Detection of circulating tumor cells (CTCs) in patients with lung carcinoma by real-time fluorescent quantitative-PCR approach before and after chemotherapy

Ming-Jian Ge, Qing-Chen Wu, Mei Wang, Li Li, Xiao-Long Zhao, Qiao-Min Huang, Liang-Bin Li

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DOI: 10.4236/health.2009.13038                

Abstract

Circulating tumour cells (CTCs) are referred to the tumour cells that disseminated from the primary tumour and survive in circulating during the pro-ceeding of tumour growth. As surgical treatment evolves and local control has improved, the failure of cancer treatment has largely remained the re-sult of systemic metastasis. Selection of patients most likely to benefit from adjuvant strategies remains problematic. In order to develop a new standard of curative effect, this study was de-signed to track the number of CTCs in patients with lung cancer during chemotherapy. Methods: Samples of peripheral blood was taken from each lung cancer patients (n=32) on the day before chemotherapy as well as the third week after the chemotherapy cycle. The samples were subjected to real-time fluorescent quantitative reverse-tran- scriptase polymerase chain reaction (fqRT-PCR). Meanwhile the tumour size was determined by chest X-ray or computed tomograghy. Results Compared to that of pre-chemotherapy, the ex-pression level of cytokeratin (CK) 19 in the pa-tients significantly declined after chemotherapy (t=4.659,P=0.000). The level of CK19 mRNA in pa-tients with small cell lung cancer (SCLC) was higher than that of patients with non-small cell lung cancer (NSCLC) (t=1.944, P=0.061). The de-crease of CK19 mRNA level correlated well with the type during the treatment. Relatively the de-crease of SCLC is more obvious (t=6.073,P=0.000). The variation of CK19 mRNA level before and after chemotherapy was positively related to the dis-parity of tumour burden (r=0.593). There was also a significant association between the type (NSCLC vs. SCLC) and the change of tumour size (t=3.686, P=0.001).The positive rate before chemotherapy Supported by grant from the Natural Science Foundation in China (No.30972961). was 71.9% (23/32), while that after chemotherapy was 37.5% (12/32), indicating that 11 patients con- verted into negative after chemotherapy. Of the 16 patients which were in Ⅳ-stage, 11 cases were po- sitive (11/16,68.8%). Surprisingly, of the remaining 16 patients which were Ⅱ/Ⅲ stage, 12 cases were regarded as positive according to the criteria (12/6,75%). Conclusions: The real-time flu- ores-cent quantitative-PCR approach is useful for mea- suring the relative number of CTCs in a patients’ peripheral blood to monitor the effectiveness of treatment, and for designing more comprehensive and reasonable therapeutic regimes at earlier dates for patients. The treatment response can be immediately assessed by serial quantitation of CTCs after chemotherapy, and therefore this method highlights an alternative approach to rapidly access the patient’s response to treatment.

Keywords

Lung Neoplasm; Blood; Polymerase Chain Reaction; Cytokeratin; Messenger RNA; Chemotherapy

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. Lankiewicz, S Zimmermann, C Hollmann, T Hillemann, T. F. Greten, (2008) Circulating tumour cells as a predictive factor for response to systemic chemotherapy in patients with advanced colorectal cancer, Mol Oncol, 2(4), 349-355.
[2]	C. Wu, H. Hao, L. Li, X. Zhou, Z. Guo, L. Zhang, X. Zhang, W. Zhong, H. Guo, R.M. Bremner, P. Lin, (2009) Pre-liminary investigation of the clinical significance of de-tecting circulating tumor cells enriched from lung cancer patients, J Thorac Oncol, 4(1), 30-36.
[3]	T. Okegawa, K. Nutahara, E. Higashihara, (2008) Im-munomagnetic quantification of circulating tumor cells as a prognostic factor of androgen deprivation responsive-ness in patients with hormone naive metastatic prostate cancer, J Urol, 180(4), 1342-1347.
[4]	M. Tewes, B. Aktas, A. Welt, S. Mueller, S. Hauch, R. Kimmig, S. Kasimir-Bauer, (2009) Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies, Breast Cancer Res Treat, 115(3), 581-590.
[5]	S. Maheswaran, L.V. Sequist, S. Nagrath, L. Ulkus, B. Brannigan, C.V. Collura, E. Inserra, S. Diederichs, A. J. Iafrate, D. W. Bell, S. Digumarthy, A. Muzikansky, D. Irimia, J. Settleman, R. G. Tompkins, T. J. Lynch, M. Toner, D. A. Haber, (2008) Detection of mutations in EGFR in circulating lung-cancer cells, N Engl J Med, 359(4), 366- 377.
[6]	M. Ignatiadis, G. Kallergi, M. Ntoulia, M. Perraki, S. Apostolaki, M. Kafousi, G. Chlouverakis, E. Stathopoulos, E. Lianidou, V. Georgoulias, D. Mavroudis, (2008) Prog-nostic value of the molecular detection of circulating tumor cells using a multimarker reverse transcription-PCR assay for cytokeratin 19, mammaglobin A, and HER2 in early breast cancer, Clin Cancer Res, 14(9), 2593-2600.
[7]	N. Xenidis, M. Ignatiadis, S. Apostolaki, M. Perraki, K. Kalbakis, S. Agelaki, E. N. Stathopoulos, G. Chlouverakis, E. Lianidou, S. Kakolyris, V. Georgoulias, D. Mavroudis, (2009) Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breast cancer, J Clin Oncol, 27(13), 2177-2184.
[8]	S. J. Cohen, C. J. Punt, N. Iannotti, B. H. Saidman, K. D. Sabbath, N. Y. Gabrail, J. Picus, M. A. Morse, E. Mitchell, M. C. Miller, G. V. Doyle, H. Tissing, L. W. Terstappen, N. J. Meropol, (2009) Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer, Ann Oncol, 20(7), 1223-1229.
[9]	M. J. Serrano, P. Sánchez-Rovira, M. Delgado-Rodriguez, J. J. Gaforio, (2009) Detection of circulating tumor cells in the context of treatment: prognostic value in breast cancer, Cancer Biol Ther, 8(8), 671-675.
[10]	H. I. Scher, X. Jia, J. S. de Bono, M. Fleisher, K. J. Pienta, D. Raghavan, G. Heller, (2009) Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data, Lancet Oncol, 10(3), 233-239.
[11]	T. Kuroda, N. Morikawa, K. Matsuoka, A. Fujino, T. Honna, A. Nakagawa, M. Kumagai, H. Masaki, M. Saeki, (2008) Prognostic significance of circulating tumor cells and bone marrow micrometastasis in advanced neuro-blastoma, J Pediatr Surg, 43(12), 2182-2185.
[12]	J. S. de Bono, H. I. Scher, R. B. Montgomery, C. Parker, M. C. Miller, H. Tissing, G. V. Doyle, L. W. Terstappen, K. J. Pienta, D. Raghavan, (2008) Circulating tumor cells predict survival benefit from treatment in metastatic castration -resistant prostate cancer, Clin Cancer Res, 14(19), 6302- 6309.
[13]	B. J. Curry, K. Myers, P. Hersey, (1998) Polymerase chain reaction detection of melanoma cells in the circulation: relation to clinical stage, surgical treatment, and recur-rence from melanoma, J Clin Oncol, 16(5), 1760-1769.
[14]	F. S. Celi, M. M. Cohen, S. E. Antonarakis, E. Wertheimer, J. Roth, A. R. Shuldiner, (1994) Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences.Genomics, 21(2), 304- 310.
[15]	C. Y. Young, B. T. Montgomery, P. E. Andrews, S. D. Qui, D. L. Bilhartz, D. J. Tindall, (1991) Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP, Cancer Res, 51(14), 3748-3752.
[16]	S. D. Qiu, C. Y. Young, D. L. Bilhartz, J. L. Prescott, G. M. Farrow, W. W. He, D. J. Tindall, (1990) In situ hybridiza-tion of prostate-specific antigen mRNA in human prostate, J Urol, 144(6), 1550-1556.
[17]	S. Hosch, J. Kraus, P. Scheunemann, J. R. Izbicki, C. Schneider, U. Schumacher, K. Witter, M. R. Speicher, K. Pantel, (2000) Malignant potential and cytogenetic char-acteristics of occult disseminated tumor cells in eso-phageal cancer, Cancer Res, 60(24), 6836-6840.
[18]	I. H. Wong, W. Y. Lau, T. Leung, W. Yeo, P. J. Johnson, (1999) Hematogenous dissemination of hepatocytes and tumor cells after surgical resection of hepatocellular carci-noma: a quantitative analysis, Clin Cancer Res, 5(12), 4021-4027.
[19]	K. Pantel, R. J. Cote, O. Fodstad, (1999) Detection and clinical importance of micrometastatic disease, J Natl Cancer Inst, 91(13), 1113-1124.
[20]	K. Peck, Y. P. Sher, J. Y. Shih, S. R. Roffler, C. W. Wu, P. C. Yang, (1998) Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients, Cancer Res, 58(13), 2761-2765.