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Clinical Investigations: LUNG CANCER |

Accurate Molecular Detection of Non-small Cell Lung Cancer Metastases in Mediastinal Lymph Nodes Sampled by Endoscopic Ultrasound-Guided Needle Aspiration* FREE TO VIEW

Michael B. Wallace, MD, MPH; Mark I. Block, MD; William Gillanders, MD; James Ravenel, MD; Brenda J. Hoffman, MD; Carolyn E. Reed, MD; Mostafa Fraig, MD; David Cole, MD; Michael Mitas, PhD
Author and Funding Information

*From the Mayo Clinic College of Medicine (Dr. Wallace), Jacksonville, FL; and the Medical University of South Carolina (Drs. Block, Gillanders, Ravenel, Hoffman, Fraig, Cole, Mitas, and Reed), Charleston, SC.

Correspondence to: Michael B. Wallace, MD, MPH, Associate Professor of Medicine, Mayo Clinic, 4500 San Pablo Rd, Jacksonville, FL 32224; e-mail: Wallace.michael@mayo.edu



Chest. 2005;127(2):430-437. doi:10.1378/chest.127.2.430
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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 (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.

Figures in this Article

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.1Standard 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)2TNM 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.

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. EUS-FNA 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 stylet in place. After insertion of the needle into the lymph node, the stylet 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.

A pathologic diagnosis of malignancy was obtained if the cytology specimen revealed cells that were consistent with malignancy. Patients without evidence of mediastinal lymph node metastases by EUS-guided FNA cytology underwent surgery. Mediastinoscopy was performed at the discretion of the operating surgeon. If N2 level lymph nodes sampled during either mediastinoscopy or thoracotomy were negative, pulmonary resection was performed along with standard mediastinal lymph node dissection. Each lymph node and the primary tumor were evaluated with a histologic methods using hematoxylin-eosin staining. Because all lymph nodes from a region (eg, the subcarina) were resected in the surgical specimen, it was assumed that the lymph node sampled by EUS-guided FNA in that region was contained within the group of resected nodes. A negative finding on EUS-guided FNA was only considered to be a true negative if all lymph nodes from the surgically resected region were negative for disease.

Negative Control Subjects

Under a separate study approved by the Institutional Review Board, patients with no prior or suspected malignancy, who were undergoing EUS for the evaluation of benign pancreatic or biliary disease, were recruited as negative control subjects. From each healthy control volunteer, a single FNA sample was obtained from the normal-appearing subcarinal lymph node. These lymph nodes were not enlarged, but were present in virtually all individuals and can be easily sampled with EUS-guided FNA. The same technique used for study subjects was employed, and the sample was used only for RNA isolation. No aspirate was obtained for cytology, and the specimen was assumed to be benign.

Primary Tumors

Primary tumor samples were obtained from the Hollings Cancer Center Tissue Procurement Center at the Medical University of South Carolina.

Cell Lines

All cell lines were obtained from American Type Culture Collection (Manassas, VA). Media and fetal bovine serum (FBS) were purchased from Gibco-BRL (Rockville, MD). The following cell lines and growth conditions were used: MCF-7 (RPMI medium with 10% FBS); MDA-MB-361, MDA-MB-453, and MDA-MB-231 (Leibovitz L-15 medium with 10% FBS in a non-CO2 environment); SkBr3 (McCoy 5a medium with 1.5 mM L-glutamine, 3.0 g/L glucose, 2.2 g/L sodium bicarbonate, and 10% FBS); MCF-10A (mammary epithelial growth medium with 100 ng/mL cholera toxin); Panc-1 (RPMI with 10% FBS); and HTB-174 (NCI-H441). Growth conditions for other lines were according to American Type Culture Collection recommendations.

Real-time RT-PCR

We have previously reported detailed quantitative methods for RT-PCR from EUS-guided FNA specimens in a pilot study,4which were also included in this study.5 The primers used for this study were previously described.45 Real-time RT-PCR was performed on a sequence detection system (Gene Amp 5700; PE Biosystems; Foster City, CA), and all reaction components were purchased from the same source (PE Biosystems). The standard reaction volume was 10 μL, and contained a 1× SYBR RT-PCR buffer (Molecular Probes; Eugene, OR), 3 mmol/L MgCl2, 0.2 mmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate, 0.4 mmol/L deoxyuridine triphosphate, 0.1 U UngErase enzyme, 0.25 U DNA polymerase (AmpliTaq Gold; Applied Biosystems; Foster City, CA), 0.35 μL complementary DNA template, and 50 to 900 nmol/L oligonucleotide primer. The initial steps of RT-PCR were 2 min at 50°C for uracil N-glycosylase erase activation, followed by a 10-min hold at 95°C. Cycles (total, 40 cycles) consisted of a 15-s melt at 95°C, followed by a 1-min annealing/extension at 60°C. The final step was a 60°C incubation for 1 min. All reactions were performed in triplicate. The threshold for the cycle of threshold (Ct) analysis of all samples was set at 0.5 relative fluorescence units.

Receiver Operator Characteristic Curve Analysis of Cancer-Associated Genes

Receiver operator characteristic (ROC) curve analyses of matched cytology-positive and control-negative data were performed using appropriate software (MedCalc Software; MedCalc; Mariakerke, Belgium). Sensitivity values corresponding to maximum sensitivity and 100% specificity at 95% confidence intervals (CIs) were extracted from data files.

The sample population for this study consisted of 144 lymph node aspirates obtained from 87 patients with NSCLC and 17 lymph node aspirates obtained from 17 healthy control patients. No complications occurred during EUS-guided FNA procedures. Six lymph nodes from NSCLC patients (4.5% of the total number of nodes) had inadequate amounts of messenger RNA extracted from the EUS-guided FNA aspirate and were excluded from the analysis. The final AJCC staging is shown in Table 1 .

Of the 127 evaluable lymph nodes that were obtained from NSCLC patients, 27 nodes (from 26 patients) had malignant cells on EUS-guided FNA cytology, while 100 lymph nodes (from 70 patients) had no malignant cells (ie, they were benign). Nine patients had at least one node that was cytologynegative and one node that was cytology positive. Thus, on a per patient basis, there were 26 cytology-positive patients and 61 cytology-negative patients. On a per-node basis, there were 27 cytology-positive nodes and 100 cytology-negative nodes. All nodes, including 17 that were derived from healthy patients, were analyzed by real-time RT-PCR (40 cycles) using a multigene marker panel consisting of genes previously used for the detection of NSCLC in mediastinal lymph nodes (ie, KS1/4, lunx, muc1, CK19, and CEA4), as well as one additional gene (PDEF). The PDEF gene encodes a transcriptional factor that is highly expressed in both prostate cancer,6 and breast cancer.5 Our rationale for including the PDEF gene in the NSCLC analysis was based on real-time PCR studies demonstrating elevated expression levels of this gene in NSCLC primary tumor samples relative to healthy lung tissue (Fig 1).

Gene expression was quantitated by determining ΔCt values. The ΔCt value is the difference between the Ct value for a NSCLC-associated gene and a β2-microglobin internal reference gene. High ΔCt values are correlated with low levels of gene expression, whereas low ΔCt values are correlated with high levels of gene expression. For genes such as KS1/4 and lunx, substantial differences in ΔCt values were observed between the samples from the healthy population and cytology-positive samples (Fig 2 ), suggesting that these two genes are useful markers of metastatic disease. For genes such as CEA and CK19, the differences between the two populations were less substantial due to wider distributions of gene expression in the negative control population. Using the 17 normal samples used as control negative samples and the 27 malignant lymph nodes used as control “gold standard” positives, the ROC curves for each marker gene were generated (Fig 3 ). The area under the curve (AUC) for each ROC is listed in Table 2 . The highest AUC value for any marker was that for the KS1/4 gene (0.96; 95% CI, 0.85 to 0.99), while the lowest was for the PSE gene (0.64; 95% CI, 0.47 to 0.79).

According to Henderson,7 when a disease is serious such that knowledge that the disease is absent is reassuring to the patient, and false-positive test results have serious psychological or economic consequences for the patient, threshold values corresponding to high specificity are preferable. For the determination of marker positivity in this study, we set the threshold for a given marker at a conservative value that corresponded to 100% specificity (ie, no normal control samples were positive) as determined from ROC curve analysis. We refer to this threshold value in the subsequent text as the clinical threshold. Using clinical thresholds for marker positivity, we observed that at least one cancer-associated gene was overexpressed in 25 of 27 cytology-positive samples (93%) [Table 2, Fig 4] . Interestingly, the KS1/4 gene overexpression was detected in all samples that were PCR positive, providing evidence that this marker was most sensitive for the detection of NSCLC. The gene with the second highest sensitivity was lunx (15 of 27 cytology-positive samples; 56% of nodes).

To determine whether real-time RT-PCR analysis was capable of detecting occult micrometastases, 100 cytologically negative mediastinal lymph nodes from NSCLC patients without cytologic evidence of metastatic cancer were analyzed. Twenty-four of the 100 nodes (24%) overexpressed at least one marker (Fig 5 ), of which 20 overexpressed KS1/4, 4 overexpressed PSE or CEA, 3 overexpressed lunx, while 1 overexpressed CK19 or muc1(Fig 4). Of the 61 cytology-negative patients in this analysis, a positive PCR signal was observed for 18 (30%), of whom 15 (25%) overexpressed the KS1/4 gene.

These results suggest that gene transcripts that are suggestive of metastatic disease may be present in approximately one third of cytology-negative samples (ie, micrometastases). These lymph nodes can be sampled safely and nonoperatively using EUS-guided FNA. The evaluation of mediastinal lymph nodes is a critical component of staging NSCLC patients. Lymph nodes > 1 cm in diameter as determined by CT scan are presumed to contain metastatic disease. However, when compared with surgical pathology, CT scans miss mediastinal lymph node metastases (false-negative test result) in approximately 13% of patients and incorrectly suggest their presence (false-positive test result) in approximately 50% of patients.8Because of this limitation, mediastinoscopy is accepted as the “gold standard.” Mediastinoscopy requires general anesthesia and specialized expertise to perform safely, and therefore is not universally employed. Recently, PET scanning has emerged as a noninvasive method for evaluating cancer stage. Some studies910 have suggested that the sensitivity and specificity of PET scanning may be in excess of 80 to 90%. Because PET scanning relies on the accumulation of a radioactive tracer in proportion to cellular metabolic activity, its ability to detect microscopic tumor deposits, and to distinguish between inflammation and cancer is limited.

EUS-guided FNA is a minimally invasive technique that enables the sampling of mediastinal lymph nodes without the need for general anesthesia or major surgery. Experience at multiple institutions has shown that EUS-guided FNA with cytology improves the accuracy of detection of mediastinal metastases compared to that for CT and PET scanning.8,1114 Ultimately, histologic analysis is used to determine the presence or absence of mediastinal lymph node metastases. However, survival statistics indicate clearly that a reliance on histology is inadequate. Following presumably curative resection, the 5-year survival rate for patients with pathologic stage I disease (no histologic evidence of lymph node metastases) is only 62%. For patients with metastatic disease identified in hilar lymph nodes but not mediastinal lymph nodes (stage II), the 5-year survival rate falls to only 42%.15These figures suggest that the histologic evaluation of mediastinal lymph nodes may miss metastatic disease in a large proportion of patients with NSCLC. Studies1619 have shown that serial sectioning and immunohistochemical staining increase the sensitivity of detection of metastatic disease, and that the presence of metastatic disease detected in this fashion is associated with worse survival. Although serial sectioning provides the ability to detect clinically significant metastatic disease, it is extremely time-consuming and expensive, and is not used on a routine basis.

Altered gene expression associated with malignant transformation provides an opportunity to identify the presence of malignant cells by detecting messenger RNA transcripts that would otherwise not be present in lymph nodes. For example, cytokeratin (CK) genes are normally expressed in epithelial cells but not in lymphoid cells, so the presence of CK messenger RNA in a lymph node suggests the presence of metastatic cells of epithelial origin. RT-PCR is a highly sensitive technique that allows for the detection of rare gene transcripts in tissue samples, and has been reported to be capable of detecting one cancer cell per 106 normal cells.2022 The clear advantages of using RT-PCR include minimal tissue requirements, sensitivity of detection, and potential cost efficiency.2324 Studies5,25with other cancers have shown that RT-PCR is capable of highly sensitive detection of metastases, and work in our laboratory has been instrumental in demonstrating the application of this technology to improve staging for patients with breast cancer. Similar work with lung cancer has been limited. RT-PCR has been used to detect MUC1 transcripts (a mucopolysaccharide gene associated with the respiratory epithelium) in histologically negative mediastinal lymph nodes from patients with resected NSCLC.26 More recently, RT-PCR for p53 and K-ras was shown to improve the detection of occult lymph node metastasis compared to immunohistochemistry for CKs.27

Although it has not been shown definitively for lung cancer, there is evidence to suggest that molecular staging is clinically relevant. Molecular staging has had considerable success in the management of hematologic malignancies, in which the monitoring of minimal residual disease in the peripheral blood has been used to guide therapy.2831 With melanoma, some studies3233 have suggested that RT-PCR is more sensitive than immunohistochemistry for the detection of metastatic melanoma in sentinel lymph nodes.

Until recently, specific genes that are highly expressed in NSCLC have not been identified. The gene muc1 was the first marker used for the detection of metastatic NSCLC, but there have been concerns about the specificity of its detection by RT-PCR. With respect to muc1, we found significant overlap in messenger RNA expression between normal and malignant lymph nodes, suggesting that muc1 lacks specificity in NSCLC. In contrast, the specificity of lunx appeared to be very high (Fig 1). This result is consistent with that of Iwao and colleagues,34 who were the first to show that lunx was highly expressed in NSCLC tumors but not in normal lymph nodes. These findings provide evidence that lunx expression might serve as a useful molecular marker for the detection of NSCLC lymph node metastases.

The molecular marker with the highest observed specificity for NSCLC was KS1/4, a gene that encodes a glycoprotein expressed on epithelial cells.35The protein is recognized by the monoclonal antibody Ber-EP4. Immunohistochemical staining with Ber-EP4 has shown that KS1/4 expression is specific for epithelial cells and is present on epithelial cancers (carcinomas).36 Interestingly, antibodies against Ber-EP4 have shown promise in clinical trials for colorectal cancer. In a study37 of 189 patients with resected stage III colorectal cancer, treatment with edrecolomab (an antibody to Ber-EP4) resulted in a 32% increase in overall survival compared with no treatment (p < 0.01) and decreased the tumor recurrence rate by 23% (p < 0.04). The results presented in this article raise the hypothesis that edrecolomab therapy may have activity in NSCLC patients.

A limitation of our study is that molecular marker overexpression alone is not definitive evidence of micrometastatic disease. By definition, micrometastatic disease is that which is below the level of detection of standard histologic and cytologic methods. Our overall hypothesis is that the overexpression of molecular markers in lymph nodes confers a poor prognosis and may indicate the need for systemic therapy (ie, chemotherapy). This hypothesis is now being tested in a longitudinal survival analysis of this ongoing cohort of patients.

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

This study was funded by the National Cancer Institute grant R21 CA97875–01.

Figure Jump LinkFigure 1. Real-time RT-PCR analysis of normal lung and NSCLC primary tumor samples. Real-time PCR analyses of 11 primary NSCLC tumor samples (samples 1 to 11) and 7 normal lung samples (samples 14 to 20) were performed using primer pairs for the indicated genes. Ct values for each gene were determined from triplicate reactions. ΔCt values were obtained by subtracting the mean Ct value of the β2-microglobin gene from the mean Ct value for each respective gene. Lg = large cell carcinoma; Br = bronchiolar carcinoma; E = epidermoid carcinoma; M = mesothelioma.Grahic Jump Location
Table Graphic Jump Location
Table 1. Characteristics of Patients and Lymph Nodes*
* 

+ = positive.

 

Patients whose EUS showed no evidence of metastases, but were unable to undergo surgery due to comorbid disease. These patients were considered “incompletely staged.”

Figure Jump LinkFigure 2. Real-time RT-PCR analysis of NSCLC in mediastinal lymph nodes obtained by EUS-guided FNA. Real-time PCR analyses of 17 negative control lymph nodes (left side of each matched data set; ♦), 27 cytology-positive lymph nodes (○), and 100 cytology-negative lymph nodes (right side of each matched data set; ▵) was performed using primer pairs for the indicated genes. Ct values for each gene were determined from triplicate reactions. ΔCt values were obtained by subtracting the mean Ct value of the β2-microglobin gene from the mean Ct value for each respective gene. Horizontal lines indicate the ΔCt threshold values obtained at a 100% specificity level using the ROC curve analysis described in the text. Note that the y-axis is inverted, reflecting the fact that lower ΔCt values correspond to higher levels of messenger RNA.Grahic Jump Location
Figure Jump LinkFigure 3. ROC curve analysis of molecular markers for NSCLC. Each matched data set shown in Figure 2 was analyzed by ROC curve analysis. The diagonal line corresponds to a coin-toss (AUC, 0.5).Grahic Jump Location
Table Graphic Jump Location
Table 2. Genes Associated With NSCLC Metastasis
* 

Based on ROC curve analysis at a specificity value of 100%.

 

AUC of the respective ROC.

 

ΔCt threshold value that defines 100% specificity.

Figure Jump LinkFigure 4. The frequency of gene expression in NSCLC subjects with cytology-positive mediastinal lymph nodes (black bars) and pathology-negative mediastinal lymph nodes (white bars). Real-time RT-PCR analyses of cytology-positive mediastinal lymph nodes (27 lymph nodes) and cytology-negative mediastinal lymph nodes (100 lymph nodes) were performed as described in the “Materials and Methods” section using primer pairs for the indicated genes. Marker positivity was determined based on ΔCt threshold values. The y-axis shows the fraction of subjects who are positive for each respective gene.Grahic Jump Location
Figure Jump LinkFigure 5. Positivity distribution of the gene expressed in NSCLC subjects with cytology-positive mediastinal lymph nodes (black bars) and pathology-negative mediastinal lymph nodes (white bars). Real-time RT-PCR analyses of cytology-positive mediastinal lymph nodes (27 lymph nodes) and cytology-negative mediastinal lymph nodes (100 lymph nodes) were performed as described in the “Materials and Methods” section using primer pairs for the indicated genes. Marker positivity was determined based on ΔCt threshold values. The y-axis shows the fraction of nodes that are positive for various numbers of marker combinations.Grahic Jump Location
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Pallisgaard, N, Clausen, N, Schroder, H, et al Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript.Genes Chromosomes Cancer1999;26,355-365. [CrossRef] [PubMed]
 
Bostick, PJ, Chatterjee, S, Chi, DD, et al Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.J Clin Oncol1998;16,2632-2640. [PubMed]
 
Zippelius, A, Kufer, P, Honold, G, et al Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.J Clin Oncol1997;15,2701-2708. [PubMed]
 
Iwao, K, Watanabe, T, Fujiwara, Y, et al Isolation of a novel human lung-specific gene, LUNX, a potential molecular marker for detection of micrometastasis in non-small-cell lung cancer.Int J Cancer2001;91,433-437. [CrossRef] [PubMed]
 
Perez, MS, Walker, LE Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker.J Immunol1989;142,3662-3667. [PubMed]
 
Latza, U, Niedobitek, G, Schwarting, R, et al Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelial.J Clin Pathol1990;43,213-219. [CrossRef] [PubMed]
 
Schwartzberg, LS Clinical experience with edrecolomab: a monoclonal antibody therapy for colorectal carcinoma.Crit Rev Oncol Hematol2001;40,17-24. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Real-time RT-PCR analysis of normal lung and NSCLC primary tumor samples. Real-time PCR analyses of 11 primary NSCLC tumor samples (samples 1 to 11) and 7 normal lung samples (samples 14 to 20) were performed using primer pairs for the indicated genes. Ct values for each gene were determined from triplicate reactions. ΔCt values were obtained by subtracting the mean Ct value of the β2-microglobin gene from the mean Ct value for each respective gene. Lg = large cell carcinoma; Br = bronchiolar carcinoma; E = epidermoid carcinoma; M = mesothelioma.Grahic Jump Location
Figure Jump LinkFigure 2. Real-time RT-PCR analysis of NSCLC in mediastinal lymph nodes obtained by EUS-guided FNA. Real-time PCR analyses of 17 negative control lymph nodes (left side of each matched data set; ♦), 27 cytology-positive lymph nodes (○), and 100 cytology-negative lymph nodes (right side of each matched data set; ▵) was performed using primer pairs for the indicated genes. Ct values for each gene were determined from triplicate reactions. ΔCt values were obtained by subtracting the mean Ct value of the β2-microglobin gene from the mean Ct value for each respective gene. Horizontal lines indicate the ΔCt threshold values obtained at a 100% specificity level using the ROC curve analysis described in the text. Note that the y-axis is inverted, reflecting the fact that lower ΔCt values correspond to higher levels of messenger RNA.Grahic Jump Location
Figure Jump LinkFigure 3. ROC curve analysis of molecular markers for NSCLC. Each matched data set shown in Figure 2 was analyzed by ROC curve analysis. The diagonal line corresponds to a coin-toss (AUC, 0.5).Grahic Jump Location
Figure Jump LinkFigure 4. The frequency of gene expression in NSCLC subjects with cytology-positive mediastinal lymph nodes (black bars) and pathology-negative mediastinal lymph nodes (white bars). Real-time RT-PCR analyses of cytology-positive mediastinal lymph nodes (27 lymph nodes) and cytology-negative mediastinal lymph nodes (100 lymph nodes) were performed as described in the “Materials and Methods” section using primer pairs for the indicated genes. Marker positivity was determined based on ΔCt threshold values. The y-axis shows the fraction of subjects who are positive for each respective gene.Grahic Jump Location
Figure Jump LinkFigure 5. Positivity distribution of the gene expressed in NSCLC subjects with cytology-positive mediastinal lymph nodes (black bars) and pathology-negative mediastinal lymph nodes (white bars). Real-time RT-PCR analyses of cytology-positive mediastinal lymph nodes (27 lymph nodes) and cytology-negative mediastinal lymph nodes (100 lymph nodes) were performed as described in the “Materials and Methods” section using primer pairs for the indicated genes. Marker positivity was determined based on ΔCt threshold values. The y-axis shows the fraction of nodes that are positive for various numbers of marker combinations.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1. Characteristics of Patients and Lymph Nodes*
* 

+ = positive.

 

Patients whose EUS showed no evidence of metastases, but were unable to undergo surgery due to comorbid disease. These patients were considered “incompletely staged.”

Table Graphic Jump Location
Table 2. Genes Associated With NSCLC Metastasis
* 

Based on ROC curve analysis at a specificity value of 100%.

 

AUC of the respective ROC.

 

ΔCt threshold value that defines 100% specificity.

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Pallisgaard, N, Clausen, N, Schroder, H, et al Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript.Genes Chromosomes Cancer1999;26,355-365. [CrossRef] [PubMed]
 
Bostick, PJ, Chatterjee, S, Chi, DD, et al Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.J Clin Oncol1998;16,2632-2640. [PubMed]
 
Zippelius, A, Kufer, P, Honold, G, et al Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.J Clin Oncol1997;15,2701-2708. [PubMed]
 
Iwao, K, Watanabe, T, Fujiwara, Y, et al Isolation of a novel human lung-specific gene, LUNX, a potential molecular marker for detection of micrometastasis in non-small-cell lung cancer.Int J Cancer2001;91,433-437. [CrossRef] [PubMed]
 
Perez, MS, Walker, LE Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker.J Immunol1989;142,3662-3667. [PubMed]
 
Latza, U, Niedobitek, G, Schwarting, R, et al Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelial.J Clin Pathol1990;43,213-219. [CrossRef] [PubMed]
 
Schwartzberg, LS Clinical experience with edrecolomab: a monoclonal antibody therapy for colorectal carcinoma.Crit Rev Oncol Hematol2001;40,17-24. [CrossRef] [PubMed]
 
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