Egbert F.

doi:10.1038/bjc.1997.331

Cited by 23 publications

(12 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1 in 100,000 cells, [12]). Most studies, in agreement with the present authors9 data, excluded the illegitimate expression of cytokeratin 20, CEA and EPG2 by normal blood cells [7,9,11,17,21], although a highly sensitive nested PCR revealed CEA expression by normal blood cells in a low percentage of normal subjects due to illegitimate transcription or to false positives [10,12], and 20% of lung adenocarcinoma can aberrantly express CK20 [15]. Therefore, before the analysis to detect circulating micrometastases, the current authors recommend that a number of blood samples from normal subjects be tested with the RT-PCR procedure used in order to exclude illegitimate transcription.…”

Section: Discussionsupporting

confidence: 77%

“…Cytokeratin 19, cytokeratin 20 and EPG2 mRNA were analysed as follows: RT: 25uC for 10 min; 42uC for 30 min; 95uC for 5 min; polymerase chain reaction (PCR): 94uC for 5 min, followed by 30 cycles (95uC for 30 s, 55uC for 30 s, 72uC for 30 s) and 72uC for 5 min, using the previously described primers cytokeratin 19 [14], cytokeratin 20 [21] and EPG2 [17]. These primers were designed to exclude amplification of known pseudogenes.…”

Section: Methodsmentioning

confidence: 99%

“…The current report studied CEA mRNA and four other mRNA species specifically expressed by human neoplasias in a novel, independent cohort of lung cancer patients, prospectively monitored for 24 months: cytokeratin 19 mRNA expressed by lung cancer and other tumour cells [5,13], cytokeratin 20, detected in circulating cells from metastatic gastrointestinal patients by RT-PCR [7,14,15], aldolase A mRNA, which discriminates between cirrhosis and hepatocarcinoma [16], and epithelial glycoprotein 2 (EPG2) mRNA that has been used to detect circulating micrometastases from colon cancer [17].…”

mentioning

confidence: 99%

See 2 more Smart Citations

Carcinoembryonic antigen mRNA analysis detects micrometastatic cells in blood from lung cancer patients: Table 1

Castaldo¹,

Tomaiuolo²,

Sanduzzi³

et al. 2003

Eur Respir J

View full text Add to dashboard Cite

The current authors previously identified circulating cells expressing carcinoembryonic antigen (CEA) messenger ribonucleic acid (mRNA) in 80% of lung cancer patients bearing distant metastases. The current study prospectively validated the data on a novel cohort and extended the study to other mRNAs expressed by neoplastic cells.CEA, cytokeratin 19 and 20, aldolase A and epithelial glycoprotein 2 (EPG2) mRNA was analysed by reverse transcriptase-polymerase chain reaction in circulating cells from 19 healthy controls, and in biopsies and blood at diagnosis from 32 lung cancer patients monitored for 24 months.Aldolase A and cytokeratin 19 mRNA occurred in circulating cells of all controls; cytokeratin 20 was not expressed by any lung cancer biopsy. EPG2 mRNA occurred in all biopsies but not in the patients9 circulating cells. CEA mRNA occurred in 29/32 (90.6%) biopsies and in 17/32 mRNA samples from circulating cells from lung cancer patients. Of these positive patients 12/17 developed metastases within 9 months of mRNA analysis. Three positive patients died, one was lost to follow-up, and one did not develop metastases within 24 months. Of the negative patients 12/15 did not develop metastases during the 24-month follow-up; one patient was lost to follow-up, one did not express CEA, and another developed metastases.Unlike in other neoplasias, cytokeratin 19 and 20, aldolase A and epithelial glycoprotein 2 messenger ribonucleic acid are not useful for the detection of circulating cancer cells in lung cancer. Carcinoembryonic antigen messenger ribonucleic acid analysis in circulating cells helps to identify lung cancer patients at a greater risk of metastases. Eur Respir J 2003; 22: 418-421.

show abstract

Section: Discussionsupporting

confidence: 77%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Carcinoembryonic antigen mRNA analysis detects micrometastatic cells in blood from lung cancer patients: Table 1

Castaldo¹,

Tomaiuolo²,

Sanduzzi³

et al. 2003

Eur Respir J

View full text Add to dashboard Cite

show abstract

“…At high RNA concentrations (1 µg), both carcinoma and lymphoblastoid lines expressed EGP-2 RNA; however, at lower concentrations (10 ng) expression was absent in lymphoblastoid lines. More recently, without immunobead selection, in terms of EGP-2 expression, it has been also demonstrated that bone marrow aspirates (Helfrich et al, 1997) or peripheral blood cells (de Graaf et al, 1997) could yield false-positive reactions by sensitive RT-PCR methods even with samples obtained from normal volunteers. By flow cytometric examination, 83% of epithelial cell lines expressed EGP-2 cell-surface protein, usually at relatively high levels, whereas B-lymphoblastoid lines were uniformly negative.…”

Section: Discussionmentioning

confidence: 99%

Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial tumour cells in effusions

et al. 1999

View full text Add to dashboard Cite

SummaryWe developed a sensitive and specific method for the detection of epithelial cancer cells in effusions with a two-stage molecularbased assay which combined enrichment for cancer cells by immunomagnetic bead selection and reverse transcriptase polymerase chain reaction (RT-PCR) detection of epithelial glycoprotein 2 (EGP-2) RNA. Preliminary experiments indicated that immunobead selection was essential to avoid occasional false-positive RT-PCR results, and this method detected ten breast cancer cells electively added to 10 7 cytologically negative effusion cells. We studied 110 cases of pleural (n = 68) and peritoneal (n = 42) effusions (30 from patients with known carcinoma and 80 from those without known carcinoma), and the results were compared with cytological findings. Of 18 effusions that were cytologically positive or suspicious for malignant cells, 17 (94%) were positive for EGP-2 RNA (the one negative sample was from a patient who recently received combination chemotherapy). Of 92 cytologically negative samples, 11 (12%) were positive for EGP-2, including six patients with a history of previous or current carcinoma. Our method appears to be highly specific and increases the sensitivity of detection of malignant cells; it may be a useful adjunct to routine cytopathological examination.

show abstract

“…Nevertheless, the immunocytochemistry method needs to be further developed before it can be used routinely in the clinic and it is not clear whether the most frequently employed reverse transcription PCR (RT-PCR) assays for cytokeratin 18 or 19 or pancarcinoma-associated tumour marker (KSA or 17-1A antigen) have the specificity to be reliably used (Kvalheim, 1996;Helfrich et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Detection of circulating tumour cells in patients with breast or ovarian cancer by molecular cytogenetics

et al. 1999

View full text Add to dashboard Cite

The detection and elimination of minimal disseminated disease in patients with solid tumours is one of the main current topics in clinical oncology (Pantel, 1996).A variety of assays of widely varying sensitivity have been utilized for the detection of circulating tumour cells such as light microscopy, cytogenetic analyses, fluorescence in situ hybridization (FISH), Southern blot analysis, immunocytochemistry, polymerase chain reaction (PCR) and flow cytometry (Kvalheim, 1996;Pantel, 1996;Vrendenburgh et al, 1996;Schoenfeld et al, 1997;Traystman et al, 1997).Because of the fact that breast and ovarian cancers do not appear to have tumour-specific chromosomal aberrations, tumour cell detection by molecular methods is based on the amplification of tissue-specific transcripts Bostick et al, 1998). In immunocytochemical assays, epithelial specific antibodies have been used to detect isolated tumour cells in bone marrow (BM) and blood (Cote et al, 1991(Cote et al, , 1995(Cote et al, , 1996Diel et al, 1996;Franklin et al, 1996).In an effort to obtain greater sensitivity, several investigators have developed techniques for the enrichment of tumour cells before their identification by immunocytochemistry, PCR or FISH (Hardingham et al, 1993(Hardingham et al, , 1995Berois et al, 1997;Eaton et al, 1997;Hildebrandt et al, 1997;Naume et al, 1997;Martin et al, 1998).Therefore, the aim of our study was to analyse the presence and frequency of circulating tumour cells in the peripheral blood of patients with breast or ovarian cancer by using a combination of magnetic activated cell sorting (MACS) and interphase FISH. MATERIALS AND METHODS PatientsIn this study we included 48 adult patients with a median age of 60.6 ± 13.3 years (range 30-86). Thirty-five had the diagnosis of breast cancer, 12 ovarian cancer and one uterine sarcoma. Twentyfive patients had primary disease (6/25 without involvement of axillary lymph nodes, 8/25 with solid metastases, 11/25 with lymph node metastases) and 23 had relapsed (20/23 with solid metastases). PB-samples were obtained following informed consent at the time of diagnosis, during or after therapy. MACSsorted tumour cells were analysed by interphase FISH, using α-satellite probes specific for the centromeric regions of chromosomes 7, 12, 17 and the region 17 q11.2-q12 (HER-2/neu). Controls were PB-samples from five normal volunteers to determine the background for each probe. Magnetic cell separationMononuclear cells were isolated from fresh blood by FicollHypaque gradient separation. After washing in PBS per 5 mm EDTA, 300 µl PBS buffer per 10 8 cells, 20 µl of CK-8 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added. After gentle mixing and incubation for 15 min at 4°C, the cells were washed once in 5 ml buffer per 10 8 cells. The buffer was completely removed, and the cells were resuspended again in 400 µl buffer. The cell suspension was then applied to a prefilled Summary Detection of micrometastases in patients with solid tumours may aid the establishment of prognosi...

show abstract

A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood

Cited by 23 publications

References 32 publications

Carcinoembryonic antigen mRNA analysis detects micrometastatic cells in blood from lung cancer patients: Table 1

Carcinoembryonic antigen mRNA analysis detects micrometastatic cells in blood from lung cancer patients: Table 1

Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial tumour cells in effusions

Detection of circulating tumour cells in patients with breast or ovarian cancer by molecular cytogenetics

Contact Info

Product

Resources

About