A combination of molecular markers accurately detects lymph node metastasis in non-small cell lung cancer patients.

Occult lymph node metastasis (micrometastasis) is a good prognostic indicator in non-small cell lung cancer (NSCLC) and could be used to direct adjuvant chemotherapy in stage I patients. This study was designed to evaluate molecular markers for detection of occult lymph node metastasis in NSCLC, define the best marker or marker combination to distinguish positive from benign lymph nodes, and evaluate these markers in lymph nodes from pathologically node-negative (pN(0)) NSCLC patients. Potential markers were identified through literature and database searches and all markers were analyzed by quantitative reverse transcription-PCR in a primary screen of six NSCLC specimens and 10 benign nodes. Selected markers were further evaluated on 21 primary NSCLC specimens, 21 positive nodes, and 21 benign nodes, and the best individual markers and combinations were identified. A combination of three markers was further validated on an independent set of 32 benign lymph nodes, 38 histologically positive lymph nodes, and 462 lymph nodes from 68 pN(0) NSCLC patients. Forty-two markers were evaluated in the primary screen and eight promising markers were selected for further analysis. A combination of three markers (SFTPB, TACSTD1, and PVA) was identified that provided perfect classification of benign and positive nodes in all sample sets. PVA and SFTPB are particularly powerful in tumors of squamous and adenocarcinoma histologies, respectively, whereas TACSTD1 is a good general marker for NSCLC metastasis. The combination of these genes identified 32 of 462 (7%) lymph nodes from 20 of 68 (29%) patients as potentially positive for occult metastasis. Long-term follow-up will determine the clinical relevance of these findings.

Prediction of lymph node metastasis by analysis of gene expression profiles in primary lung adenocarcinomas.

PURPOSE: Lymph node status is a strong predictor of outcome for lung cancer patients. Recently, several reports have hinted that gene expression profiles of primary tumor may be able to predict node status. The goals of this study were to determine if microarray data could be used to accurately classify patients with regard to pathologic lymph node status, and to determine if this analysis could identify patients at risk for occult disease and worse survival. EXPERIMENTAL DESIGN: Two previously published lung adenocarcinoma microarray data sets were reanalyzed. Patients were separated into two groups based on pathologic lymph node positive (pN+) or negative (pN0) status, and prediction analysis of microarray (PAM) was used for training and validation to classify nodal status. Overall survival analysis was performed based on PAM classifications. RESULTS: In the training phase, a 318-gene set gave classification accuracy of 88.4% when compared with pathology. Survival was significantly worse in PAM-positive compared with PAM-negative patients overall (P < 0.0001) and also when confined to pN0 patients only (P = 0.0037). In the validation set, classification accuracy was again 94.1% in the pN+ patients but only 21.2% in the pN0 patients. However, among the pN0 patients, recurrence rates and overall survival were significantly worse in the PAM-positive compared with PAM-negative patients (P = 0.0258 and 0.0507). CONCLUSIONS: Analysis of gene expression profiles from primary tumor may predict lymph node status but frequently misclassifies pN0 patients as node positive. Recurrence rates and overall survival are worse in these "misclassified" patients, implying that they may in fact have occult disease spread.

Molecular staging of lymph nodes from patients with esophageal adenocarcinoma.

PURPOSE: This study was designed to evaluate molecular markers for the detection of micrometastasis in esophageal adenocarcinoma, define algorithms to distinguish positive from benign lymph nodes and to validate these findings in an independent tissue set and in patients with p(N0) esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Potential markers were identified through literature and database searches. All markers were analyzed by quantitative reverse transcription (QRT)-PCR on a limited set of primary tumors and benign lymph nodes. Selected markers were further evaluated on a larger tissue set and classification algorithms were generated for individual markers and combinations. Algorithms were statistically validated internally as well as externally on an independent set of lymph nodes. Selected markers were then used to identify occult disease in lymph nodes from 34 patients with p(N0) esophageal adenocarcinoma. RESULTS: Thirty-nine markers were evaluated, six underwent further analysis and five were analyzed in the external validation study. Two markers provided perfect classification in both the screening and validation sets, although parametric bootstrap analysis estimated 2% to 3% optimism in the observed classification accuracy. Several marker combinations also gave perfect classification in the observed data sets, and estimates of optimism were lower, implying more robust classification than with individual markers alone. Five of thirty-four patients with esophageal adenocarcinoma had positive nodes by multimarker QRT-PCR analysis and disease-free survival was significantly worse in these patients (P = 0.0023). CONCLUSIONS: We have identified novel QRT-PCR markers for the detection of occult lymph node disease in patients with esophageal adenocarcinoma. The objective nature of QRT-PCR results, and the ability to detect occult metastases, make this an attractive alternative to routine pathology.

Prognostic significance of micrometastasis in non-small-cell lung cancer.

Accurate staging of non-small-cell lung cancer (NSCLC) determines prognosis and facilitates decisions regarding treatment options. Unfortunately, even after an apparently complete resection in patients with stage I disease, the recurrence rates range from 25% to 50%, and overall survival is not encouraging. One possible reason for this may be that those patients with a poor outcome actually have more extensive disease, with occult locoregional and/or distant metastasis than originally identified by routine pathologic staging techniques. There is now a sizable body of literature on the detection and possible prognostic role of occult disease in lung cancer. The majority of these studies are based on immunohistochemical analysis of lymph nodes and/or bone marrow, but a handful of studies use molecular approaches. The purpose of this review is to summarize and critique the current literature on occult tumor cell spread to lymph nodes and bone marrow in patients with NSCLC. Based on this literature, we believe that the prognostic significance of bone marrow micrometastasis remains unclear. However, the majority of studies indicate that occult lymph node disease is associated with a poor outcome. Thus, our ability to detect individual tumor cells could result in more accurate staging of NSCLC in patients and would potentially lead to the development of novel therapies, as well as influence decisions regarding the use of appropriate multimodality treatment strategies, the choice of surgical technique, and extent of dissection. As data accumulate, the presence or absence of occult nodal involvement should probably be considered at the next revision of the staging system for NSCLC.