Abstract
Reverse transcriptase polymerase chain reaction (RT-PCR) is increasingly used to detect small numbers of circulating tumour cells, though the clinical benefit remains controversial. The largest single contributing factor to the controversy of its value is the different approaches to sample processing. The aim of this study was to compare the sensitivity and reproducibility of RT-PCR for the detection of tumour cells after four commonly used different methods of sample processing. Using RT-PCR, one tumour cell spiked in 2 ml of whole blood was detected after analysis of separated mononuclear cell RNA, whole blood total or poly-A+RNA. No false positives were identified with any method. However, the reproducibility of tumour cell detection was reduced after isolation of the mononuclear cell fraction. Only analysis of poly-A+RNA had a sensitivity of 100% in all the cell spiking experiments. In patient blood samples, analysis of poly-A+RNA increased the number of blood samples positive for tyrosine hydroxylase (TH) mRNA compared with those positive after analysis of total RNA. This may reflect high levels of cDNA reducing the efficiency of the PCR. Isolation of poly-A+RNA increases the sensitivity and reproducibility of tumour cell detection in peripheral blood. © 1999 Cancer Research Campaign
Keywords: reverse transcriptase polymerase chain reaction, poly-A+RNA, tumour cell detection
Full Text
The Full Text of this article is available as a PDF (261.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brodeur G. M., Seeger R. C., Barrett A., Berthold F., Castleberry R. P., D'Angio G., De Bernardi B., Evans A. E., Favrot M., Freeman A. I. International criteria for diagnosis, staging, and response to treatment in patients with neuroblastoma. J Clin Oncol. 1988 Dec;6(12):1874–1881. doi: 10.1200/JCO.1988.6.12.1874. [DOI] [PubMed] [Google Scholar]
- Burchill S. A., Bradbury F. M., Selby P., Lewis I. J. Early clinical evaluation of neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. Eur J Cancer. 1995;31A(4):553–556. doi: 10.1016/0959-8049(95)00053-l. [DOI] [PubMed] [Google Scholar]
- Burchill S. A., Bradbury F. M., Smith B., Lewis I. J., Selby P. Neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. Int J Cancer. 1994 Jun 1;57(5):671–675. doi: 10.1002/ijc.2910570510. [DOI] [PubMed] [Google Scholar]
- Fidler I. J. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990 Oct 1;50(19):6130–6138. [PubMed] [Google Scholar]
- Gläser R., Rass K., Seiter S., Hauschild A., Christophers E., Tilgen W. Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: a clinical two-center study. J Clin Oncol. 1997 Aug;15(8):2818–2825. doi: 10.1200/JCO.1997.15.8.2818. [DOI] [PubMed] [Google Scholar]
- Johnson P. W., Burchill S. A., Selby P. J. The molecular detection of circulating tumour cells. Br J Cancer. 1995 Aug;72(2):268–276. doi: 10.1038/bjc.1995.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jung F. A., Buzaid A. C., Ross M. I., Woods K. V., Lee J. J., Albitar M., Grimm E. A. Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients. J Clin Oncol. 1997 Aug;15(8):2826–2831. doi: 10.1200/JCO.1997.15.8.2826. [DOI] [PubMed] [Google Scholar]
- Keilholz U., Willhauck M., Rimoldi D., Brasseur F., Dummer W., Rass K., de Vries T., Blaheta J., Voit C., Lethé B. Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: a quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer. 1998 Apr;34(5):750–753. doi: 10.1016/s0959-8049(97)10105-8. [DOI] [PubMed] [Google Scholar]
- Kuroda T., Saeki M., Nakano M., Mizutani S. Clinical application of minimal residual neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction. J Pediatr Surg. 1997 Jan;32(1):69–72. doi: 10.1016/s0022-3468(97)90097-x. [DOI] [PubMed] [Google Scholar]
- Miyajima Y., Horibe K., Fukuda M., Matsumoto K., Numata S., Mori H., Kato K. Sequential detection of tumor cells in the peripheral blood and bone marrow of patients with stage IV neuroblastoma by the reverse transcription-polymerase chain reaction for tyrosine hydroxylase mRNA. Cancer. 1996 Mar 15;77(6):1214–1219. doi: 10.1002/(sici)1097-0142(19960315)77:6<1214::aid-cncr31>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
- Miyajima Y., Kato K., Numata S., Kudo K., Horibe K. Detection of neuroblastoma cells in bone marrow and peripheral blood at diagnosis by the reverse transcriptase-polymerase chain reaction for tyrosine hydroxylase mRNA. Cancer. 1995 Jun 1;75(11):2757–2761. doi: 10.1002/1097-0142(19950601)75:11<2757::aid-cncr2820751120>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
- Moss T. J., Reynolds C. P., Sather H. N., Romansky S. G., Hammond G. D., Seeger R. C. Prognostic value of immunocytologic detection of bone marrow metastases in neuroblastoma. N Engl J Med. 1991 Jan 24;324(4):219–226. doi: 10.1056/NEJM199101243240403. [DOI] [PubMed] [Google Scholar]
- Moss T. J., Sanders D. G. Detection of neuroblastoma cells in blood. J Clin Oncol. 1990 Apr;8(4):736–740. doi: 10.1200/JCO.1990.8.4.736. [DOI] [PubMed] [Google Scholar]
- Rogers D. W., Treleaven J. G., Kemshead J. T., Pritchard J. Monoclonal antibodies for detecting bone marrow invasion by neuroblastoma. J Clin Pathol. 1989 Apr;42(4):422–426. doi: 10.1136/jcp.42.4.422. [DOI] [PMC free article] [PubMed] [Google Scholar]