Detection of disseminated lung cancer cells in regional lymph nodes by assay of CK19 reverse transcriptase polymerase chain reaction and its clinical significance.

Purpose: To set up a molecular method (i.e. RT-PCR) that can be used to detect disseminated tumor cells (DTCs) in regional lymph nodes (LNs) in patients with lung cancer and to evaluate its clinical significance.

Methods: Cytokeratin 19 (CK(19)) was used as marker. Serial dilution study for LC-5 cells (a lung squamous cell line) was performed to detect sensitivity of the molecular protocol. Regional LNs (n = 261) and primary lung cancer tissue (n = 40) were obtained from 40 patients with lung cancer who underwent lobectomy or pneumonectomy. They were randomly categorized into two groups: group I (LN-based study, n = 20) and group II (patient-based study, n = 20). Each LN was halved. One half of a LN was subjected to histological examination (HE) and the other half was subjected to RT-PCR amplification of CK(19) mRNA. The effect on survival was analyzed. The cumulative survival was calculated by the Kaplan-Meier method and compared by the log rank test. The Cox model analyzed the prognostic factors.

Results: CK(19) mRNA expressed in all tumor tissues as well as LC-5, PAa cells (a lung adenocarcinoma cell line), but not in normal control LNs. Serial dilution study for LC-5 cells demonstrated that CK(19) mRNA was detectable at a concentration as low as 10 LC-5 cells in 1x10(7) LN cells. There was no significant difference between the detecting result of single LN and that of mixed LNs (P > 0.05). In 18 of 40 patients, the metastasis in regional LNs was found by both HE and RT-PCR. Of 22 patients without pathologically involved nodes, six (27%) were found to express CK(19) mRNA in regional LNs. According to the results of regional LNs in 40 patients by molecular assay, the presence of the CK(19) product in LNs was related to tumor size (chi(2) = 5.76, P < 0.025) as well as cell differentiation of the tumor (chi(2) = 7.08, P < 0.01). Following a median observation time of 26 months (range, 4 to 60 months), patients with DTCs in nodes showed significant shorter disease-free survival duration than node-negative patients (log-rank test, P = 0.001). The independence of this prognostic significance was demonstrated by a multivariate analysis (Cox regression model, P = 0.004). The results diagnosed by HE had no significant effect on prognoses (P = 0.455).

Conclusions: Comparing with HE, RT-PCR can make more accurate assessment of metastatic status in LNs, which is helpful for screening the patients in whom the early subclinical metastasis exists and disclosing the intrinsic regulation of malignant metastasis. The presence of DTCs in LNs is an independent factor for prognosis. Molecular detection of DTCs in LNs is a supplement for current tumor staging in lung carcinoma.