| 1.A study on the feasibility of RT-PCR detecting micrometastasis in the peripheral blood of NSCLC patientsPurpose: To explore the sensitivity and specificity of nested CK19 RT-PCR and Lunx RT-PCR for detecting micrometastasis in NSCLC peripheral blood. Method: 10ml blood samples were taken from 30 healthy volunteers after discarding initial 2ml blood to avoid potential epithelial cell contamination, whereas 10ml peripheral blood were directly withdrawn from another 30 normal donors without throwing away the first 2ml blood. In vitro NSCLC micrometastsis model systems were created through adding 105,104,103,102,101,100and 0 A549 line cells to 107 PBMN by 10-fold serial dilution and micro-tube injection under a microscope. Micrometastases were measured by nested CK19 RT-PCR and Lunx RT-PCR, respectively, in both the normal blood samples and in vitro micrometastasis model system in order to determine their sensitivity and specificity. Result: Of the all normal blood samples collected after discarding the initial 2ml blood, one had positive CK19 expression while the others showed negative CK19 expression. Thus false positive rate got 3.3% of nested CK19 RT-PCR; On the other hand, of the all normal blood sample taken directly without throwing away the first 2ml blood, 6 were positive and the rest negative. Consequently, the false positive rate was 20%. The sensitivity of nested CK19 RT-PCR was 10-7 to detect NSCLC micrometastasis in peripheral blood samples. All of 60 control blood samples demonstrated negative Lunx expression, thus the false positive rate got 0 of Lunx RT-PCR. However, the sensitivity of Lunx RT-PCR was only 10-5 for measuring micrometastases in NSCLC peripheral blood. Conclusion: Nested CK19 RT-PCR was a very good method for detecting lung cancer cells in the peripheral blood with the sensitivity of 10-7 and the specificity of 96.7%. It was not proven that Lunx was a suitable marker for measuring NSCLC micrometastasis due to relatively lower sensitivity of Lunx RT-PCR.
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