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Eur J Cardiothorac Surg 2005;28:26-32
© 2005 Elsevier Science NL

Detection of disseminated tumor cells in mediastinoscopic lymph node biopsies and lymphadenectomy specimens of patients with NSCLC by quantitative RT-PCR

Xiao Tao Wang a , b , c , Wulf Sienel a , * , Stephan Eggeling a , Corina Ludwig a , Erich Stoelben a , Johannes Mueller d , Christoph A. Klein e , Bernward Passlick a

a Department of Thoracic Surgery, Albert-Ludwigs-University Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany
b Department of Surgery, Chirurgische Klinik und Poliklinik—Innenstadt, University of Munich, 80336 Munich, Germany
c Department of Respiratory Medicine, The First Affiliated Hospital of Dalian Medical University, 116011 Dalian, China
d Department of Thoracic Surgery, Asklepios Fachkliniken Mü;nchen-Gauting, 82131 Gauting, Germany
e Institute for Immunology, Ludwig-Maximilian-University Munich, 80336 Munich, Germany

Received 15 November 2004; received in revised form 25 February 2005; accepted 22 March 2005.

* Corresponding author. Tel.: +49 761 270 2455; fax: +49 761 270 2459. (Email: wulf.sienel{at}uniklinik-freiburg.de).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Objective: Detection of disseminated tumor cells in mediastinoscopic biopsies could improve staging and might be helpful concerning indications for neoadjuvant therapy regimens. This prospective study was performed to evaluate a simple and observer-independent polymerase chain reaction (PCR)-based method for the detection of disseminated tumor cells in regional lymph nodes. Methods: Lymph nodes of 32 consecutive patients without neoadjuvant therapy were removed by systematic lymphadenectomy during resection of primary NSCLC. One hundred of these lymph nodes were cut into two equal halves which were examined using either routine histopathology or quantitative reverse transcriptase PCR (qRT-PCR). qRT-PCR amplification of cytokeratin 19 (CK19) transcripts was applied for the detection of tumor cell-specific RNA. We differentiated between illegitimate marker gene transcription and cancer-specific expression by using a cut-off value that was obtained from the analysis of 18 lymph nodes of patients with benign lung diseases. Subsequent to the evaluation of qRT-PCR, a pilot project with five additional patients was conducted to examine 19 mediastinoscopic biopsies, which were cut into two equal halves and proceeded as described above. Results: Ninety-four (94%) lymph nodes were tumor-free by histopathology. qRT-PCR detected disseminated tumor cells in 26 (28%) of these lymph nodes. All of the remaining six lymph nodes that were judged by the pathologist to contain tumor cells exhibited CK19 transcripts. Twenty-three patients had a pN0 status. qRT-PCR detected disseminated tumor cells in 13 (56%) of these pN0 patients. The mediastinoscopic biopsies showed disseminated tumor cells in four (21%) out of 19 histopathologically tumor-free samples. Conclusions: CK19 qRT-PCR is a sensitive and specific tools for the detection of disseminated tumor cells in regional lymph nodes of patients with operable NSCLC. Further studies are required to asses if this molecular method might improve mediastinoscopic staging.

Key Words: Reverse transcriptase polymerase chain reaction • Lymph nodes • Staging • Lung neoplasm


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Multimodal concepts of anticancer therapy become increasingly popular even in early stages of non-small cell lung cancer (NSCLC). Previously, it has been shown that NSCLC patients with histologically proven N2/N3-status could benefit from neoadjuvant therapy [1,2]. Therefore, mediastinoscopical diagnosis of N2/N3-status may indicate neoadjuvant therapy regimens including chemotherapy. Cervical mediastinoscopy with lymph node biopsy remains the criterion standard for staging of the N2/N3 region [3,4].

The sensitivity of assessment of the N2/N3 region remains between 79 and 87% at a specificity of 100% using conventional histopathologic examination of mediastinoscopic biopsies [5,6]. Fluorodeoxyglucose-positron-emission tomography (FDG-PET) may increase the sensitivity up to 92% [7]; however, this method is hampered by a lower specificity of 76–78% [7,8]. Using serial sectioning and immunohistochemical staining of lymph-node biopsies improves the sensitivity of mediastinoscopy to 90% without limiting the specificity [9]. However, this method is laborious, time consuming and observer-dependent and therefore did not become a diagnostic standard. In consequence, alternative methods are required to improve the sensitivity of mediastinoscopy. Detection of individual disseminated tumor cells that are not discovered by conventional histopathologic methods potentially might increase the sensitivity of mediastinoscopy. Such individual tumor cells in mediastinal lymph nodes are of clinical impact since they correlate with poor prognosis [10–12].

Reverse transcriptase polymerase chain reaction (RT-PCR)-based tumor cell detection is an observer-independent, automated, rapid and versatile method for the detection of disseminated cancer cells [13]. Detection of vital cancer cells in clinical samples is approached by the amplification of specific mRNA transcripts, which are selectively expressed in the cancer cells of interest, but not in normal tissues. The presence of such transcripts is taken as indirect evidence for the presence of intact vital tumor cells within the sample, since once released from cells, RNA is rapidly degraded [14]. The major problem associated with RT-PCR is illegitimate transcription, a transcription of marker genes at a minimal basic level in normal tissues without necessarily being translated into detectable amounts of protein [15]. Quantitative RT-PCR (qRT-PCR) has the potential to solve this problem by setting a cut-off value using control samples to differentiate illegitimate marker gene transcription and cancer-specific expression.

Well-evaluated marker genes for the PCR-based detection of individual tumor cells that are expressed in NSCLC are carcinoembryonic antigen (CEA) [16] and cytokeratin 19 (CK19) [12,17]. Since only 73% of NSCLC are CEA positive [16] and on the other hand, CK19 has been reported to be expressed in 88–94% of primary tumors in NSCLC [18], CK19 was chosen as RT-PCR marker for the present study. So far, quantitative assessment of cytokeratin 19 (CK19) transcripts has not been used for the detection of individual tumor cells in lymph nodes of lung cancer patients. The CK19 qRT-PCR presented here exhibited a high sensitivity and specificity and detected individual cancer cells in lymph nodes of 56.5% out of 23 consecutive patients with pN0 NSCLC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
2.1. Patients
From April 2003 through March 2004 all patients with operable non-small cell lung cancer (NSCLC) and without neoadjuvant treatment regimens were enrolled into the study. All patients were operated at the department of Thoracic Surgery, Asklepios Fachkliniken Mü;nchen-Gauting, Munich, Germany and had given written informed consent. The study was approved by the Ethical Committee of the University of Munich.

The preoperative staging of all patients had resulted in cT1–T3 tumors without evident distant metastasis (cM0) and without mediastinal or supraclavicular lymph node involvement (cN0–N1). In general, a lobectomy or pneumonectomy with systematic mediastinal lymphadenectomy was performed. Nodes completely removed in the right chest included the 2R, 4R, 7, 8, 9, and 10R stations along with the rest of the N1 nodes contained in the part of the lung that was resected (11, 12, 13, and 14) [19]. In the left chest the stations completely resected included 5, 6, 7, 8, 9, and 10L stations, and the rest of the N1 nodes contained in the part of the lung resected (11, 12, 13, and 14). Generally, three lymph nodes per patient were cut into two equal halves which were examined using either routine histopathology or qRT-PCR. In five patients, four lymph nodes were obtained and included into the study. Exclusion of one patient whose primary tumor did not express the marker cytokeratin 19 (CK19) resulted in 100 eligible lymph nodes of 32 patients. The median weight of lymphaden-ectomy lymph node halves submitted to the study was 165mg (range 30–250mg).

Following tumor resection, 0.25cm3 samples of the primary tumors were excised for study purposes. The local pathologist classified the tumors according to the International Union Against Cancer's TNM-Classification. Primary tumors of 24 patients were available to control the expression of the marker gene. Of nine small primary tumors, no samples could be obtained for this study in order to prevent hampering of conventional histopathological assessment. The median weight of primary tumor samples analyzed in this study was 200mg (range 50–250mg).

Subsequent to the evaluation of CK19 qRT-PCR in lymhadenectomy specimens, a pilot project with five additional patients was conducted to examine mediastinoscopic biopsies. For this purpose, cervical mediastinoscopy was performed in patients with computertomographically enlarged mediastinal lymph nodes and the 2R, 4R, 2L, 4L and proximal 7 lymph nodes stations were biopsied resulting in 19 lymph node biopsies being cut into two equal halves which were examined using either routine histopathology or qRT-PCR. The median weight of mediastinoscopic biopsy halves submitted to the study was 33mg (range 9–69mg). All lymph node and tumor samples were stored in a RNA-stabilizing reagent (RNA-later, QIAGEN, Hilden, Germany) at –20°C.

2.2. Quantitative RT-PCR
For extraction of total RNA, 30–250mg tumor or lymph node tissue was frozen in liquid nitrogen, grinded thoroughly with a mortar and pestle and mixed with the ß-mercaptoethanol RNA extraction buffer of a commercial RNA isolation kit (RNeasy Midi Kit, QIAGEN, Hilden, Germany). Subsequently, RNA was extracted from the homogenate according to the manufacturer's protocol (RNeasy Midi Kit, QIAGEN, Hilden, Germany) and RNA concentration was detected using a Biophotometer. cDNA was produced in 20µl reactions from 500ng of RNA. Reaction mixtures consisting of 20 units reverse transcriptase, 1x reaction buffer, 1mM deoxynucleotide, 3mM MgCl2 and 4.0µg of random hexamers were used for reverse transcription of RNA into cDNA. The reactions were incubated at 25°C for 10min, 42°C for 1h, and 95°C for 5min. All reagents were obtained from a commercially available cDNA synthesis kit (1st strand cDNA synthesis kit for RT-PCR (AMV)+ (ROCHE DIAGNOSTICS, Mannheim, Germany)).

Subsequent to reverse transcription, quantitative PCR was performed in a LightCycler TM instrument (ROCHE DIAGNOSTICS, Mannheim, Germany) using a commercially available CK19 primer set (SEARCH-LC GmbH, Heidelberg, Germany). The intron-spanning primers were designed to maximize sequence differences between CK19 and its pseudogenes CKa and CKb [20]. For amplification of a stably expressed house keeping gene a commercially available human Cyclophilin B (CPB) primer set (SEARCH-LC GmbH, Heidelberg, Germany) was applied. Each 20µl quantitative PCR reaction contained 4µl of cDNA, 2µl of the primer mix and 2µl of the ‘FastStart-DNA Master Sybr Green’ (ROCHE DIAGNOSTICS, Mannheim, Germany) containing Sybr green, dNTPs, MgCl2 and reaction buffer as described in the manufacturer's data sheet. In the LightCycler instrument, each reaction capillary underwent a 10min incubation at 95°C before 40 cycles of 95°C for 10s, 68°C for 10s, and 72°C for 16s. The PCR run was finished with a melting curve analysis. The PCR reactions for CK19 and CPB were performed in separate capillaries. The absence of contamination was routinely controlled by quantitative PCR amplification of negative samples, in which RNA and cDNA was replaced with sterile water. The detected Sybr green fluorescence was analyzed by the LightCycler Analysis Software. The beginning of the detectable exponential PCR phase for each reaction were determined by the ‘fit point’ algorithm and arithmetic baseline adjustment as described by the LightCyclerTM manufacturer. CK19 concentrations were calculated in relation to the concentration of the reference house keeping gene CPB from the same sample material omitting the need for a standard with known concentrations and balancing potential differences of lymph node sample quality or cell count [21].

To determine PCR efficiency at different target RNA concentrations, standard curves for CK19 and CPB were created using serial dilutions (500, 100, 20, 8ng) of tumor RNA derived from 5x106 cells of the non-small-cell lung carcinoma cell line A549. Triplicate determinations were performed for each dilution step and each RNA sample. Five hundred nanograms of total A549 RNA was used in each LightCycler run as positive control and as calibrator for the comparison of different quantitative PCR runs. Calibrator-normalized relative CK19 quantities were obtained by dividing the CK19/CPB ratio of the patient sample by the CK19/CPB ratio of the calibrator sample as suggested by the LightCycler manufacturer [21]. PCR efficiency for calculation of CK19 quantities was determined using the Relative Quantification Software (ROCHE DIAGNOSTICS, Mannheim, Germany).

Specificity of the detection method was ensured by setting the cut-off value which differentiates between illegitimate marker gene transcription and cancer-specific expression above the level of CK19 transcript quantification in 18 lymph nodes of 13 patients with benign lung diseases. There were two smokers among these patients with operations for causes other than cancer. The highest relative CK19 ratio in those control samples was 0.179. Therefore, a relative CK19 ratio of 0.180 was set as cut-off value to differentiate between negative and positive CK19 qRT-PCR results.

2.3. Statistical analysis
Statistical analysis was performed using the SPSS software package, version 11.0 (SPSS, Inc., Chicago, USA). A comparison between patient parameters and the detection of disseminated tumor cells in lymph nodes was performed using cross-tables and the two-tailed Pearson's Chi-square test. The threshold for statistical significance was chosen at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Conference...
 References
 
3.1. Primary tumors and negative controls
Analysis of the 24 primary tumors showed CK19 expression ratios between 0.026 and 220.12 (Fig. 1 ). The CK19 expression of primary tumors did neither correlate with tumor type nor with grading. Relative CK19 quantities in 10 squamous cell carcinomas ranged from 0.34 to 220.12 (Mean 25.13; SD 68.57). In 11 adenocarcinomas, the CK19 expression ratio was insignificantly lower (Range 0.21–4.33; Mean 1.69; SD 1.16; P=0.27; t-test). The three examined large cell carcinomas showed CK19-RNA quantities of 0.026–3.60 (Mean 1.72; SD 1.79). Grading showed that in G1–G2 tumors CK19 was expressed at ratios between 0.21 and 8.62 (Mean 2.84; SD 2.73). Analysis of G3–G4 tumors revealed expression ratios of 0.32–220.12 (Mean 23.86; SD 68.97) that were not significantly increased in comparison to G1–G2 tumors (P=0.32; t-test). According to the qRT-PCR cut-off value which differentiates between illegitimate marker gene expression and cancer-specific expression, the large cell carcinoma with the relative CK19 expression ratio of 0.026 was considered as CK19-negative (Fig. 1) resulting in 23 (95.8%) CK19-positive primary tumors. The cut-off value was set at a CK19 expression ratio of 0.18 (Fig. 1) according to the qRT-PCR values of lymph nodes of patients without malignancy resulting in negative CK19 qRT-PCR results in all 18 lymph nodes of patients without malignancy (Table 1 ). The patient with a CK19-negative primary tumor was excluded from further analyses resulting in 100 eligible lymph nodes of 32 patients.



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  		Fig. 1. Relative ratios of CK19 expression in histopathological tumor-free lymph nodes of lymphadenectomy specimens ‘LNp-NSCLC’, histopathological tumor-involved lymph nodes of lymphadenectomy specimens ‘LNp+NSCLC’, mediastinal lymph nodes of patients with benign lung disease ‘LN neg. control’, tumor-free lung tissue ‘Lung’ and primary NSCLC tumors ‘Tumor’. Relative CK19 ratios were calculated by dividing the CK19/CPB ratio of the patient sample by the CK19/CPB ratio of the constant calibrator sample deriving from an A549 lung cancer cell line. According to the quantitative PCR values of negative control lymph nodes, a relative CK19 ratio of 0.180 was set as cut-off value to differentiate between negative and positive CK19 qRT-PCR results (dashed line).

 

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  		Table 1. Detection of cytokeratin 19 (CK19) transcripts a in lymph nodes and in primary NSCLC
 
3.2. Lymphadenectomy specimens
One-hundred lymph nodes collected of 32 patients at time of tumor resection with systematic lymphadectomy were eligible for CK19 qRT-PCR. Among 94 histopathological tumor-free lymph nodes, qRT-PCR detected CK19 transcripts in 26 (28%) lymph nodes. All six available histopathologically tumor-positive lymph nodes exhibited positive CK19 qRT-PCR results. Nineteen (59.4%) patients harbored CK19 transcripts in their lymphadenectomy specimens. Analysis of patient characteristics revealed that there was no association of pT-status with CK19 qRT-PCR results in lymph nodes: 59.3% of patients with pT1–pT2 tumors and 60.0% of patients with pT3–pT4 tumors showed CK19 transcripts in their lymph nodes (P=1.00; Fisher's exact test; Table 2 ). Conventional histopathological examination of lymphadenectomy specimens showed that 23 (72%) patients had a pN0 status. qRT-PCR detected individual disseminated tumor cells in 13 (56.5%) of these pN0 patients. Thus, CK19 qRT-PCR would lead to an up-staging of 13 patients, if molecular lymph node assessment was considered in the stage classification (Table 3 ). In patients with pN1–pN2 disease, disseminated tumor cells were observed in all six patients from whom histopathologically positive lymph nodes were available for qRT-PCR (Table 2). The remaining 21 available lymph nodes of the nine pN1–pN2 patients were negative using conventional histopathology and using qRT-PCR. Therefore, no up-staging would occur in the pN1–pN2 group if qRT-PCR was considered in the stage classification (Table 3). Analysis of tumor histology showed that 50.0% of squamous cell carcinomas, 63.6% of adenocarcinomas and 80.0% of large cell carcinomas caused a positive CK19 RT-PCR in the lymphadenectomy specimens (P=0.46; Fisher's exact test; Table 2). According to primary tumors' grade of differentiation, disseminated tumor cells in lymph nodes were observed in 56.3% of G1–G2 tumors and in 54.5% of G3–G4 tumors (P=0.62; Fisher's exact test; Table 2). Furthermore, there was no correlation between detection of CK19 transcripts in lymphadenectomy specimens and patient gender or age (P=0.45 and 0.72, respectively; Fisher's exact test; Table 2).


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  		Table 2. Patient characteristics according to the detection a of cytokeratin 19 (CK19) transcripts in lymph nodes of 32 patients with operable NSCLC b
 

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  		Table 3. Comparison between staging basing on conventional histopathology and on molecular lymph node assessment of lymphadenectomy specimens deriving from tumor resections
 
3.3. Mediastinoscopic biopsies
So far, mediastinoscopic biopsies of 19 lymph nodes were obtained from five patients with computertomographically enlarged mediastinal lymph nodes and without neoadjuvant therapy. Histopathological examination of all examined mediastinoscopic biopsies showed tumor-free lymph nodes. However, CK19 qRT-PCR detected disseminated tumor cells in four (21%) of the mediastinoscopic samples. All positive CK19 qRT-PCR results derived from one patient with a primary adenocarcinoma of the right lower lobe and were observed in the 2R (2x) 2L (1x) and 4L (1x) lymph nodes stations. Subsequent to mediastinoscopy, all five patients underwent lobectomy with systemtic lymphadenectomy since conventional histopathology reported tumor-free mediastinoscopic lymph node biopsies. Interestingly, the patient with CK19 transcripts in mediastinoscopic biopsies of the N2- and N3-region harbored histopathologically visible tumor cells in the lymphadenectomy specimen resulting in a pT3 pN2 M0 R0 G2 stage (Table 4 ). The patients with a negative CK19 qRT-PCR in their mediastinoscopic biopsies showed no histopathological involvement of their mediastinal lymph nodes at time of tumor resection. Histopathologic examination of tumor resection specimens revealed involvement of the N1 region in one patient, however, this lymph node region was not reachable by cervical mediastinoscopy. Therefore, CK19 qRT-PCR in mediastinoscopic biopsies resulted neither in false-positive nor in false-negative predictions of involvement of mediastinal lymphadenectomy samples. According to the pN-status at time of tumor resection, qRT-PCR-based examination of mediastinoscopic biopsies was more sensitive than conventional histopathology at the same level of specificity.


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  		Table 4. Correlation between conventional histopathological and molecular lymph node assessment at mediastinoscopy and at tumor resection
 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Conference...
 References
 
Regional lymph node metastasis is one of the most important risk factors for tumor recurrence in patients with completely resected NSCLC [19]. However, the frequency of lymph node involvement is underestimated in many cases, since single metastatic tumor cells will not be detected by routine histopathological examination. Various studies have demonstrated that the detection of such disseminated tumor cells in the lymph nodes of patients with histopathologically ‘node-negative’ disease is of prognostic significance in NSCLC [22,23]. This occult tumor spread most likely arises early during cancer evolution, a long time before diagnosis of apparently localized primary tumors [24]. Detection of single disseminated tumor cells prior to resection is important since it might indicate to study neoadjuvant therapy regimens with the aim of eliminating these occult tumor deposits. Such multimodal concepts of anticancer therapy become more and more popular even in early stages of NSCLC since it has been shown that adjuvant chemotherapy in operable NSCLC may result in an approximately 4% increased 5-years survival rate [25]. For the assessment of lymphatic tumor spread to the N2/N3 region prior to tumor resection cervical mediastinoscopy is the criterion standard [3,4]. Detection of individual disseminated tumor cells that are not discovered by conventional histopathologic methods potentially might increase the sensitivity of mediastinoscopy.

Several techniques are available for the detection of tumor cells in regional lymph nodes of NSCLC patients. Immunohistochemical techniques using antibodies against epithelial-specific proteins are well established [9,26,27]. However, immunohistochemical approaches require to screen a large number of specimens [9], and the examined lymph node sections probably represent random samples of a lymph node. Since immunohistochemistry is relatively laborious, time consuming and observer-dependent, it did not become a diagnostic standard to clinicians. In contrast to immunohistochemistry, RT-PCR provides the possibility to analyze whole lymph nodes in one reaction instead of screening a large number of lymph node sections. It is an automated, rapid and versatile method for the detection of disseminated cancer cells [13]. However, its specificity might be hampered by illegitimate expression of the respective marker gene in normal lymph nodes [15]. Illegitimate transcription of marker genes occurs at a minimal basic level in normal tissues without necessarily being translated into detectable amounts of protein [15]. Quantitative RT-PCR (qRT-PCR) has the potential to solve this problem by setting a cut-off value using control samples to differentiate illegitimate marker gene transcription and cancer-specific expression. In the present study, qRT-PCR was applied and low level transcription in lymph nodes of patients without malignancy was observed up to a CK19 expression ratio of almost 0.180 (Fig. 1). This value was set as cut-off value for the detection of cancer-specific expression reducing the risk of false-positive assessment of illegitimate transcripts. This is the first CK19 qRT-PCR study in lymph nodes from NSCLC patients setting a cut-off value for the detection of single disseminated tumor cells to guarantee a high specificity of the method.

CK19 was chosen as RT-PCR marker for the present study. CK19 is a specific cytoskeletal structure of simple epithelia, including bronchial epithelial cells. CK19 is a stably and abundantly expressed polypeptide of epithelial cells. At least 20 different types of cytokeratins have been identified on the basis of differences in molecular weight, and pH. CK19 qRT-PCR had already been used to detect disseminated tumor cells in blood and bone marrow of breast cancer patients [28], but no related research is reported in lymph nodes of operable lung cancer using qRT-PCR. Therefore, CK19 qRT-PCR was evaluated in lymphadenectomy specimens of tumor resections for pT1–4 pN0–2 cM0 NSCLC. Tumor-associated transcripts were detected in 19 (59.4%) operable patients. Even 13 (56.5%) of the patients with histopathological tumor-free lymphadenectomy specimens harbored transcripts of single disseminated tumor cells in their regional lymph nodes. These frequencies of disseminated individual tumor cells are comparable to the detection rate of other qRT-PCR methods (43–70%) [16,29] and were higher than those reported in immunohistochemical studies (21–30%) [9,26,27]. Therefore, the applied CK19 qRT-PCR is of high sensitivity. According to the cut-off value of the present study, no tumor-associated transcripts were present in 18 lymph nodes of patients without malignancy demonstrating a high specificity of the method. Since the specificity of the method depends on the cut-off value which differentiates between detection cancer-specific transcripts and false-positive illegitimate transcripts, the specificity of the described method should to be proven in a further independent control study.

Since CK19 is not expressed in all NSCLCs [18], primary tumors of the patients in the present study were analyzed to control expression of the used marker transcript CK19. The relative CK19 expression ratio of one large cell lung carcinoma was lower than the cut-off value indicating a negative qRT-PCR result. CK19-positive disseminated tumor cells theoretically may occur in CK19-negative primary tumors if the tumor lost CK19 expression during its evolution subsequent to early dissemination. So far, no evident data exist concerning this issue and to prevent any misinterpretation in CK19-negative primary tumors, such cases were excluded from the present study (n=1). This experience shows that for the detection of disseminated tumor cells by qRT-PCR in NSCLC, currently analysis of the marker gene expression in primary tumors should be performed to prevent false-negative results.

No correlation between the presence of disseminated tumor cells in lymphadenectomy specimens and clinicopathological parameters were observed (Table 2). This result goes conform with previous immunohistochemical studies in other populations of operable NSCLC [22,26]. It strengthens the hypothesis, that dissemination of single tumor cells is an early event in the evolution of NSCLC disease [24], because it is not influenced by later tumor progress leading to tumor extension or dedifferentiation.

In the present study, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of molecular examination of mediastinoscopic biopsies may neither be calculated nor be compared with the respective parameters of conventional histopathologic lymph node assessment due to the small study population in the mediastinoscopy group. Nevertheless, conventional lymph node examination at mediastinoscopy in Patient 2 did not predict the pN2-status at tumor resection whereas molecular assessment of the same mediastinoscopy samples showed involvement of the N2 and even of the N3 region. This observation suggests that molecular examination of mediastinoscopic biopsies tends to increase the sensitivity of mediastinoscopy. However, this conclusion relies on a limited population in the mediastinocopy group and has to be verified by larger studies. By evaluation of CK19 qRT-PCR in lymphadenectomy specimens this study generated the prerequisites for the screening of mediastinoscopic biopsies for occult disseminated tumor cells.


   Appendix A. Conference discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Conference...
References
 
Dr M. Dusmet (London, United Kingdom): I have a comment and a question. First the comment. As to sensitivity of mediastinoscopy, one of the major problems is that unfortunately not everybody does a complete, thorough staging mediastinoscopy, and the best way to improve the sensitivity of mediastinoscopy is to turn it into a very systematic approach to staging of the mediastinum.

Now, my question. All cytokeratins are expressed to a variable degree in adenocarcinoma, squamous cell carcinoma, and poorly differentiated non-small cell cancer. What percentage of squamous cell, adeno, and large cell undifferentiated cancers express cytokeratin 19?

Dr Sienel : Concerning the first comment, we completely agree that a systematic mediastinoscopy is absolutely important.

According to your question, I must state that, of course, some cytokeratins are expressed differently in the different types, like adenocarcinoma and squamous cell carcinoma of the lung. For example, cytokeratin 20 is not expressed in adenocarcinoma of the lung and can be used for the differentiation between colorectal and pulmonary adenocarcinoma, but cytokeratin 19 was expressed in almost all kinds of tumor types that we examined. Our results showed that adenocarcinoma and squamous cell carcinoma were all cytokeratin-19-positive. Only one large cell carcinoma out of three examined large cell carcinomas was cytokeratin-negative.


   Footnotes
 
{star} Presented at the joint 18th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual Meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12–15, 2004.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 

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