PCR detection of circulating tumor cells in nasopharyngeal carcinoma patients with distant metastasis: effect of enzyme and sampling.
BACKGROUND
Nasopharyngeal carcinoma (NPC) has a high potential to develop distant metastasis after radiotherapy. Cytokeratin 19 (CK-19) mRNA has been frequently used as a marker in the detection of circulating tumor cells of epithelial origin, but has rarely been investigated in NPC. This study was performed to evaluate the effect of blood sampling and different Taq DNA polymerase on the results of nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay.


METHODS
Peripheral blood samples from a total of 37 NPC patients with well-documented distant metastasis (M1) were collected before treatment. Eighteen patients had more than one blood sampling. Five different Taq DNA polymerases were used to test the blood from 17 patients. Peripheral blood of 37 nonmetastatic (M0) NPC patients was tested by the same nested RT-PCR system using multiple Taq DNA polymerases to evaluate the impact of multienzyme assay in the prediction of subsequent distant metastasis.


RESULTS
Among M1 NPC patients, the accumulative positive rates of CK-19 mRNA were 22.2%, 44.4%, 70.6%, 75.0%, and 80.0% when one, two, three, four, or five blood sampling were taken, respectively. The accumulative positive rates increased as the numbers of different enzymes increased-from 35.5% by one enzyme to 82.4% by five enzymes. Six of 37 M0 patients had distant metastasis develop after a median follow-up time of 20 months. The detection sensitivity for four-enzyme test (5 of 6 = 83.3%) is better than that of one-enzyme test (2 of 6 = 33.3%).


CONCLUSIONS
Our data demonstrate that multiple blood sampling or using multiple enzymes for nested RT-PCR assay significantly enhances the sensitivity in the molecular diagnosis of NPC metastasis.
Lin
Jin-Ching
JC
Institute of Clinical Medicine, College of Medicine, National Yang-Ming University, Taipei, Taiwan. jclin@vghtc.vghtc.gov.tw
Chen
Kuang Y
KY
Wang
Wen-Yi
WY
Jan
Jian-Sheng
JS
Wei
Yau-Huei
YH
D000293:Adolescent; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis; D004259:DNA-Directed DNA Polymerase; D005260:Female; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009303:Nasopharyngeal Neoplasms / Q000097:blood* / Q000473:pathology*; D009360:Neoplastic Cells, Circulating; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D007633:Keratins; C108542:DyNAzyme polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase