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Detection of Telomerase Expression in Mediastinal Lymph Nodes of Patients with Lung Cancer

Division of Gastroenterology, Digestive Disease Center; Department of Surgery, Division of Pulmonary Medicine and Critical Care, Medical University of South Carolina and Ralph H. Johnson Veterans Administration Medical Center; Department of Pathology; and Department of Radiology, Medical University of South Carolina, Charleston, South Carolina

Correspondence and requests for reprints should be addressed to Michael B. Wallace, M.D., M.P.H., Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 210 CSB, P.O. Box 250327, Charleston, SC 29425. E-mail: wallacem{at}musc.edu

	ABSTRACT

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Mediastinal lymph nodes are the most common site of tumor spread in non–small cell lung cancer (NSCLC). We hypothesized that micrometastatic disease could be detected by reverse transcription-polymerase chain reaction (RT-PCR) for expression of human telomerase reverse transcriptase (hTERT) in mediastinal lymph nodes and that a minimally invasive technique (endoscopic ultrasound-guided fine-needle aspiration [EUS-FNA]) is capable of sampling lymph nodes for PCR analysis without surgery. Mediastinal lymph nodes were sampled with EUS-FNA in patients with NSCLC and negative control subjects undergoing EUS for benign disease. Total RNA was harvested from samples, and RT-PCR was performed to detect telomerase gene expression. RNA was available from 87 of 100 lymph node aspirates from 39 patients with NSCLC and from 12 negative control patients. hTERT was expressed in 0 of 14 negative control lymph nodes in 18 of 57 pathologically negative lymph nodes from cancer patients and in 10 of 16 pathologically positive lymph nodes (p < 0.05). Five of 18 (28%) patients with no pathologically evident mediastinal disease expressed telomerase in at least one lymph node. Minimally invasive EUS-FNA with RT-PCR is capable of detecting expression of cancer specific mRNA in lymph nodes. Approximately one-third of pathologically negative mediastinal lymph nodes in NSCLC patients express hTERT mRNA. The clinical significance of this observation is yet to be determined.

Key Words: non–small cell lung cancer • staging • biomarkers • endoscopic ultrasound • micrometastases

Non–small cell carcinoma of the lung (NSCLC) is the most common cause of cancer death in the United States (1). The most common sites of tumor metastases are the lymph nodes in the mediastinum. Patients with involvement of mediastinal lymph nodes are at higher risk of failure from surgical resection alone and are generally treated with radiochemotherapy (2, 3). Non–invasive imaging methods such as computed tomography (CT) and positron emission tomography have imperfect accuracy for detecting mediastinal lymph node involvement but may be useful in guiding where to obtain pathologic confirmation. Approximately 20% of patients with a "normal" CT of the mediastinum eventually have malignant mediastinal adenopathy detected at surgery (4). Better preoperative staging may reduce this rate of unnecessary surgical exploration and morbidity.

Endoscopic ultrasound (EUS) is a minimally invasive guidance mechanism that is capable of sampling mediastinal lymph nodes by a transesophageal approach by fine-needle aspiration and can be performed under conscious sedation on an outpatient basis. Others and we have shown that EUS-guided fine-needle aspiration (EUS-FNA) is a safe and accurate method of staging lymph nodes in the mediastinum of NSCLC patients (5–8).

There is growing evidence that early "micrometastases" are present in lymph nodes that cannot be detected with standard cytologic or histologic methods. Many of these tumor cells can be detected using molecular analysis for lung epithelial cell–specific proteins or mRNA in lymph nodes. Detection of these micrometastases using molecular techniques such as immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to improve the detection of metastatic epithelial cells in the lymph nodes and bone marrow of patients with lung carcinoma (9–14). These studies of micrometastases suggest that up to 50% of patients with histologically normal lymph nodes have micrometastases detected by molecular techniques. These also demonstrate that the presence of micrometastases has a significant impact on long-term survival even among patients with pathologically "normal" lymph nodes.

Although those studies used epithelial markers, other markers of malignant transformation also show promise. For example, K-ras codon 12 mutations have been reported in up to 50% of adenocarcinomas of the lung (15), and P53 mutations have been reported in 24–50% of primary tumors from patients with all histologic types of NSCLC (16–18). Telomerase has been reported to be expressed in up to 93% of all lung cancers, making it a potentially useful marker for different histologic types of lung cancer (19, 20). Increased expression of telomerase is not limited to one histologic type of lung cancer. In one study, analysis of 136 primary tumors from patients with NSCLC identified telomerase activity in 78.6% compared with 4% of adjacent normal tissues (21). More recently, telomerase activity was identified in 85% of NSCLC specimens, and the presence of telomerase activity in these specimens correlated with increased cell proliferation rates and advanced pathologic stage (22, 23). Finally, detection of telomerase in cells obtained by bronchial lavage has been shown to be a sensitive and specific test for the presence of lung cancer (24, 25).

The purpose of this study was determine whether EUS-FNA combined with RT-PCR could detect micrometastatic disease in mediastinal lymph nodes of patients with NSCLC.

	METHODS

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The study was approved by the Institutional Review Board of the Medical University of South Carolina, and all patients completed informed consent. Patients were eligible for the study if they had pathologically proven NSCLC and were considered 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 prior history of other malignancy other than basal cell carcinoma of the skin, an age of less than 18 years, were unfit for surgical resection due to concurrent cardiac disease, insufficient pulmonary reserve, or refusal to consent.

All patients were reviewed at a multidisciplinary thoracic tumor board. A helical CT was performed of the chest and upper abdomen. If no distant metastases (contralateral lung, abdomen, etc.) were found, the patient underwent EUS-FNA. EUS-FNA was performed under conscious sedation with midazolam (0–5 mg) and meperidine (0–200 mg). The entire liver, left adrenal gland, celiac trunk, and mediastinum were surveyed for focal, hypoechoic lymph nodes, or tumor metastases. All patients had some detectable lymph nodes in the subcarinal region, as is common in normal patients. This lymph node, as well as any other visualized lymph nodes in the aortopulmonary window (American Joint Committee on Cancer [AJCC] level 5), inferior mediastinum (AJCC level 8–9), and superior mediastinum (AJCC levels 2 and 4), was sampled with FNA for cytology and to obtain a sample for RT-PCR (Figure 1) . The site sampled depended on which site would give the most advanced stage. 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.

View larger version (139K): [in this window] [in a new window]   Figure 1. (A) Technique for EUS-FNA of mediastinal lymph nodes. The echoendoscope is located in the mid esophagus. A 22-gauge needle is passed under real-time ultrasound imaging into a lymph node located in the aortopulmonary window (AP window). After entry into the lymph node, an occluding stylet is removed, and material is aspirated with suction and to-and-fro movement of the needle. (B) Diagram of the mediastinal lymph node stations with numbering according to the American Thoracic Society. EUS can typically access stations 2 R/L, 4 R/L, 5 (aortopulmonary window), 7 (subcarina), 8, and 9. AO = aorta; PA = pulmonary artery.

Each lymph node was sampled with a minimum of four needle passes unless a definitive diagnosis of malignancy was rendered before the fourth pass. Each fine-needle sample was expressed, using a 10-cc air filled syringe, onto a separate glass slide, and a direct smear was made by an onsite 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). Onsite evaluation of smears was performed to assess cellular adequacy. Final interpretation of all of the material consisted of reviewing the slides prepared onsite and stained later and examination of thin-layer cytology or cytospin/cell block material prepared from the cellular material kept in the Hank's solution.

A pathologic diagnosis of malignancy was obtained if the cytology specimen revealed cells consistent with malignancy. Patients who had no evidence of mediastinal lymph node metastases by EUS-FNA cytology underwent surgical exploration, including lymphadenectomy of all mediastinal lymph node stations. Each lymph node and the primary tumor were evaluated with histologic methods using hemotoxylin and eosin staining. Because all lymph nodes from a region (e.g., subcarina) were resected in the surgical specimen, it was assumed that the lymph node sampled by EUS-FNA in that region was contained within the group of resected nodes. A negative EUS-FNA was only considered a true negative if all lymph nodes from the surgical resected region were negative.

Patients without cytology-proven mediastinal metastases were considered for surgical resection. Because of the extensive preoperative staging (with CT, EUS, and in some cases, positron emission tomography), we performed mediastinoscopy or thoracoscopy only if a lymph node appeared malignant (based on CT or positron emission tomography) but was inaccessible to EUS-FNA. All other patients underwent thoractomy with complete mediastinal lymph node resection.

Negative Control Subjects
Under a separate institutional review board–approved study, patients with no prior or suspected malignancy, who were undergoing EUS for evaluation of benign pancreatic or biliary disease, were recruited ("negative control subjects"). Virtually all persons have lymph node tissue in the subcarina, which is readily identified with EUS. For each negative control, a single FNA sample was obtained for RNA using the identical technique as was used for patients. No specimen was obtained for cytology, and the specimen was assumed to be benign.

RT-PCR
At the time of EUS-FNA, each lymph node was also sampled for RT-PCR after completion of the three to four passes for cytology, except each needle pass was performed for 2 minutes with 10 cc of suction applied to the needle. Approximately 0.5 cc of nodal aspirate was obtained with this method. Aspirated cells were immediately placed in a sterile tube containing ethylenediaminetetraacetic acid. After centrifugation, total cellular RNA was harvested by adding RNAzol (Teltest. Inc., Friendswood, TX) to the cell pellet followed by phenol/chloroform extraction and ethanol precipitation. Average RNA yield from each sample was 7.8 ± 3.3 µg. RNA was then further purified with an RNeasy column (Qiagen, Valencia, CA), and equal amounts (1 µg) were used in reverse-transcription reactions with avian myeloblastosis virus-reverse transcriptase according to the manufacturer's instructions (Promega, Madison, WI). Equal amounts of the complimentary DNA (2 µl) products were used in hot-start PCR with primers based on the human telomerase reverse transcriptase (hTERT) sequence: hTERT (forward, bp 1790–1811) 5'-AGTGTCTGGAGCAAGTTGCAAA-3' and hTERT (reverse, bp 1970–1987) 5'-CGTTCTGGCTCCCACGAC-3'. The amplification reactions were performed at 95°C for 10 minutes, 92°C for 30 seconds, 60°C for 1 minute, and 70°C for 1 minute for a total of 40 cycles. The amplified fragments were separated in 2% agarose gels and visualized by ethidium bromide staining. As control subjects, rRNA levels in each sample were determined by amplifying equal amounts of complimentary DNA (1 µl) using these primers: forward 5'-CTCCGGTCCGTGCCTCCAAG-3' and reverse 5'-CAGAGAATAGCCTGTCTTCAGTC-3'. Amplification reactions were performed at 95°C for 10 minutes, 92°C for 30 seconds, 57°C for 1 minute, and 72°C for 1 minute for a total of 30 cycles. RT-PCR assays were determined as positive or negative by independently by two investigators (A.B. and S.M.) blinded to the clinical and histopathologic information.

Statistical Analysis
The prevalence of telomerase overexpression in each group (negative) of control subjects (pathologically negative and pathologically positive NSCLC patients) was tabulated and compared using Fisher's exact test.

	RESULTS

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A total of 83 lymph nodes from 44 patients with NSCLC and 17 lymph nodes from 14 negative control subjects (patients undergoing EUS for benign pancreatic or biliary disease with no history or suspicion of cancer) were evaluated by EUS-FNA and RT-PCR. A summary of the pathologic diagnoses is shown in Table 1 . RNA sufficient for telomerase evaluation was obtained from 87 of 100 (87%) of lymph nodes and is the subject of this study (73 lymph nodes from 39 NSCLC patients and 14 lymph nodes from 12 negative control patients).

View larger version (30K): [in this window] [in a new window]   Figure 2. RT-PCR analysis of hTERT expression lymph node specimens obtained by EUS-FNA. The mRNA levels of hTERT (upper panel) and rRNA (control, lower panel) were determined using RT-PCR. Fragments were resolved on 2% agarose gels and visualized by ethidium bromide staining. Lanes 1 and 8 show expression in the positive control, a lung adenocarcinoma cell line. Lanes 2 and 3 show the negative control, distilled water rather than cDNA in PCR reaction mixture. Lanes 6, 7, and 9–14 contain specimens from patients with benign disease. Of note, the sample in lane 7 came from a patient with granulomatous disease. Samples 3–5 were obtained from patients with known lung cancer. Cytologic analysis of the samples obtained from the same lymph nodes as lanes 4 and 5 was negative for the presence of malignant cells, whereas cytologic analysis of three was positive. MW = molecular weight.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Using a minimally invasive method of sampling, we have identified evidence of telomerase expression in both pathologically positive and negative mediastinal lymph nodes from patients with NSCLC and the absence of telomerase expression in all negative control lymph nodes. Almost one-third of pathologically negative lymph nodes express telomerase. The clinical relevance of these findings remains to be determined by survival studies, but the pattern of expression suggests that analysis of hTERT expression may prove useful in detecting micrometastatic disease.

Standard therapies for patients with NSCLC include surgery, chemotherapy, and radiation therapy, and the stage of disease, especially the presence of mediastinal and distant metastases, dictates choice of therapy. Surgery is generally performed on patients in whom disease is confined to the lung and hilar lymph nodes (stages I and II). Patients with mediastinal lymph nodes metastases (stage III) are rarely cured by surgery, and combined chemoradiotherapy is most appropriate (26).

EUS-FNA is a relatively new and minimally invasive method of cancer staging. EUS was developed to stage gastrointestinal tumors. However, its access to mediastinal lymph nodes and excellent safety, cost, and convenience (outpatient) make it well suited for staging the mediastinum in NSCLC patients. EUS-FNA has been shown to be an accurate and safe method of evaluating patients with enlarged mediastinal lymph nodes detected by CT scan (5–8). EUS can be performed within approximately 30 minutes under conscious sedation, on an outpatient basis, with a procedural risk of major complications of less than 0.5% (27). Tissue samples obtained by EUS-FNA are typically analyzed by standard cytopathology techniques (thin prep, Papanicolau staining), but can also be analyzed for molecular markers of cancer cells that may not be detectable by cytologic methods. Cost-effectiveness studies suggest that EUS-FNA is more cost effective than other surgical methods of lymph node sampling such as mediastinoscopy (28, 29), although no randomized control subject trials have compared these modalities.

Ultimately histologic analysis is used to determine the presence or absence of mediastinal lymph node metastases. Survival statistics indicate clearly that reliance on histology is inadequate. After 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% (30). These figures suggest that histologic evaluation of mediastinal lymph nodes misses metastatic disease in a large proportion of patients with NSCLC.

Other studies 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 (31–34). Although this technique 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 mRNA transcripts that would otherwise not be present in lymph nodes. Furthermore, metastatic spread produces ectopic expression of tissue-specific genes. RT-PCR is highly sensitive for detection of rare gene transcripts in tissue samples and has been reported to be capable of detecting one cancer cell per 10⁶ normal cells (35–37). Clear advantages include minimal tissue requirements, sensitivity of detection, and potential cost efficiency (38, 39). Studies with other cancers have shown that RT-PCR is capable of highly sensitive detection of metastases. Similar work with lung cancer has been limited. RT-PCR has been used to detect MUC1 transcripts (a mucopolysaccharide gene associated with respiratory epithelium) in histologically negative mediastinal lymph nodes from patients with resected NSCLC (9).

Although it has not been shown definitively for lung cancer, there is considerable evidence to suggest that molecular staging is clinically relevant. Molecular staging has had considerable success in hematologic malignancies, where monitoring of minimal residual disease in the peripheral blood has been used to guide therapy (40–44). Telomerase represents a unique target for molecular staging in lung cancer. Not only is telomerase expressed in the majority of lung cancers, but all histologic types of lung cancer have been shown to express it; expression in NSCLC has been shown to be independently predictive of a worse prognosis (45). However, recent studies in animal models of lung inflammation have put into question the specificity of telomerase as a marker of malignancy. Injury to rat lungs caused by both silica and bleomycin has been shown to be associated with the development of increased telomerase activity in lung fibroblasts (46, 47), raising the question as to whether telomerase might be increased in lungs inflamed by other agents such as cigarette smoke. Yet in two studies of patients with known NSCLC, analysis of matched pairs of tumor and normal tissue showed no evidence of telomerase gene expression in any of the normal tissue specimens (total of 218 samples for both studies) (45, 48). Evaluation of lymph nodes for the presence of metastatic disease using telomerase remains controversial. Although high levels of telomerase activity in hyperplastic lymph nodes and tonsils have been reported (25), others have found a significant difference in telomerase activity between benign and malignant lymph nodes, with high levels of activity in malignant and only low levels of activity in benign nodes (23). Similarly, in our study, none of the lymph node biopsies from patients with benign disease showed hTERT expression, but the majority of cytologically positive lymph nodes (63%) did have evidence of expression.

Activation of the telomerase gene has been analyzed using a variety of techniques. Most commonly, the activity of the telomerase is measured by using telomerase harvested from the cell extract to add telomeric repeats to the end of a primer followed by amplification of the product using the polymerase chain reaction. However, this technique requires in the order of 10³ cells (49). Because of the small size of the samples and potentially the few malignant cells in samples obtained by EUS, we instead chose to analyze our specimens using RT-PCR to detect expression of the hTERT. hTERT encodes a protein that is the rate-limiting determinant of the enzymatic activity of human telomerase, and expression appears to develop early in the course of tumorigenesis (50). In addition, expression of this gene has been shown to correlate with telomerase activity (51, 52), metastatic disease (53), and shorter survival in NSCLC (52, 54, 55). hTERT expression has been shown to correlate better with survival than the telomere repeat amplification protocol assay (56). Finally, hTERT has been shown to maintain cell survival and proliferation via both telomerase enzymatic activity-dependent telomere lengthening and enzymatic activity-dependent intermolecular interactions involving p53 and poly(ADP-ribose) polymerase (57).

A limitation of our study is the fact that only 10 of the 16 pathologically malignant lymph nodes had evidence of hTERT. Although hTERT was highly specific (none found in negative control lymph nodes), it has limited sensitivity. We are unable to determine whether this is due to sampling error within the lymph node (which could be overcome by whole lymph node evaluation) or whether other markers or a combination of markers is needed to increase sensitivity. The evaluation of other markers and their survival implications are the subjects of active study. Other limitations of EUS-FNA include inability to sample lymph nodes in the anterior mediastinum (American Thoracic Society levels 2, 4, and 6) and a lack of widespread availability. The ability to determine telomerase or other gene expression in FNA samples may be less than from whole lymph node samples. The advantage of FNA, however, is the ability to obtain these specimens using nonoperative, minimally invasive techniques. We are however comparing, in ongoing studies, the accuracy of FNA versus whole node specimens for detection of genetic markers of micrometastases.

We have shown that detection of tumor specific genes in FNA samples of pathologically normal lymph nodes is feasible with minimally invasive techniques. In addition, these data suggest that detection of hTERT gene expression in lymph nodes may identify micrometastatic disease when normal cytologic and histologic techniques fail. Further study is ongoing to determine the clinical significance of expression of hTERT in pathologically negative lymph nodes on survival and whether chemoradiotherapy is beneficial to patients with RT-PCR evidence but not histologic evidence of mediastinal lymph node metastases (RT-PCR stage III).

	FOOTNOTES

 
Supported by National Institutes of Health grant R21/R33 CA97875–01.

Received in original form November 7, 2002; accepted in final form February 17, 2003

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