Accurate molecular detection of non-small cell lung cancer metastases in mediastinal lymph nodes sampled by endoscopic ultrasound-guided needle aspiration

Objectives: The recurrence of disease after the complete resection of early stage non-small cell lung cancer (NSCLC) indicates that undetected metastases were present at the time of surgery. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a highly sensitive technique for detecting rare gene transcripts that may indicate the presence of cancer cells, and endoscopie ultrasound (EUS)-guided fine-needle aspiration (FNA) is a minimally invasive technique for the nonoperative sampling of mediastinal lymph nodes. The aim of this study was to determine whether these two techniques could enhance the preoperative detection of occult metastases. Methods: Patients with NSCLC were evaluated with chest CT and positron emission tomography scans. Those patients without evidence of metastases (87 patients) underwent EUS-guided FNA. Lymph nodes from levels 2, 4, 5, 7, 8, and 9 were sampled and evaluated by standard cytopathology and real-time RT-PCR. Normal control FNA specimens were obtained from patients without cancer who were undergoing EUS for benign disease (17 control specimens). For each sample, messenger KNA was extracted and real-time RT-PCR was used to quantitate the expression of six lung cancer-associated genes (ie, CEA, CK19, KS1/4, lunx, muc1, and PDEF) relative to the expression of an internal control gene (β₂-microglobulin). Results: Clinical thresholds of marker positivity were set at 100% specificity, as determined by the receiver operating characteristic curve analysis. Of the cytology-positive lymph nodes (27 lymph nodes), the expression of the KS1/4 gene was above its respective clinical threshold in 25 of 27 samples (93%), making this the most sensitive marker for the detection of metastatic NSCLC. At least one of the six lung cancer-associated genes was overexpressed in 18 of 61 cytology-negative patients (30%), of which KS1/4 was overexpressed in 15 of 61 patients (25%). Conclusions: Based on the high accuracy of EUS-guided FNA/RT-PCR, we predict that some of the patients in the cytology-negative/ marker-positive category will have high NSCLC recurrence rates. Among the genes used in our marker panel, KS1/4 appears particularly useful for the detection of overt or occult metastatic disease.