Non small cell lung cancer

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 endoscopic 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 RNA 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 ([[beta].sub.2]-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.

Key words: cancer staging; cytology; endosonography; fine-needle aspiration; lung neoplasm

Abbreviations: AJCC = American Joint Committee on Cancer; AUC = area under the curve; CI = confidence interval CK = cytokeratin. Ct = cycle of threshold. EUS = endoscopic ultrasound. FBS fetal bovine serum; FNA = fine-needle aspiration; NSCLC = non-small cell lung cancer; PET = positron emission tomography; ROC = receiver operating characteristic. RT-PCR = reverse transcriptase-polymerase chain reaction

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Non-small cell lung cancer (NSCLC) is the most common cancer-related cause of death for both men and women in the United States. Each year there are > 170,000 new cases and approximately 160,000 deaths due to NSCLC. (1) Standard therapies for patients with NSCLC include surgery, chemotherapy, and radiation therapy, with the stage of disease dictating the choice of therapy. The current staging system for lung cancer uses the American Joint Committee on Cancer (AJCC) (2) TNM system, and its goal is to classify patients into groups based on the extent of disease. This system relies heavily on the pathologic evaluation of the primary tumor (T), regional nodes (N), and distant metastases (M). Surgery is most appropriate for patients in whom disease is confined to the lung and hilar lymph nodes (stages I and II, respectively). For patients with metastatic disease to mediastinal lymph nodes (stage III), the benefit of surgery as primary therapy isquestionable, and combined chemotherapy/radiotherapy may be the most appropriate. (3) Thus, the reliable staging of mediastinal lymph nodes is essential for choosing for the most appropriate therapy for each patient.

In this study, we describe a new technique for the detection of mediastinal lymph node metastases. We have combined the use of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA), a minimally invasive approach to tissue biopsy, with the sensitive detection technology of quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The aims of this study were to determine the following: (1) whether tissue procured by EUS-FNA was suitable for multimarker RT-PCR analysis, and (2) whether the relative sensitivity and specificity of molecular markers of NSCLC micrometastasis in known malignant lymph nodes (positive controls) and in pathologically negative mediastinal lymph nodes of patients with NSCLC were appropriate.

MATERIALS AND METHODS

Patients and Clinical Procedures

The study was approved by the Institutional Review Board of the Medical University of South Carolina, and all patients completed informed consent forms. Patients were eligible for the study if they had pathologically proven NSCLC and were considered to be medically fit for surgical resection in the event that no metastatic disease was found (including mediastinal lymph node metastases). Patients were excluded if they had a history of malignancy other than basal cell carcinoma of the skin, were < 18 years of age, were unfit for surgical resection due to concurrent cardiac disease, had insufficient pulmonary reserve, or refused to consent.

All patients were reviewed at a multidisciplinary thoracic tumor board. A helical CT scan was performed of the chest and upper abdomen. Positron emission tomography (PET) scans were performed in selected cases but not as a routine part of the protocol. If no distant metastases (eg, contralateral lung or abdomen) were found, the patient underwent EUS-FNA. EUSFNA was performed under conscious sedation with midazolam (0 to 5 mg), and meperidine (0 to 200 mg). The liver, left adrenal gland, celiac trunk, and posterior mediastinum were surveyed for focal, hypoechoic lymph nodes or tumor metastases. All visualized lymph nodes in the subcarina (AJCC level 7) aortopulmonary window (AJCC level 5), inferior mediastinum (AJCC levels 8 to 9), and superior mediastinum lateral to the trachea (AJCC levels 2L/R and 4L/R) were sampled with FNA for cytology, and to obtain a sample for RT-PCR (Fig 1). The site sampled depended on which site would yield the most advanced stage of disease. If an N3 (contralateral to tumor) lymph node was present, it was sampled first. If nonmalignant, the procedure was continued until at least one lymph node from all accessible sites was sampled. If multiple lymph nodes were present in the same AJCC station, the largest lymph node was chosen for FNA.

Fine-needle sampling was performed with a 22-gauge needle (Echo-tip; Wilson-Cook Co; Winston Salem, NC) under EUS and color Doppler with a curved linear array echoendoscope (model GF-UC30P or GF-UCT30; Olympus America; Melville NY). All lymph nodes were punctured with an occluding styler in place. After insertion of the needle into the lymph node, the styler was removed, the needle was moved to and fro within the lymph node for approximately 30 s, and then was withdrawn.

Each lymph node was sampled with a minimum of four needle passes unless a definitive diagnosis of malignancy was rendered prior to the fourth pass. Each fine-needle sample was expressed, using a 10-mL air-filled syringe, onto a separate glass slide, and a direct smear was made by an on-site cytotechnician. Each slide was air-dried and/or alcohol-fixed (95% ethanol), and direct smears were prepared for immediate interpretation by staining with a Romanowsky stain (Diff-Quik; Sigma-Aldrich Chemical; St. Louis, MO). On-site evaluation of smears was performed to assess cellular adequacy. The final interpretation of all of the material consisted of reviewing the slides prepared on-site and stained later and examining the thin-layer cytology or cytospin/ cell block material prepared from the cellular material kept in the Hank solution.