Detection of circulating tumor cells in the peripheral blood of nasopharyngeal carcinoma patients by a nested reverse transcriptase polymerase assay for cytokeratin 19 mRNA

Abstract

Nasopharyngeal carcinomas (NPC) are tumors of nasopharynx origin with high rate of distance metastases after radiotherapy. Therefore, detection of micrometastasis will be an important issue in the prognostic and the choice of the systemic treatment. Our aim was early detection of circulating tumor cells in the blood of NPC patients by an RT-PCR assay for cytokeratin19 mRNA. Patients and methods used are venous blood samples obtained from 30 NPC patients with biopsy-proven NPC and 20 healthy blood donors which were tested using a previously reported assay. Identity of products was confirmed by sequencing. Results obtained show that 10 of the 30 NPC were positive for CK19 transcripts in peripheral blood, suggesting the presence of tumour epithelial cells. No CK19 positive cells were detected in the 20 healthy volunteers. Nevertheless, we encountered the problem of CK19 pseudogene interference due to genomic DNA contamination of RNA preparations. Four of the 10 positive patients were proven to have CK19 mRNA, as assessed by sequencing. In the remaining six, the presence of the CK19 pseudogene may has masked the CK19 mRNA in RT-PCR and sequencing. In conclusion, we described an improved RT-PCR assay that is sensitive and has high clinical specificity to detect minimal metastatic disease in NPC patients. Nevertheless, one should be aware about the necessity to optimize the analytical specificity of the assay.

Keywords: Nasopharyngeal carcinomas, micrometastasis, cytokeratin 19, nested reverse transcriptase polymerase

African Journal of Biotechnology, Vol. 13(3), pp. 378-384, 15 January, 2014