Ruey-Ho Kao1, Li-Chih Huang. 1. Division of Hematology-Oncology, Tzu Chi General Hospital, Hualien, Taiwan, ROC. rueykao@mail.tcu.edu.tw
Abstract
BACKGROUND: Nasopharyngeal carcinoma (NPC) harbors a higher metastatic potential than other head and neck cancers. In order to seek a possible surrogate marker for early detection of recurrent or metastatic disease, we tested the feasibility of cytokeratin-19 (CK-19)-nested reverse-transcription polymerase chain reaction (RT-PCR) for detecting circulating tumor cells in NPC patients. METHODS: Two tubes of blood were sequentially collected in individual draws from 7 NPC patients and 15 healthy persons. Total ribonucleic acid (RNA) was extracted from blood cells and treated with deoxyribonuclease. The RNA was then subjected to RT and nested PCR with specific CK-19 primers. The reaction products were run on an agarose gel and visualized under UV light. The sequences of the products were determined using an ABI377 automatic sequencer. RESULTS: Among the 7 NPC cases, 4 cases presented CK- 19 expression with 2 in both tubes, 1 in the first tube, and 1 in the second tube. In the control group, 8 of 15 cases also presented CK-19 expression with 6 in both tubes and 2 in the second tube resulting in a 53.3% false-positive rate. Incidentally, an aberrant splicing product lacking exon 4 of CK-19 messenger RNA was discovered. CONCLUSION: Results of the present study indicate that the CK-19-nested RT-PCR is not suitable for detecting circulating tumor cells in NPC patients because of a high false-positive rate in the control group. The reason for the high rate of false-positives may be attributed to pseudogenes, different blood cell separation methods, or illegitimate expression of CK-19 in blood cells.
BACKGROUND:Nasopharyngeal carcinoma (NPC) harbors a higher metastatic potential than other head and neck cancers. In order to seek a possible surrogate marker for early detection of recurrent or metastatic disease, we tested the feasibility of cytokeratin-19 (CK-19)-nested reverse-transcription polymerase chain reaction (RT-PCR) for detecting circulating tumor cells in NPCpatients. METHODS: Two tubes of blood were sequentially collected in individual draws from 7 NPCpatients and 15 healthy persons. Total ribonucleic acid (RNA) was extracted from blood cells and treated with deoxyribonuclease. The RNA was then subjected to RT and nested PCR with specific CK-19 primers. The reaction products were run on an agarose gel and visualized under UV light. The sequences of the products were determined using an ABI377 automatic sequencer. RESULTS: Among the 7 NPC cases, 4 cases presented CK- 19 expression with 2 in both tubes, 1 in the first tube, and 1 in the second tube. In the control group, 8 of 15 cases also presented CK-19 expression with 6 in both tubes and 2 in the second tube resulting in a 53.3% false-positive rate. Incidentally, an aberrant splicing product lacking exon 4 of CK-19 messenger RNA was discovered. CONCLUSION: Results of the present study indicate that the CK-19-nested RT-PCR is not suitable for detecting circulating tumor cells in NPCpatients because of a high false-positive rate in the control group. The reason for the high rate of false-positives may be attributed to pseudogenes, different blood cell separation methods, or illegitimate expression of CK-19 in blood cells.