Potential for intraoperative molecular staging of the mediastinum in NSCLC patients

Abstract Introduction: Accurate staging of mediastinal lymph nodes guides decisions in the choice of treatment of NSCLC. Unfortunately, reported sensitivity for mediastinoscopy ranges from 40%–89%, using standard pathologic techniques. Our objective was to develop molecular techniques to improve sensitivity and to apply these methods intraoperatively such that mediastinoscopy and tumor resection can potentially be performed in one procedure when the mediastinum is negative for disease. Methods: Approximately 40 markers were screened for expression in 6 primary tumors and 10 benign lymph nodes. From these, 6 markers that showed high expression in primary tumors and at least 500 fold lower expression in benign nodes were chosen for a secondary screening in 22 primary tumors, 21 positive lymph nodes and 22 benign lymph nodes to assess sensitivity and specificity. In collaboration with Cepheid, we have developed and tested a fully integrated, automated instrument (the GeneXpert) that is capable of performing both RNA isolation and QRT-PCR in less than 35 minutes. To demonstrate the feasibility of intraoperative staging, 5-micron sections were cut from OCT embedded tissues from a set of 6 positive, 6 negative, and 6 benign nodes and analyzed on the GeneXpert. Results: We identified 6 markers with good potential for lymph node analysis. Using an expression level cutoff equal to the highest expression in the benign lymph nodes and thus yielding 100% specificity, the following sensitivities were achieved for each marker respectively: carcinoembryonic antigen (CEA) (95.2%), cytokeratin 19 (CK19) (100%), lung-specific X protein (LUNX) (61.9%), novel marker LC-1 (61.9%), surfactant protein B (SFTPB) (57.1%), and TACSTD-1 (100%). Using TACSTD-1 as the marker, the fully automated GeneXpert assay (completed in 35 minutes) clearly discriminated positive from negative nodes. Conclusions: We evaluated molecular markers for their ability to detect nodal disease in NSCLC. In addition, we have developed instrumentation that allows rapid, automated molecular analysis of lymph nodes. The combination of the appropriate markers with this technology could potentially provide the ability to detect occult nodal disease, allowing for accurate intraoperative analysis of mediastinal lymph nodes, and enabling the surgeon to determine operability at the time of the original procedure.