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(Chest. 2005;128:1539-1544.)
© 2005 American College of Chest Physicians

Quantitative Real-time Polymerase Chain Reaction Detection of Lymph Node Lung Cancer Micrometastasis Using Carcinoembryonic Antigen Marker*

Mario Nosotti, MD; Monica Falleni, MD; Alessandro Palleschi, MD; Caterina Pellegrini, MD; Francesca Alessi, MD; Silvano Bosari, MD and Luigi Santambrogio, MD

* From the Division of Thoracic Surgery (Drs. Nosotti, Palleschi, and Santambrogio), IRCCS Ospedale Maggiore Policlinico, Milan; and Division of Pathology (Drs. Falleni, Pellegrini, Alessi, and Bosari), University of Milan, Milan, Italy.

Correspondence to: Mario Nosotti, MD, Via Salomone 21, 20138 Milano, Italy; e-mail: m.nosotti{at}policlinico.mi.it


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: The survival of patients with surgically resected stage I non-small cell lung cancer (NSCLC) is not optimal, probably because of unsuspected systemic occult tumor dissemination. The current applied technologies and methods for scanning the body and examining lymph nodes for tumor cells have broadly recognized limitations. Several studies have reported that it is possible to detect occult lymph node metastases (micrometastases) using more sensitive methods such as immunohistochemistry or molecular technology. The aim of our study was to evaluate the utility of quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) messenger RNA (mRNA) for detection of lymph node micrometastases and its impact on disease-free interval.

Methods: Quantitative real-time RT-PCR for CEA mRNA was performed on primary lung tumors and regional lymph nodes from 44 surgically resected NSCLC patients classified as clinical stage I. Fourteen lymph nodes from five patients without malignancy were used as controls. The end point of clinical analysis was cancer recurrence. Average follow-up was 22.5 months.

Results: CEA mRNA was detected in all but four lymph nodes used as controls. All primary tumors were positive for CEA mRNA. Of 261 lymph nodes analyzed, 35 lymph nodes (13.4%) showed CEA mRNA levels higher than those detected in control lymph nodes and were considered positive for micrometastasis. Survival analysis by micrometastases showed less cancer recurrences in patients with lymph nodes negative for CEA mRNA (log rank, 5.3; p = 0.021). Among tumor type, tumor grading, age, sex, and molecularly detected lymph node micrometastases, the most powerful predictor of cancer recurrences was the presence of micrometastases (Cox proportional hazard, 3.3; p = 0.027).

Conclusion: Quantitative real-time RT-PCR for CEA mRNA can be applied for detection of micrometastases in lymph nodes. This technique may be an appropriate tool in predicting cancer recurrences, and further studies are warranted to determine the most useful clinical applications.

Key Words: lung cancer • micrometastasis • molecular staging • real-time reverse-transcriptase polymerase chain reaction


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The most significant prognostic factors in non-small cell lung cancer (NSCLC) are clinical and pathologic stages. Nevertheless, the postoperative pathologic stage does not provide completely correct information about survival after surgical resection. Several studies123 point out that NSCLCs, considered to be an early stage by standard pathologic criteria, are correlated with disseminate tumor cells (micrometastases). Moreover, an association between presence of micrometastases in lymph nodes and a poor prognosis has been observed.45 Therefore, the detection of micrometastases might identify those patients at increased risk, which could draw advantage by adjuvant systemic therapy.

Sensitive immunocytochemical tests have been developed to identify micrometastases,6 but immunocytochemistry has not been routinely used in cancer staging procedure for a combination of factors: loss of antigen expression in poorly differentiated tumors, cytokeratin and epithelial membrane antigen positivity in nonepithelial cells, and the necessity to standardize methods and the subjective readout of immunostained cells. The need for the development of a better method for the detection of micrometastases has been satisfied by the advent of the polymerase chain reaction in the late 1980s. Polymerase chain reaction amplification may be preceded by reverse transcription of messenger RNA (mRNA) into DNA; this technique is known as reverse-transcription polymerase chain reaction (RT-PCR). This procedure offers several advantages: mRNA may be detected in poorly differentiated tumors that do not express tissue-specific proteins, RT-PCR is much more sensitive than immunocytochemistry (1 cancer cell on 106–7 normal cells, vs 1 on 104–5), the ability to analyze the entire specimen decreases sampling errors, and a minimal tissue amount is sufficient.7 Real-time RT-PCR is a development of the RT-PCR procedure used to accurately estimate gene expression levels.89 The advantages of the real-time RT-PCR procedure are the capability to differentiate between baseline levels of gene expression in normal tissue vs increased levels in cancer cells, and the benefit of a simple, rapid, and automated procedure.

It is reported that the quantitative real-time RT-PCR procedure for carcinoembryonic antigen (CEA) can exactly estimate micrometastatic cells in the lymph nodes of surgically resected NSCLC patients.10 To confirm previous observations and to evaluate the impact of the micrometastases on cancer recurrence, we started this study based on molecular staging of patients with clinical stage I NSCLC.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Tissue Specimens
Forty-four lung cancer patients treated with pulmonary lobectomy at our Division of General Thoracic Surgery from 2000 to 2003 were prospectively studied. Eligible patients had NSCLC classified as clinical stage I by routine imaging and cytologic assessments. Patients with history of previous malignancy were excluded. Consent was obtained from each patient.

At operation, all the accessible mediastinal lymph nodes were gathered and divided in two parts. The first part of each specimen was instantaneously frozen in liquid nitrogen and stored for the molecular study at – 80°C. The second part was formalin fixed and paraffin embedded for routine histopathologic examination (at least three nonconsecutive hematoxylin-eosin–stained sections per block were considered). Lobectomy was then carried out, and the pulmonary lymph nodes were treated as the mediastinal. Tissue specimens were immediately obtained from primary tumors and frozen for molecular studies. Control lymph node specimens were obtained from five patients, without evidence of cancer, undergoing surgery for thoracic benign pathology (11 lymph nodes) and inflammatory bowel disease (3 lymph nodes). Tumor histotype, grading, and staging was performed according to the World Health Organization and TNM staging systems, respectively.

After surgery, the patients were examined every 2 months in the first year and afterwards every 3 months. CT 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 was the study end point.

Cell Lines and Standard Curve
Eight cell lines were obtained from American Type Culture Collection (Rockville, MD) and cultured as recommended. Cell lines were from pharyngeal carcinoma (Detroit 562), laryngeal carcinoma (Hep2), breast cancers (MDA-MB-231, SKBR-3, T47D, MCF-7), and colon cancers (HCT-15, SW1116). Cells were harvested at approximately 80% of confluence and prepared for mRNA extraction. CEA-expressing MCF-7 cells (10-fold serial dilution, from 105 cells to 1 cell) were mixed with 106 lymphocytes from a healthy CEA-negative donor for the construction of the standard curve.

mRNA Isolation and DNA Synthesis
Total cellular mRNA was isolated from cell lines, control lymph nodes, lymph nodes from cancer patients, and primary tumors. Tissue specimens were homogenized (TRIzol solution; Invitrogen; Carlsbad, CA) according to the instructions of the manufacturer. The quantity and purity of mRNA were established by spectrophotometry.

Total mRNA was reverse transcribed in DNA as previously described.11 The amounts of reverse-transcribed mRNA were 100 ng for cell lines and tumor specimens, 1 µg for lymph nodes, and standard curve points. All reagents were from Applied Biosystems (Foster City, CA).

Real-time RT-PCR
The ABI Prism 7700 Sequence Detection System (Applied Biosystems) was used for the quantitative assessment of CEA. During the DNA polymerization, the TaqMan (Applied Biosystems) probe was hydrolyzed and fluorescence emitted. The charge-coupled device camera on the Prism 7700 device continuously collected the fluorescence emissions during the amplification cycles. When the fluorescence signal reached 10 SDs of background, the threshold cycle (Ct) was noted.

CEA mRNA expression in cell lines and lung carcinomas was obtained with the {Delta}{Delta}Ct method. ß-actin (ACTB) and an arbitrary selected lung carcinoma of the series were used as intemRNA1 control gene for normalization and as calibrator, respectively.

CEA mRNA quantification in lymph nodes was achieved with the standard curve method. The number of CEA-positive cells detected was obtained from the linear regression of the standard curve.

All PCRs were performed in duplicate (Universal TaqMan 2X PCR Mastermix; Applied Biosystems) in a volume containing 0.3 µmol/L of each primer and 0.1 µmol/L of TaqMan probe. The thermal profile included 2 min at 50°C, 10 min at 95°C followed by 40 cycles (50 for lymph nodes and standard curve points) at 95°C for 15 s, and 1 min at 60°C.

Primer Sequences
Intron-spanning primers and TaqMan probes for CEA and ACTB mRNAs were designed using Primer express software (Applied Biosystems). The sequences of primers used in the present study are the following: forward 5'-ATT CCA TAG TCA AGA GCA TCA CA-3', reverse 5'-GCA AAT GCT TTA AGG AAG AAG-3', TaqMan probe 5'-(6-FAM) TGA AAT GAA GAA ACT ACA CCA GGG CTG CTA TAT (TAMRA)-3' for CEA (National Center for Biotechnology Information accession NM 004363), and forward 5'-TCC TTC CTG GGC ATG GAG-3', reverse 5'-AGG AGG AGC AAT GAT CTT GAT CTT-3', TaqMan probe 5'-(6-FAM) CCT GTG GCA TCC ACG AAA CTA CCT TC-(TAMRA)-3' for ACTB (National Center for Biotechnology Information accession NM_001101).

Statistical Analysis
Survival functions were estimated using the Kaplan-Meier method, and the differences were evaluated by log-rank or Wilcoxon test as appropriate. The Cox proportional hazards test was used to identify the factors that influenced the cancer recurrence. The regression analysis was applied to standard curves, and the analysis of variance was used where indicated.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The standard curve, obtained from MCF-7 cell line, established the relation between CEA mRNA Ct and number of cancer cells in the sample. The equation y = – 3.4x + 42.2 used for linear regression analysis determined a correlation coefficient of 0.97. ß-actin mRNA Ct < 23 was considered indicative of appropriate mRNA quality and quantity in the specimens. All the cell lines were adequate for mRNA integrity, whereas four lymph node specimens were inadequate. A delay in freezing process was the major reason of mRNA inadequacy; all the patients with deteriorated mRNA were not included in the evaluation.

All the 44 patients enrolled in the study underwent pulmonary lobectomy for clinical stage I NSCLC. There were 33 men and 11 women (mean age, 70.2 years; range, 39 to 80 years). Tumor histotype included 30 adenocarcinomas, 12 squamous cell carcinomas, and 2 undifferentiated carcinomas. Patients characteristics are shown in Table 1 .


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  		Table 1.. Patient Characteristics

 
Investigations were first performed to determine the presence of CEA mRNA in the 44 primary tumors. CEA transcript levels were detected in all tumor specimens, with values ranging from 0.044 to 1782.88. The highest levels were observed in adenocarcinomas, whereas all the squamous cell carcinomas under study showed the lowest CEA mRNA levels. The difference was statistically significant (p = 0.004).

The 14 control lymph nodes from the five patients without any malignancies were tested: CEA mRNA was identified in 10 lymph nodes. The highest CEA mRNA was observed in a patient with pulmonary fibrosis and a Ct value of 37.42. Therefore, Ct values < 37.42 were considered significant for the presence of cancer cells in the lymph nodes. This choice allowed us to avoid interferences by illegitimate transcription or by the presence of dendritic cells in the lymph nodes.

Afterwards, we performed quantitative real time RT-PCR on the 261 lymph nodes from NSCLC patients: 35 lymph nodes were positive for CEA mRNA (13.4%). The median cell number was 213 tumor cells per lymph node (range, 54 to 36 x 105). The lymph nodes with micrometastases were encountered in 16 of the 44 patients (36.3%). Contingency analysis did not reveal any correlation between the presence of micrometastases and the tumor mass characteristics (pathologic T1 or T2), histotype, tumor grade, age, gender, and levels of CEA mRNA in the primary cancer.

The patients were classified according to the current American Joint Committee on Cancer criteria for NSCLC, and Table 2 details the distribution of the N status according to conventional histologic staining (hematoxylin-eosin) vs quantitative real-time RT-PCR.


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  		Table 2.. Pathologic N Factor Staging by Hematoxylin-Eosin Staining vs Quantitative Real-time RT-PCR for CEA mRNA*

 
The mean follow-up of the 44 enrolled patients was 22.5 months, no patients were unavailable for follow-up, and recurrence developed in 7 patients. Figure 1 shows the actuarial curves for cancer recurrence by hematoxylin-eosin nodal status (pathologic N0 vs N1). There were no significant differences in survival function; however, the graph demonstrates two separate curves, and the lack of significant differences might depend from the small population. Figure 2 illustrates the different actuarial curves for cancer recurrence by nodal status according to real-time RT-PCR results (patients without micrometastases [pathologic Nmi–] vs patients with micrometastases [pathologic Nmi+]). Although the sample size was small, the differences were statistically significant (p = 0.021). The Cox proportional hazards test demonstrated a higher risk of cancer recurrence in patients with micrometastases (hazard ratio, 3.3; confidence interval, 1.14 to 9.51; p = 0.027). The same test denied the correlation between cancer recurrence and pathologic N factor, pathologic T, histologic types, tumor grading, age, and gender. The number of metastatic cancer cells in lymph nodes documented by real-time RT-PCR was not related to cancer recurrence.



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  		Figure 1.. Disease-free interval analysis of 44 patients grouped by hematoxylin-eosin nodal status.

 


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  		Figure 2.. Disease-free interval analysis of 44 patients showing significant stratification by presence or absence of micrometastases in the lymph nodes.

 
Finally, the N status of each patient was reclassified according to the location of micrometastases: pathologic N0mi, no micrometastases; pathologic N1mi, micrometastases in the hilar lymph nodes; and pathologic N2mi, micrometastases in mediastinal lymph nodes. Figure 3 shows the significant differences in the actuarial survival curves for cancer recurrence of patients reclassified for micrometastases locations (p = 0.015).



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  		Figure 3.. Disease-free interval analysis of 44 patients showing significant stratification by N status according to the location of micrometastases.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is growing evidence that disseminate tumor cells (micrometastases) in regional lymph nodes, bone marrow, and blood might have an independent clinical meaning. Reporting the results of an international workshop held in 2002, Lugo and co-workers12 stressed the necessity of definitive clinical studies on micrometastases. In effect this item, described for the first time by Ashworth in 1869, has been under extensive examination since the 1980s.613 In 2003 Vollmer et al14 stated that immunohistochemical technique detects greater than twice as many positive regional lymph nodes as does hematoxylin-eosin staining. Probably, this was the best result of 20 years of studies on micrometastases, but unfortunately this technique has been considered too expensive, cumbersome, and variable to be used as a routine basis. The molecular oncology developments grant access to new opportunities, and real-time RT-PCR is now largely available.15 D’Cunha et al10 demonstrated that quantitative real time RT-PCR for CEA might offer greater sensitivity than any other technique for detecting lymph node micrometastases.

The current study confirms the data of D’Cunha et al10 on the capability of quantitative real-time RT-PCR for CEA mRNA to identify and estimate the number of micrometastatic cells per lymph node. The correlation between the amount of CEA mRNA and the number of micrometastatic cells depends on the type of cell line used for the standard curve. Our observed rate of micrometastatic lymph nodes was 13.4% vs 25% of the study by D’Cunha et al.10 Such differences may depend on different patient populations. Identification of CEA mRNA threshold level in nonneoplastic lymph node parenchyma may be important as well. In our study, we evaluated 14 lymph nodes from five patients without cancer to define the highest level of CEA mRNA expression in normal and reactive nodes from both thoracic and abdominal districts. The threshold level identified has allowed us to avoid the classification of a significant number of lymph nodes in the metastatic category. The low level of CEA mRNA expression in nonmetastatic lymph nodes may depend on illegitimate CEA gene transcription16 and/or CEA mRNA expression from dendritic cells.

We report, for the first time, a high variability of the CEA mRNA expression level in the primary lung cancer despite the normalization for ß-actin levels. The difference between adenocarcinomas and squamous cell carcinomas was highly significant, but the CEA mRNA expression in primary lung cancer did not relate to the disease-free interval, and its possible clinical significance requires further investigations. Anyway, it is remarkable that CEA was found in all the primary cancers, among general low expression of individual oncogenic markers in NSCLC.

Our data, based on a small number of cases, reveal a significant stratification of the patients by the presence or absence of micrometastases in the regional lymph nodes, suggesting that quantitative real-time RT-PCR for CEA mRNA might have a relevant prognostic value in NSCLC. Moreover, grouping the patients by the location of the micrometastatic lymph nodes, the disease-free interval analysis shows distinct survival curves between patients without micrometastatic disease (pathologic N0mi), patients with micrometastatic hilar lymph nodes (pathologic N1mi), and patients with micrometastatic mediastinal lymph nodes (pathologic N2mi). The Cox proportional hazards regression analysis points out the presence of micrometastases as the primary factor influencing the cancer recurrence in our cohort of patients. Considering the low correlation between survival and micrometastases detected by molecular methods in previous studies (using markers as k-Ras, p-53, bcl-2, KI67, CD-44, Rb, EGFr, angiogenesis factor viii, STN, and erbB2),171819 our preliminary results with CEA are encouraging.

It is important to note that use of quantitative real-time RT-PCR for detecting micrometastases in clinical sample may provide significant savings compared to both histopathologic routine examination and an immunohistochemical staining.20 If our results will be confirmed by extensive prospective investigation, real-time RT-PCR for CEA mRNA could become the most effective technique with remarkable cost saving as compared to currently available pathologic staging in NSCLC.

In conclusion, quantitative real-time RT-PCR for CEA mRNA can reveal the presence, and estimate the number, of micrometastatic cells in lymph nodes of patients submitted to lung resection for NSCLC. Furthermore, this sensitive and cost-effective technique provides prognostic information in low-stage patients; on-going clinical trials will address this relevant subject.


   Footnotes
 
Abbreviations: CEA = carcinoembryonic antigen; Ct = threshold cycle; mRNA = messenger RNA; NSCLC = non-small cell lung cancer; RT-PCR = reverse-transcription polymerase chain reaction

Received for publication January 15, 2005. Accepted for publication March 15, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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