Annals of Oncology Advance Access published online on July 29, 2008
Annals of Oncology, doi:10.1093/annonc/mdn538
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Expression of molecular markers in mediastinal nodes from resected stage I non-small-cell lung cancer (NSCLC): prognostic impact and potential role as markers of occult micrometastases
1 Research Unit, Hospital General Universitario de Alicante, Alicante
2 Pathology, Hospital General Universitario de Alicante, Alicante
3 Thoracic Surgery, Hospital General Universitario de Alicante, Alicante
4 Medical Oncology, Hospital General Universitario de Alicante, Alicante
5 Thoracic Surgery, Hospital de La Ribera, Alzira
6 Preventive Medicine and Public Health,Universidad Alcalá de Henares, Madrid, Spain
* Correspondence to: Dr S. Benlloch, Hospital General Universitario de Alicante, Unidad de Investigación, Avda Pintor Baeza 12, Alicante 03010, Spain. Tel: +34935460122; Fax: +34935460172; E-mail: sbenlloch{at}pangaeabiotech.com
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Abstract |
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Background: Occult lymph node (LN) metastases are clinically relevant and confer a worse prognosis in non-small-cell lung cancer (NSCLC) patients. Current staging methods are unable to identify patients with poor outcome. Their detection requires both a more sensitive and specific technique. We aimed to assess the role of messenger RNA expression in pathologically negative LNs (pN0) of stage I NSCLC patients as markers of occult micrometastases and to correlate the results with local or distant tumor recurrence and survival.
Patients and methods: Potential molecular markers were evaluated in 344 LNs and 38 tumors by quantitative real-time RT-PCR. Only CEACAM5 and PLUNC showed high expression in lung tumor tissue and null expression in RNA from benign LNs.
Results: Thirteen per cent of the LNs were positive for CEACAM5 and 16% for PLUNC. Eight of 38 NSCLC patients had positive expression in pN2 nodes by CEACAM5 and/or PLUNC and disease-free survival (P = 0.028) and overall survival time was significantly worse in these patients compared with those with negative expression (P = 0.0083).
Conclusions: Quantitative real-time RT-PCR of CEACAM5 and PLUNC can estimate the presence of micrometastatic cells in LNs with greater precision than current staging method used for assessing tumor recurrence risk.
adjuvant therapy, disease progression, NSCLC, occult micrometastases, prognosis, quantitative real-time RT-PCR
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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In cancer types, the identification of the lymphatic spread of the tumor is a key factor that influences the therapy and prognosis of the disease [1]. In non-small-cell lung cancer (NSCLC), lymph node (LN) involvement is a strong predictor of disease recurrence and survival. A review of the literature indicates that occult LN metastases (micrometastases) are indeed clinically relevant and confer a worse prognosis [2]. ‘Micrometastasis’ has been defined in the AJCC Cancer Staging Manual, sixth edition as ‘when the tumor deposits in the LNs are larger than 0.2 mm but not larger than 2.0 mm’. Nodes with micrometastasis are considered positive for staging.
The 5-year survival rate for patients with surgically resected stage I NSCLC is 60%–70%, whereas for patients with affected LNs it is <50%. Recurrent disease might develop within an early interval after the operation, even in patients in stage I of NSCLC, without an apparent metastasis in the regional LNs [3]. Adjuvant therapies are not routinely given to patients in stage I [4]. Current staging methods are unable to identify those patients with poor outcome [5]. Histopathologic analysis can miss occult micrometastases, especially in nodal tissues. A recurrence of the disease is probably due to undetected systemic occult micrometastases in the histopathologic study carried out at the time of the initial diagnosis. The detection of micrometastases requires both a more sensitive and more specific technique, and it would be useful to define a high-risk population and select patients for postoperative adjuvant treatment. In the last few years, new immunologic and molecular analytic techniques have been developed to diagnose and characterize these occult disseminated tumor cells. In recent studies, real-time quantitative RT-PCR (RT-QPCR) has proven powerful enough to detect disseminated tumor cells [2, 6–9]. We are, therefore, in need of a clinically useful approach to better stratify patients with respect to the risk of recurrence and their survival rate.
In the present study, we examined the presence of micrometastases in LNs by means of both RT-QPCR and immunohistochemistry (IHC) in the same pathologically negative LNs (pN0) NSCLC patients, who underwent curative surgery. The aim of this study was to assess the role of messenger RNA (mRNA) expression of several genes in pN0 from resected stage I NSCLC patients in a prospective study as markers of occult micrometastasis and to correlate the results with both local or distant cancer recurrence and global survival.
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patients and methods |
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patients
Surgery for NSCLC was carried out on 38 consecutive patients (28 men, 10 women; median age 66 years old, range 44–79) at the University General Hospital of Alicante, Spain, from 1 April 2003 to 2 February 2006 (Table 1). Eligible patients had stage I NSCLC [10] classified by histopathological analysis after surgery and mediastinal lymphadenectomy. All patients signed the informed consent and the study was accepted by the hospital ethics committee. Patients with a history of previous malignancy were excluded. Thirty four patients underwent a lobectomy and four of them a pneumonectomy. After a median follow-up of 24 months (range 9–46), 11 patients had died from NSCLC and one patient had died without recurrent disease. None of the living patients had tumor recurrence.
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clinical evaluation of patients
During the first year after surgery, the patients were examined every 2 months and afterwards every 3 months. Computed tomography scans of the thorax, abdomen, and brain were obtained every 6 months. If recurrence was suspected, the appropriate investigations were undertaken. Local or distant cancer recurrence and overall survival (OS) were the study end points.
pathological evaluation
After the lung resection, a complete LN dissection (removal of all mediastinal nodes found at the common nodal sites) was carried out. The LNs were then classified to understand the nodal descriptors pN1, pN2, and pN3 using the international staging system of the American Thoracic Society[10]. In this system, LNs are placed into well-defined stations based on clearly defined anatomic boundaries.
Surgical specimens (primary tumor and LN) were immediately evaluated by a pathologist. LN dissection was carried out and each LN was divided into two parts. The first one was formalin fixed and paraffin embedded for routine histopathologic examination and the second one was instantaneously frozen in isopentane and stored for the molecular study at –80°C. Tumor and normal tissue were also snap frozen. Routine histopathological classification, grading, and staging of the tumor were carried out according to the tumor–node–metastasis (TNM) staging systems. After routine evaluation of the specimen and final diagnostic, additional examination of each LN was made. For each LN, at least three nonconsecutive hematoxylin–eosin (H&E)-stained sections were examined and immunohistochemical analysis for CK7 (clone COU-TL12/30, Dako, 1:50) was carried out in at least three more sections. A mean of 14 LNs were studied for H&E and IHC analyses.
identification and validation of potential markers
An extensive literature and public database survey was conducted to identify any potential marker relevant to lung cancer. SAGE [11] (http://cgap.nci.nih.gov/Catalog) was the main resource for this survey. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project uses serial analysis of gene expression (SAGE) [12]. SAGE Genie offers a collection of gene expression data across many different cancer and tissue types. Our survey criterion was to identify genes with high expression in lung tissue and low or null expression in benign LNs. We looked for specific markers that showed no expression in benign LNs and positive expression in primary lung tumors and pathology-positive LNs. We tested CEACAM5, CDX1, CK19, CK20, EVA1, Ephrine A1, Mesothelin, MGB2, MAGE A10, PLUNC, SFTPC, E-cadherin, and TACSTD2. Expression of all potential markers was tested in complementary DNA (cDNA) from commercial RNA from benign LNs obtained from a woman without cancer (Stratagene, La Jolla, CA) and a standard curves of them were constructed in order to check levels of expression through a dilution series of cDNA being the first point of dilution 100 ng. The 13 potential markers were tested in 15 primary tumors and 19 histologically positive LNs. Following these statements, only CEACAM5 and PLUNC were chosen for LN metastasis detection in our study because, with a GAPDH mRNA Ct 
26 corresponding to 1 ng of cDNA, no expression of both markers was found in cDNA from benign LNs. So we considered a GAPDH mRNA Ct 26 indicative of maximum appropriate mRNA quantity and quality in the specimens. With respect to the other potential markers tested, we found expression in benign LNs until 10 ng point dilution of CK19, Mesothelin, and E-cadherin; 1 ng point dilution of CDX1, TACSTD2, and SFTPC; 0.1 ng point dilution of Ephrine A1 and EVA1; no expression was found for CK20, MGB2, and MAGE A10 benign LNs or in lung primary tumor of 10 NSCLC patients.
molecular evaluation of frozen LNs
A total of 344 frozen LNs (median 9.1 per patient) were analyzed by RT-QPCR. The TNM system advocates a pN0 (mol+) designation for LN found to be positive only by molecular methods and pN0 (mol–) denotes a nonmetastatic LN. In our study, LNs were classified as pN1 (mol+), pN2 (mol+) when were found to be positive for CEACAM5 and/or PLUNC in the triplicates of RT-QPCR 96-well plates. The nodal station N1 are intralobar and hilar nodes confined to the pleural envelope; N2 nodes are metastasis to ipsilateral mediastinal LNs and subcarinal LNs.
RNA isolation and cDNA synthesis
Total RNA was isolated from frozen LNs and primary tumors of cancer patients. Tissue specimens were homogenized (Nucleic Acid Purification Lysis Solution; Applied Biosystems; Foster City, CA) according to the manufacturer's instructions. The RNA was extracted by Prep Station 6100 (Applied Biosystems) including an additional step for DNase-treatment. The quantity and quality of RNA were established by Bioanalyzer 2100 (Agilent, Santa Clara, CA). Total RNA was reverse transcribed in DNA by Archive kit (Applied Biosystems).
real-time RT-PCR
The 7500 Sequence Detector (Applied Biosystems) was used for the quantitative assessment of the potential markers. The primers, probes, and TaqMan Universal PCR Master Mix were purchased from Applied Biosystems. We used 2.5 µl of cDNA for PCR amplification. Analyses were carried out for each sample in triplicate and the mean result was used for further analysis. Nontemplate control was included in all plates. The cycling conditions used were as follows: initial incubation at 50°C for 2 min to activate AmpErase UNG, subsequently at 95°C for 10 min to activate the AmpliTaq Gold polymerase, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. Primers and probes were purchased from Applied Biosystems as Assay-on-demand (CEACAM5 Hs00237075_m1, CDX1 Hs00950423_g1, CK19 Hs01051611_gH, CK20 Hs00962995_m1, EVA1 Hs01083647_m1, E-cadherin Hs00170423_m1, Ephrine A1 Hs 00358886_m1, Mesothelin Hs 00245879_m1, MGB2 Hs01014212_m1, MAGE A10 Hs01560792_m1, PLUNC Hs 00213177_m1, SFTPC Hs 00161628_m1, and TACSTD2 Hs 00242741_s1). Amplicons spanned exon junction in order to provide cDNA specificity. Tumor tissue of each patient served as positive expression control for genes tested. GAPDH and β-glucuronidase were used as endogenous genes as indicative of appropriate mRNA quality and maximum quantity in the specimens (see ‘identification and validation of potential markers’). The tumor tissue was not used for calculation of relative expression.
statistical analysis
To evaluate the prognostic capacity of molecular marker expressions in LNs, we calculated and analyzed survival curves using the Kaplan–Meier method. We carried out the log-rank test for the comparison of the survival functions. A multivariate stepwise procedure Cox regression analysis was used to asses the association between each potential prognostic factor and survival functions (event-free survival and OS). In order to determine the concordance between both molecular markers on assessing pN2 (mol+/–) status, a chi-square test was carried out (P > 0.05) allowing to use CEACAM5 and/or PLUNC to assess pN2 (mol+/–) status in following analysis. The hazard ratios and 95% confidence intervals (CIs) for covariates were calculated from the Cox model. Analyses were carried out using SPSS version 11.0 (SPSS Inc, Chicago, IL). The statistical significance was set at P < 0.05.
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Thirty-eight consecutive NSCLC stage I patients took part in our survey (Tables 1 and 2). Thirty-four underwent a pulmonary lobectomy and four of them a pneumonectomy. Histopathological classification included 14 adenocarcinomas, 14 squamous cell carcinomas, and 10 other carcinomas. Three hundred and forty-four LNs were obtained by systematic mediastinal lymphadenectomy. H&E and IHC for CK7 were negative for tumor cells in all LNs evaluated. After a median follow-up of 24 months (range 0.4–46), the median disease free-survival and OS were calculated and recurrence developed in 11 patients. Eleven patients died from NSCLC and one patient due to a noncancer-related disease. None of the living patients had tumor recurrence when we closed the study.
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Tumor tissues and LNs were adequate except for six LNs that were not included in the evaluation. Expression of 13 genes was analyzed in cDNA from commercial RNA from benign LNs. Our survey criterion was to identify genes with high expression in lung tissue and low or null expression in normal LNs. Eight of them (CK19, CDX1, Mesothelin, Ephrine A1, E-cadherin, EVA1, TACSTD2, SFTPC) had high expression in benign LNs. Three of them (MAGE A10, MGB2, CK20) had null or low expression in benign nodes, but also had undetectable or very low expression in the primary tumors. Two of them (CEACAM5 and PLUNC) had null or low expression in benign nodes and detectable expression in the primary tumors. On the basis these results, CEACAM5 and PLUNC were chosen as potential markers. So, we consider those LNs in which expression of CEACAM5 or PLUNC was detected to be molecular positive.
A total of 344 pathologically negative LNs were tested. Thirteen per cent (44 of 344) were positive for CEACAM5 and 16% (54/344) for PLUNC. Nodes were classified as molecular-positive node when an expression of one of the two markers was detected. The expression patterns in samples were very similar for both markers. For the prognostic assessment, molecular-positive LNs were classified as pN1 (mol+) and pN2 (mol+) (see ‘molecular evaluation of frozen LNs’). Median event-free survival was 15 ± 11.74 months in patients with pN2 (mol+) nodes and has not yet reached in cases with pN2 (mol–) LNs (P = 0.028) (Figure 1). Cox proportional hazard modeling showed that pN2 (mol+) was not predictor of event-free survival in these patients (Table 3). Median OS time of patients with pN2 (mol+) nodes was 17.3 ± 5.7 months and has not yet reached in cases with pN2 (mol–) LNs (P = 0.0083) (Figure 2). There was no statistical association between gender, age, pN1 (mol+) nodes, tumor size, visceral pleural invasion, tumor location, or pathological diagnosis and disease-free survival and global survival time. Therefore, pN1 (mol+) micrometastases did not show prognostic significance. Fifty per cent (four of eight) of the patients with pN2 (mol+) nodes died compared with 20% (6 of 30) of the patients with pN2 (mol–) nodes. The Cox proportional hazards test demonstrated a higher risk of death in patients with pN2 (mol+) (hazard ratio 6.14; 95% CI 1.49–25.34; P = 0.01). The same test denied the correlation between survival and tumor size, visceral pleural invasion and histologic types, and gender and age (Table 4).
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discussion |
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LN metastases are indicative of a poor prognosis in several cancer types because they indicate clear evidence of metastasis, a factor that influences the prognosis and the therapy. In the case of primary lung cancer, the presence of LN metastasis is an important prognostic factor: an accurate assessment of the presence or absence of tumor cells in the regional LNs is therefore critical for making an accurate prognosis [13].
Surgical intervention remains the gold standard treatment for locoregional disease of NSCLC [14]. Despite complete resection, NSCLC patients still have a poor 5-year survival rate, most likely because some metastases are undetected at the time of surgery [15]. With the recent progress in surgical techniques, mortality has been increasingly linked to early metastases. The early detection of such tumor dissemination might be a promising approach to assess recurrence risk and to identify specific patients who would benefit from chemotherapy treatment.
The standard technique to evaluate LN metastases from solid tumors is histopathologic analysis [16]. Multiple investigators have used IHC to detect submicroscopic metastases in bone marrow and LNs in patients with NSCLC [17–20]. Various authors demonstrated the presence of these metastases in patients with breast [21], colon [7], prostate [22], and gastric cancers [23]. Other investigators have demonstrated that the immunohistochemical detection of micrometastases in NSCLC is possible and correlate with a higher recurrence rate and poorer survival rate [24].
More recently, efforts have been made to detect micrometastases in LNs at molecular levels. Reverse transcriptase–quantitative polymerase chain reaction (RT-QPCR) has been shown to be more sensitive than IHC for the detection of micrometastases in patients with colorectal, breast, and prostate cancer [7, 25, 26].
In our study, we have explored the possibility of detecting tumor cells in regional LNs using RT-QPCR as an alternative to the classical methods. A yet unresolved question is which biomarker to use for such detection. A major goal is to determine which properties of a tumor marker are of importance for the detection of micrometastases of NSCLC in regional LNs. We mainly identified the potential tumor markers according to the data obtained from SAGE [11] (see ‘identification of potential markers’). Our survey criterion was to identify genes with high expression in lung tissue and low or null expression in benign LNs. Thirteen genes were tested and two of them were finally chosen: CEACAM5 and PLUNC. CEACAM5 is carcinoembryonic antigen-related cell adhesion molecule 5 and it has been reported to support the growth of primary tumors. PLUNC expression has been found in lung, upper airways, and nasopharyngeal regions, including trachea and nasalepithelium. It was isolated by differential-display mRNA analysis [27]. It is specifically expressed in the secretory ducts and submucosal glands of tracheobronchial tissues, but it is also expressed in lung cancers and some other types of cancer.
In localized tumors, intrathoracic LN metastases are the most powerful predictors of survival and curability, and the prognosis of patients with apparently resecable NSCLC is substantially influenced by the extent of mediastinal LN involvement [28]. The nodal station N1 (intralobar and hilar nodes confined to the pleural envelope) [29] classifies the tumors as stage II; in stages I and II, surgery remains the cornerstone of treatment. However, the existence of N2 nodes (metastasis to ipsilateral mediastinal LNs and subcarinal LNs) is an independent prognostic factor [30]. The predictable sequence of metastases begins in the intrapulmonary nodes (N1) and progress to the mediastinal nodes (N2). pN2 (mol+) patients are pN1 (mol+) as shown in Table 2. Tumor extension is disseminated through lymphatic vessels following a nodal net from the closest to tumor (pN1) to those in mediastine (pN2). As we know, the prognosis of NSCLC is directly related to the completeness of resection and the status of the regional nodes.
In our study, the Kaplan–Meier disease-free (Figure 1) and survival curves (Figure 2) showed statistically significant differences between the two groups of patients, those with pN2 molecular positive (mol+) or negative (mol–) LNs. The fact that tumor cells are present in these LNs (pN2) represents a high metastatic potential which might not be curable by surgery alone. Therefore, we think that our results established the worst prognosis when pN2 LNs are detected by molecular methods when affected by the tumor. The Cox proportional hazards regression analysis points out the presence of micrometastases in pN2 (mol+) LNs as the primary prognostic factor influencing the survival in our cohort of patients. This fact could explain why a group of patients classified as stage I after surgery by means of conventional methods progress with a worse outcome. All patients were classified as T1 or T2 in the pathological study, except three of them. The most frequently diagnosed histological types were squamous and adenocarcinomas, and pN2 (mol+) nodes were detected in both. This fact, added to the idea of the occult tumor load, is the major reason for the high mortality rate and the shorter disease-free survival time in this group of patients.
Molecular diagnosis may be a powerful tool for identifying patients with worst outcome. In conclusion, real-time RT-QPCR of CEACAM5 and PLUNC can estimate the presence of micrometastatic cells in LNs with greater precision than the current staging method used for assessing tumor recurrence risk. Comparing the prognostic values for pN2 (mol+) LNs with those for pN2 (mol–) ones is very important for the prognosis and to establish adjuvant therapies. A controlled, randomized study will be necessary to ascertain whether patients identified by this method as high risk can benefit from adjuvant therapy.
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Fundación para la Investigación del Hospital General Universitario de Alicante and Dirección General de Ordenación, Evaluación e Investigación Sanitaria, Conselleria de Sanitat (0022/2005); Spanish Ministry of Health, Instituto de Salud Carlos III (CM04/00035 and 01/3080) to CA and SB; Fundación para la Investigación del Hospital General Universitario de Alicante and Dirección General de Ordenación, Evaluación e Investigación Sanitaria, Conselleria de Sanitat (0022/2005) to ER.
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We want to thank the technical support of pathology staff, especially Juan Pulido and Monica Medina. This study was presented in part at the IASLC 12th World Conference on Lung Cancer, Seul, Korea, September 2007. The authors have no conflict to disclose.
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Present address: USP Instituto Universitario Dexeus, Pangaea Biotech, Laboratorio Oncología, Sabino Arana 5, 08028, Barcelona, Spain. 
Received for publication May 14, 2008. Revision received June 30, 2008. Accepted for publication July 1, 2008.
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