Journal of Thoracic Oncology:
MAGE qPCR Improves the Sensitivity and Accuracy of EBUS-TBNA for the Detection of Lymphatic Cancer Spread
Cucuruz, Beatrix MD*,†; Dango, Sebastian MD†; Jurinovic, Vindi MSc‡; Mayer, Olga†; Follo, Marie PhD§; Böhm, Joachim MD║; Freudenberg, Nikolaus MD║; Elze, Mirjam MD†; Sienel, Wulf MD†; Klein, Christoph A. MD*; Passlick, Bernward MD†; Polzer, Bernhard MD*
*Department of Pathology, University of Regensburg, Regensburg, Germany
†Department of Thoracic Surgery, University Medical Center Freiburg, Freiburg, Germany
‡Institute for Medical Informatics, Biometry and Epidemiology, University of Munich, Munich, Germany
§Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany
║Department of Pathology, University Hospital Freiburg, Freiburg, Germany
Disclosure: Beatrix Cucuruz, MD, Sebastian Dango, MD, and Bernhard Polzer, MD, report that their employing organizations received funding from the Deutsche Krebshilfe for this project. Christoph A. Klein, MD, reports that funding was received from the Bavarian State Ministry of Science, Research and Art (BayGene). The other authors declare no conflicts of interest.
Address for correspondence: Bernhard Polzer, MD, Chair of Experimental Medicine and Therapy Research, Department of Pathology, University of Regensburg, 93053 Regensburg, Germany. E-mail: email@example.com and Bernward Passlick, MD, Department of Thoracic Surgery, University Medical Center Freiburg, 79106 Freiburg, Germany. E-mail: firstname.lastname@example.org
B. Cucuruz is currently at Department of General, Thoracic and Vascular Surgery, Klinikum Landshut, 84034 Landshut, Germany.
W. Sienel is currently at Department of Cardio-Thoracic Surgery, Klinikum Augsburg, 86156 Augsburg, Germany.
B. Cucuruz and S. Dango contributed equally to this work.
Introduction: Microscopic examination of histologic slides or cytologic specimens of mediastinal lymph node samples obtained by diagnostic mediastinoscopy or endobronchial ultrasound-guided fine-needle aspiration (EBUS-TBNA) is routinely used for the staging of lung cancer patients. Therefore, we explored whether the detection of tumor-associated mRNA in lymph node samples from patients with suspected lung cancer adds diagnostic accuracy to conventional histopathological staging.
Methods: We examined 202 lymph nodes obtained by EBUS-TBNA or mediastinoscopy from 89 patients with lung cancer. Lymph node samples from patients with nonmalignant disease were available as controls (60 samples from 31 patients). Real-time quantitative mRNA analysis was performed for melanoma antigen-A genes (MAGE-A 1–6, MAGE-A 12) using a LightCycler 480 instrument.
Results: MAGE transcript levels in control and cancer patients differed widely, and the 95% confidence interval served to define the threshold between negative and positive samples. MAGE 1 to 6 transcripts were detected in 35 of 122 (28.7%) lymph nodes obtained by EBUS-TBNA and 16 of 80 (20.0%) lymph nodes obtained by mediastinoscopy. MAGE 12 transcripts were detected in 10 of 122 (8.2%) lymph nodes obtained by EBUS-TBNA and 9 of 80 (11.3%) lymph nodes obtained by mediastinoscopy. Although the accuracy of histopathological diagnosis after EBUS-TBNA and mediastinoscopy was 69.6% and 84.1%, respectively, it increased to 81.2% and 86.4%, respectively, when combined with MAGE-quantitative polymerase chain reaction.
Conclusions: The combination of EBUS-TBNA and MAGE-quantitative polymerase chain reaction increases the accuracy of tumor cell detection to the level seen with mediastinoscopy.
Lung cancer is the leading cause of tumor-related deaths worldwide. The 5-year survival rate ranges from 3% to 89% depending on the extent of tumor spread.1 Surgery remains the cornerstone of early stage non-small cell lung cancer treatment, and its indication highly depends on the accuracy of mediastinal staging. Patients with ipsilateral lymph node metastasis are treated with an multimodal approach including surgery, whereas patients with contralateral mediastinal lymph node involvement are addressed by primary radiochemotherapy or chemotherapy alone as a first-line treatment.1,2 Therefore, adequate staging is highly important to define accurate treatment strategies for patients with non-small cell lung cancer.
At present, mediastinoscopy is most often used to rule out or confirm mediastinal lymph node involvement. Overall mortality ranges from 0% to 0.08%3,4 in large series, whereas complications occur in up to 3% of cases, primarily described as massive hemorrhage after injury to the great vessels (0.4%)5 or palsy of the left recurrent laryngeal nerve (1–2%).6 Hence, it is an invasive diagnostic technique and less invasive staging methods, such as the endobronchial ultrasound-guided fine-needle aspiration (EBUS-TBNA), may be indicated. EBUS-TBNA is a minimally invasive method to examine mediastinal and hilar lymph nodes with a reported sensitivity of 79 to 99%.7–11 However, the negative predictive value (60–99%) seems to be lower than for mediastinoscopy (80–99%), and therefore, a confirmation of negative EBUS-TBNA findings by mediastinoscopy has been recommended.12
To improve the sensitivity of the preoperative diagnostic procedures, especially EBUS-TBNA, we looked for molecular markers to detect disseminated tumor cells in lymph node samples. Recently, we established a quantitative polymerase chain reaction (qPCR) assay to detect transcripts of cytokeratin 19, a well-known marker for epithelial cancer cells. We showed that cytokeratin 19 is detected in 100% of EBUS-TBNA lymph node samples, excluding its diagnostic use. This lack of specificity of cytokeratin 19 assays may be explained by contamination of the samples with epithelial cells from the bronchial tubes.13 Other highly sensitive markers such as ks1/4 (EpCam, CD326) or lunx (also known as palate, lung, and nasal epithelium carcinoma-associated gene, PLUNC) are also expressed in normal bronchial epithelium and therefore might be unsuitable for EBUS-TBNA.14 However, a highly specific class of markers to detect disseminated tumor cells are the melanoma antigens (MAGE). The MAGE proteins belong to the large family of human tumor-associated antigens recognized by T cells and are known to be expressed in a large variety of neoplasms but not in normal tissues with the notable exception of testis.15,16 Because of this they are also known as cancer-testis antigens.17 Recently, we could show that MAGE transcripts detected in bone marrow, blood, and lymph nodes are a suitable marker for the detection of disseminated tumor cells.18–20 In primary lung cancers, MAGE expression has been reported to range between 30% and 85%.19,21–25 To capture the various MAGE transcripts with high probability, we decided to use primers that amplify MAGE 1 through MAGE 6, which are highly homologous.26 In addition, to detect MAGE 12 mRNA, which is frequently expressed in lung cancer,18,19 but poorly amplified by the universal primers, we added MAGE 12-specific primers.
Using this molecular biological approach, we investigated the presence of disseminated tumor cells in EBUS-TBNA samples and mediastinoscopic biopsies for a refinement of the staging procedure.
PATIENTS AND METHODS
Patients and Sample Preparation
The study population consisted of 120 patients, which were screened and prospectively included in the trial. Eighty-nine patients had a primary lung cancer (clinical stages IIA–IIIB) and 31 patients presented with nonmalignant diseases (sarcoidosis, tuberculosis, anthracosilicosis, or reactive lymphadenopathy). Study population’s age ranged from 33 to 88 years. All patients were treated in the Department of Thoracic Surgery at the University Medical Center Freiburg between December 2007 and May 2009 after giving informed consent. The study was approved by the Ethical Committee of the University Freiburg (ethics vote 168/04). Consecutive patients routinely underwent staging with high-resolution spiral computed tomography-scan and bronchoscopy followed by EBUS-TBNA and/or mediastinoscopy. For EBUS-TBNA, we used an Olympus ViziShot system (Olympus Ltd, Tokyo, Japan) equipped with an ultrasonic 7.5-mHz longitudinal transducer. A 21-gauge needle was used for 3 to 5 needle passes for each lymph node, as described by Herth et al.9 Needles were cleaned between different lymph nodes according to a specific cleaning procedure as given by the manufacturer and then reused. The specimen was judged visually by the surgeon and then analyzed directly using liquid cytology and microscopy. In case of negative lymph node samples, patients were primarily subjected to thoracotomy including lymph node extirpation. Pathological assessment of surgically removed lymph nodes was used as a benchmark. The samples were taken from mediastinal lymph node stations 4R, 4L, and 7 by EBUS-TBNA and 2R, 2L, 4R, 4L, and 7 by diagnostic mediastinoscopy. The detection of tumor cells in ipsilateral lymph nodes classified the patients as pN2 stage, whereas detection in contralateral lymph nodes classified the patients as pN3 stage. The sixth edition of the tumor, node, metastasis classification was used.27
RNA Preparation and cDNA Synthesis
Before isolation, mediastinoscopy samples were frozen in RNA later (QIAGEN, Hilden, Germany) at –20°C and before use were ground with a mortar before transferring them to RLT buffer (QIAGEN). EBUS-TBNA samples were directly collected in RLT buffer with β-mercaptoethanol and stored at –80°C. Subsequently, total RNA was extracted from the homogenate according to the manufacturer’s protocol (RNeasy mini kit, QIAGEN). RNA concentration was measured using a spectral photometer. cDNA was prepared in 20 µl of reactions using 500 ng of total RNA. All reagents were obtained from a commercially available cDNA synthesis kit (1st strand cDNA synthesis for RT-PCR [AMV], Roche Diagnostics, Mannheim, Germany). The outcome was tested with a control—PCR for the β-actin gene. Only positive samples were further analyzed.
The human colon cancer cell line HT29 has abundant expression of MAGE-A transcripts and was used as a positive control for the qPCR assay. The cell line was maintained in Dulbecco’s modified Eagle’s medium (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The cells were harvested when they became subconfluent.
For the first round of PCR (preamplification), we used 100 µl of reactions containing 1 µl of random-primed cDNA, 10 µl of 103 PCR buffer (200 mM Tris [pH 8.4], 500 mM KCl), 2 µl of 10 mM dNTP, 3 µl of 50 mM MgCl2, 5 µl of 5 µM each of the outer primer, and 0.6 U Taq DNA polymerase (Invitrogen, Darmstadt, Germany). The cycling parameters were as follows: initial denaturation at 95°C for 5 minutes, followed by 15 cycles (20 cycles for MAGE 12) of 95°C for 30 seconds, 60°C for 45 seconds, and 72°C for 45 seconds. The final extension incubation was performed at 72°C for 10 minutes. After the first PCR, qPCR was performed in a LightCycler 480 instrument (Roche Diagnostics). Each 20 µl of qPCR contained 2 µl of the first reaction, 2 µl of 5 µM each of the inner primer and 14 µøl of SYBR Green I master (Roche Diagnostics) containing SYBR green, dNTPs, MgCl2, and reaction buffer as described in the manufacturer’s data sheet. Cycling parameters were 5 minutes at 95°C for initial activation of the enzyme, 15 seconds at 95°C for denaturation, 10 seconds at 61°C for annealing, and 20 seconds at 72°C for elongation for 40 cycles. After completion of the reaction, the PCR products were subjected to a melting curve analysis spanning the temperature range from 65 to 95°C with a ramping rate of 0.1°C/sec. The specificity of the amplification was further confirmed by electrophoresis on 2% ethidium bromide-stained agarose gels.
The combination of primers MMRP3 and MMRP4 was used for the gene expression of all MAGE 1 to 6 genes together, as described by Park et al.26 For MAGE 12 a specific primer was used, because of its relatively high expression in lung cancer.18 The sequences of all oligonucleotide primers are listed in Supplementary Table 1. For the quantitative analysis of the reference housekeeping gene porphobilinogen desaminase (PBGD), specific primers were not added to the MAGE preamplification but only for the qPCR, that is., PBGD was measured in an effective dilution of 1:10 of the initial cDNA. The samples were each measured in triplicate, a negative control was also included for each sample, and an internal standard curve was measured in each run.
Calculations and Statistical Analysis
The quantification of gene expression was based on the cycle number at which the fluorescence of a sample rises above the background fluorescence (crossing point) and was calculated by a standardized algorithm of the software. Relative quantification of MAGE expression was calculated in relation to the concentration of the reference housekeeping gene PBGD. To determine PCR efficiency at different target RNA concentrations, standard curves using serial dilutions of HT29 cDNA (1:1, 1:10, 1:100, 1:200, 1:1000) were performed for each experiment. In addition, for normalization, an internal calibrator was included in each run. Investigators performing qPCR were blinded concerning the histopathological results. All statistical analyses were performed using SPSS software version 17.0 for Windows (SPSS Inc., Chicago, IL). The values of p below 0.05 were considered statistically significant.
Figure 1 depicts the study design and how we evaluated the diagnostic procedures. We focused on the reported negative predictive value of EBUS-TBNA and therefore controlled each negative result after a diagnostic algorithm. In summary, the negative findings in EBUS-TBNA and mediastinoscopy were then assessed by open surgery, which is considered the gold standard.
We first analyzed the expression of MAGE transcripts in 60 lymph node samples from 31 patients with various nonmalignant disease conditions, as several studies had reported the expression of MAGE genes in chronic nonmalignant disease.28–31 Forty samples obtained by EBUS-TBNA and 20 lymph node samples obtained by mediastinoscopy were analyzed by qPCR. Fifty-six samples (93.3%) showed no specific expression of mRNA transcripts for MAGE 1 to 6, and 54 samples (90.0%) were free of MAGE 12 transcripts. Four samples showed low levels of MAGE 1 to 6 transcripts (relative quantification values 0.0008, 0.011, 0.060, and 0.07; two samples were from patients with sarcoidosis, one with anthracosilicosis, and one with reactive lymphadenopathy) and six samples showed MAGE 12 expression (relative quantification values 0.055, 0.061, 0.100, 0.130, 0.211, and 0.616; four samples were from patients with sarcoidosis, one with tuberculosis, and one with reactive lymphadenopathy). There was no overlap between samples, which were positive for MAGE 1 to 6 and MAGE 12. Given this low expression in control samples, we decided to define a cutoff level for the qPCR result. For this, we used a relative quantification value that we calculated from the 95% confidence interval of positive values in the control group. We obtained a threshold value of 0.091 for the MAGE 1 to 6 primers and 0.420 for the MAGE 12 primers, above which MAGE expression was considered to be cancer specific (Table 1).
Next, we assessed the expression of MAGE transcripts in 202 lymph nodes of 89 patients with lung cancer (for clinical characteristics of the patients see Table 2). One hundred twenty-two lymph node samples from 69 patients were obtained by EBUS-TBNA and 80 lymph nodes from 44 patients were collected by mediastinoscopy before operation. We detected MAGE 1 to 6 mRNA in 35 of 122 (28.7%; median, 1.36; range, 0.100–30.4) and MAGE 12 mRNA in 10 of 122 EBUS-TBNA samples (8.2%; median, 4.55; range, 0.60–24.5). In the lymph node samples obtained by mediastinoscopy, MAGE 1 to 6 mRNA was detected in 16 of 80 (20.0%; median, 3.24; range, 0.100–2878) and MAGE 12 mRNA in 9 of 80 (11.3%; median, 3.00; range, 0.570–25.2) lymph nodes. A correlation with clinicopathological parameters revealed a significant correlation of MAGE 1 to 6 or MAGE 12 transcripts in EBUS-TBNA and mediastinoscopic samples with the lymph node status (p = 0.004 and 0.033, respectively), whereas age, histology, and tumor size did not show any significant correlation (Table 2). Furthermore, we observed a highly significant positive correlation between high transcript levels of MAGE 1 to 6 and MAGE 12 levels (Figures 2A, B; Spearman’s [rho] r = 0.422, p < 0.001 for EBUS-TBNA and r = 0.257, p = 0.004 for mediastinoscopy samples).
Detection of tumor-specific MAGE expression correlated with the finding of malignant cells in histopathology for mediastinoscopy samples (p = 0.010 for MAGE 1 to 6 expression and p = 0.044 for MAGE 12 expression, Figure 3A and Table 3) and EBUS-TBNA samples (p < 0.001 for MAGE 1 to 6 expression and p = 0.048 for MAGE 12 expression, Figure 3B and Table 3).
The extent of regional lymph node metastasis at diagnosis is important for therapy selection of lung cancer patients. Therefore, we tested whether the combination of MAGE qPCR and histopathology more accurately stratifies lung cancer patients than histopathology alone. In total, we included 69 patients who underwent preoperative EBUS-TBNA and 44 patients from whom lymph nodes were obtained by mediastinoscopy. MAGE 1 to 6 mRNA was detected in 21 of 69 patients (30.4%) and 11 of 44 patients (25.0%), respectively. Cancer-specific MAGE 12 transcripts were detected in 8 of 69 (11.6%) and 7 of 44 (15.9%) patients. For EBUS-TBNA samples, all eight MAGE 12-positive patients also showed MAGE 1 to 6 transcripts, whereas three of seven MAGE 12-positive patients who underwent mediastinoscopy showed no MAGE 1 to 6 transcripts. All but one of the patients, who displayed cancer-specific MAGE transcript levels before operation, were postoperatively diagnosed with extensive regional lymph node metastasis (pN2–3). Given these observations, we combined MAGE qPCR with histopathology of the EBUS-TBNA and mediastinoscopy samples. We reasoned that a more sensitive preoperative diagnostic procedure might prevent nonrecommended operation of patients with extended lymph node spread (pN2 or pN3 disease). Among our patients, cytopathological evaluation of preoperative EBUS-TBNA alone correctly identified 27 of 48 patients as pN2 or pN3 (accuracy 69.6%) and histopathological evaluation of mediastinoscopy 21 of 28 patients (accuracy 84.1%), respectively. The addition of cancer-specific MAGE expression resulted in increased sensitivity in tumor cell detection, providing a correct diagnosis of pN2 or pN3 in 35 of 48 EBUS-TBNA samples (accuracy 81.2%; Table 4) and 23 of 28 mediastinoscopy samples (accuracy 86.4%; Table 4).
In this study, we investigated the impact of qPCR on the detection of disseminated tumor cell mRNA in mediastinal lymph node samples and on its accuracy when combined with conventional histopathology. We established a highly sensitive and tumor-specific real-time PCR for the quantitative assessment of disseminated tumor cells in lymph node samples of lung cancer patients.
Among our control patients, we detected MAGE 12 transcripts in four lymph nodes from patients with sarcoidosis. Although MAGE expression has not been linked to sarcoidosis until to date, these results are in agreement with previous studies, which detected expression of MAGE mRNA in patients with chronic lung damage.28,31 In our study, the relative expression of MAGE transcripts in samples from patients with a nonmalignant bronchial disease was low, and cutoff levels for cancer-specific MAGE 1 to 6 and MAGE 12 expression could be defined. By these cutoff levels based on the 95% confidence interval of control samples, we were able to clearly separate MAGE expression values from control and cancer patients. By this, our assay was designed for high specificity to exclude false-positive samples, while on the other hand resulting in a decrease of sensitivity. Therefore, we consider our MAGE qPCR assay to be additive to cytological/histological analysis of samples investigated by EBUS-TBNA or mediastinoscopy.
In patients with primary lung cancer, we detected MAGE 1 to 6 expression in 28.7% of EBUS-TBNA and 20.0% of mediastinoscopy samples, and MAGE 12 expression in 8.2% of EBUS-TBNA and 11.3% of mediastinoscopy samples (Table 4). We could not find any correlation between MAGE gene expression and the histological tumor type, confirming the results of several previous studies.<sup>19,23–25</sup> The rather low detection rate of MAGE 12 may be related to the intratumoral heterogeneity of MAGE expression, which has also been reported for melanoma.32,33 Differences in promoter methylation, which regulate MAGE expression on a transcriptional level in different cellular subtypes,34,35 were suggested as an underlying mechanism. For example, a recent study described MAGE transcript up-regulation by genomic hypomethylation after incubation of normal tissue with cigarette smoke concentrate for up to 9 months.30 This finding supports the view that MAGE gene expression is linked to early events in carcinogenesis, especially in its role to inhibit the pro-apoptotic functions of wild-type p53.36–38
Furthermore, we tested whether MAGE qPCR could increase the diagnostic accuracy of the currently used methods EBUS-TBNA and mediastinoscopy. In our hands, routine cytology of EBUS-TBNA samples was less sensitive for the detection of lymph node involvement in lung cancer patients than histopathology of mediastinoscopy samples (accuracy 69.6% and 84.1%, respectively). The addition of the quantitative MAGE qPCR assay to cytologic diagnosis improved the accuracy to 81.2% and 86.4%, respectively. The increase was more pronounced for EBUS-TBNA, resulting in a comparable accuracy as for mediastinoscopy alone. The sensitivity of EBUS-TBNA in our study is 56.3% and therefore lower compared with published literature (79–95%).7 This is probably due to the relatively low number of patients and the recent establishment of EBUS-TBNA as a diagnostic procedure in our department at the time of the study. EBUS-TBNA is a challenging procedure that requires considerable expertise and training, and the reported false-negative rate in most published studies indicates that EBUS-TBNA is largely operator dependent.7 However, because we show that a quantitative MAGE qPCR assay supplements EBUS-TBNA in the diagnostic setting and ensures high diagnostic accuracy of EBUS-TBNA from the beginning, it could provide significant advantages during the necessary training periods of clinicians.39,40
Several studies indicate that detection of few disseminated cancer cells in a pN0 staged lymph node has an impact on survival.41,42 Whether MAGE positive lymph nodes predict poor survival has not been analyzed so far and demands larger diagnostic studies. Taken together, molecular-pathological detection of MAGE transcripts in lymph nodes may be used in mediastinal staging, especially as addition to the less invasive EBUS-TBNA. As mediastinoscopy is an invasive method, we suggest future prospective studies on larger cohorts of patients to investigate whether EBUS-TBNA in combination with MAGE qPCR could replace mediastinoscopy for routine staging of lung cancer and therapy planning.
Supported by Deutsche Krebshilfe (GZ 108433).
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Non-small cell lung cancer (NSCLC); Lymph node metastasis; Melanoma antigen (MAGE); Endobronchial ultrasound-guided fine-needle aspiration (EBUS-TBNA); Mediastinoscopy
© 2012International Association for the Study of Lung Cancer
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