| BackgroundLung cancer is worldwide the most frequent cause of cancer-related mortality. The most effective treatment for non-small cell lung cancer (NSCLC) is surgical resection, but recurrent lung cancer usually appears quickly with 50–90% appearing within 24 months of surgery; 90–95% will occur within the first 5 years. There is little availability to predict recurrence in NSCLC patients, except for evaluating with a history and physical examination along with a chest imaging procedure every 6 months for the first 2 years then yearly thereafter. This dismal survival could be improved through earlier detection or through identification of prognostic biomarkers, which could identify subsets of patients with worse prognosis who might benefit from a more effective treatment strategy. Therefore, the development of more useful laboratory diagnostic methods for lung cancer is urgently required. Taking into account the biological properties of a tumor, like the very long time for the development from a single transformed cell into a clinically detectable tumor, but the tumor-associated nucleic acids can be released in bloodstream by apoptosis, necrosis, active release and otherwise. Circulating DNA alterations are detectable ahead of cancer diagnosis, raising the possibility of exploiting them as biomarkers for prognostication and monitoring cancer occurrence.ObjectiveThe purpose of this study was to validate and evaluate the quantitation of plasma DNA and methylated APC, RASSF1A in predicting short-term recurrence of resectable NSCLC patients.MethodsFifty-four NSCLC patients with a median age of 63 years (range: 38-79 years), 38 males and 16 females, from the Department of Thoracocardiac Surgery of The First Affiliated Hospital of Nanjing Medical University, were staged I–IIIA. Two-milliliter blood samples were withdrawn from a peripheral vein and placed into EDTA-containing tubes from all patients pre-surgery (median: 6 days; range: 2-14 days) and post-surgery (median: 8 days; range: 6-17 days), followed by 1 600×g centrifugation at room temperature for 10 min. The plasmas were then transferred and followed an additional 16 000×g centrifugation step at 4℃for 10 min. Control venous blood samples were obtained from 50 healthy volunteers for an annual health check with a median age of 58.5 years (range: 36-77 years), 30 males and 20 females. DNA was extracted from cell-free plasmas of patients and controls based on principle of magnetic beads binding, then analyzed for the amounts of DNA by duplex real-time quantitative PCR with internal control, and for the quantity of methylated genes APC and RASSF1A by duplex real-time methylation-specific PCR. Patients were called back at the 6th month after operation to perform a CT scan and physical examination for evaluating the status of recurrence.ResultsValue reported of plasma DNA quantity, methylated APC and RASSF1A quantity was median and interquartile range. Fifteen patients occurred recurrence in 6-month reexamination after operation. The level of plasma DNA in control group was 18.3 (16.1~27.2) ng/ml without any correlations to gender (P = 0.593) and age (P = 0.270). On the whole, the levels of plasma DNA were significantly higher in patients both before (64.1 ng/mL, 46.4~79.3) and after (56.1 ng/mL, 41.9~90.6) operation than in controls, not presenting significant decrease after operation. The level of plasma DNA in pre-surgery was significantly associated with gender (P = 0.027), but not with age, tumor size, lymph node invasion, AJCC stage, pathologic subtype and grade, while in post-surgery was only correlated to recurrence. The quantity of methylated APC was 101.3 (0~1 060.0) copies/ml after operation, which was significantly lower than 401.8 (0~2 038.2) copies/ml before operation (P = 0.016), while this reduction was not seen in RASSF1A (P = 0.060). The quantity of methylated RASSF1A was 377.8 (0~1 283.9) copies/ml before operation and was 0 (0~1 062.8) copies/ml after operation. As well as these aberrant methylations in controls were only 1 for APC (211.4 copies/ml) and 2 for RASSF1A (218.0 copies/ml and 338.5 copies/ml respectively), which were far less than NSCLC patients (APC: P < 0.001; RASSF1A: P < 0.001). Plasma APC and RASSF1A methylation were correlated to recurrence either before or after operation, but not to age, gender, lymph node invasion, pathologic subtype and grade. The aberrant methylation level after operation was significantly different between groups by AJCC staging (APC: P = 0.020; RASSF1A: P = 0.022).The quantities of plasma DNA, methylated APC and RASSF1A after operation were all presented a similar mode of reduction in non-recurrences and unchanging in recurrences. The levels of the three indices after operation were all significantly higher in recurrences than in non-recurrences and all had a superior predictive value for recurrence than those before operation (plasma DNA quantitation: P < 0.001; methylated APC quantitation: P = 0.011; methylated RASSF1A quantitation: P = 0.033). By selecting≥73.3 ng/ml (sensitivity: 80.0%, specificity: 82.0%) of plasma DNA quantity,≥810.7 copies/ml (sensitivity: 86.7%, specificity: 87.2%) of methylated APC quantity,≥698.7 copies/ml (sensitivity: 93.3%, specificity: 89.7%) of methylated RASSF1A quantity as cut-off values to achieve the best accuracy, the plasma methylated RASSF1A was the only independent factor related to recurrence after logistic multivariate modeling.ConclusionThe quantitation of plasma DNA, methylated APC and RASSF1A during 6 to 17 days after operation are useful prognostic biomarkers for predicting recurrence in NSCLC patients after curative-intent surgery, which can signify progress of neoplastic malignancy prior to clinical findings. These approaches with its high accuracy and facilitation in monitoring progresses on patients by serial blood samplings can be applicable in clinical settings for the management of NSCLC patients. The plasma methylated RASSF1A was the only independent factor related to recurrence in this study, whose prognostic usefulness needs to be further assessed. |
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