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Eur J Cardiothorac Surg 2004;25:131-134
© 2004 Elsevier Science NL

Melanoma associated antigen (MAGE)-A3 expression in Stages I and II non-small cell lung cancer: results of a multi-center study

^a Klinik für Thoraxchirurgie, Asklepios Fachkliniken München-Gauting, Gauting, Germany
^b Department of Surgery, Chirurgische Klinik und Poliklinik – Innenstadt, University of Munich, Nussbaumstrasse 20, 80336 Munich, Germany
^c Abteilung für Thoraxchirurgie, Lungenklinik Hemer, Hemer, Germany
^d Abteilung für Thoraxchirurgie, Zentralklinik Emil von Behring, Berlin, Germany
^e Klinik für Thoraxchirurgie, Zentralkrankenhaus Bremen Ost, Bremen, Germany
^f Glaxo SmithKline Biologicals, Rixensart, Belgium
^g Zentrum für Thoraxchirurgie, Ruhrlandklinik, Essen, Germany

Received 28 April 2003; received in revised form 17 August 2003; accepted 22 September 2003.

^* Corresponding author. Tel.: +49-89-5160-2511; fax: +49-89-5160-4437
e-mail: wulf.sienel{at}ch-i.med.uni-muenchen.de

	Abstract

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
Objectives: Adjuvant immunotherapy is an innovative therapeutic option that might potentially improve outcome of early-stage non-small cell lung cancer. Melanoma associated antigen (MAGE)-A3 is a promising target for immunotherapy because it is exclusively presented on the cell surface of cancer cells and might be associated with an aggressive cancer phenotype. The present study was performed to determine the rate of MAGE-A3 expression in early-stage non-small cell lung cancer (NSCLC). Patients and methods: Primary tumor samples from 204 patients with operable clinical Stages I or II NSCLC were collected between March and November 2001. Pathological Stage was determined by the local pathologist in each of the 16 participating institutions. Tissue samples were stored immediately after surgery in a RNA-stabilizing solution and were frozen at -20°C. MAGE-A3 expression was analyzed by detection of MAGE-A3 transcripts using reverse-transcriptase polymerase chain reaction. Results: MAGE-A3 expression was observed in 80 out of the 204 (39.2%) examined Stages I–II primary tumors. Stratification into UICC-Stages showed that 31 out of 105 (29.5%) Stage I non-small cell lung cancers and 49 out of 99 (49.5%) Stage II non-small cell lung cancers expressed MAGE-A3. In comparison to Stage I, the rate of MAGE-A3 positive tumors was significantly increased in Stage II (P=0.004; Chi-square test). Conclusion: The MAGE-A3 expression rate showed that a promising proportion of operable patients with early-stage non-small cell lung cancers are possible candidates for trials investigating adjuvant therapy with MAGE-A3 immunization. Currently, a phase two trial of adjuvant MAGE-A3 vaccination is in progress.

Key Words: Immunotherapy • Non-small-cell cancer • Melanoma associated antigen-A3 expression

	1. Introduction

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
Despite immense medical progress, 5-year survival rates in operable non-small cell lung cancer (NSCLC) remain at only 50–60% in Stages I and II [1,2]. Adjuvant immunotherapy is an innovative therapeutic option that might potentially improve outcome [3,4]. A better understanding of tumor immunology [5,6] lead to multiple ongoing Phases I, II or III immunotherapy trials in NSCLC [7]. Tumor biologic considerations suggest that immunotherapy might be more effective in patients with low tumor burden [3,8] like present in Stages I and II NSCLC.

The melanoma associated antigen (MAGE)-A gene family is a promising target for a specific immunotherapy because MAGE-A is expressed in cancer cells, but not in normal tissue, except male germ line cells, which are devoid of major histocompatibility complex molecules and therefore do not present MAGE-A antigens [9,10]. MAGE-A3 codes for an antigenic nonapeptide that is recognized by cytolytic T cells on the human leukocyte antigen (HLA)-A1 molecule [11]. About 26% of the European population carries the HLA-A1 allele [8] and are therefore potential candidates for an anti-MAGE-A3 immunotherapy.

The major prerequisite for adjuvant anti-MAGE-A3 immunotherapy in operable NSCLC is the expression of MAGE-A3 in NSCLC cells. Previous studies investigating the rate of MAGE-A3 expression in NSCLC were only performed in a few patients with early-stage NSCLC [12,13], and thus were of limited value for planning an anti-MAGE-A3 immunotherapy trial. Therefore, the present multi-center study was initiated to determine the rate of MAGE-A3 expression in a large population of Stages I and II NSCLC patients.

	2. Patients and methods

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
2.1. Patients
Sixteen European Departments of Thoracic Surgery participated in this multi-center study (listed in the Appendix A). After approval by the ethical committee and after written informed consent, 204 consecutive patients with completely resected Stage I (T1N0) or Stage II (T1/2N0/1 or T3N0) NSCLC were admitted to the study. Eligibility criteria were limited to Stages I and II because tumor biologic considerations suggest that immunotherapy might be more effective in patients with low tumor burden [3,8]. Primary tumor specimens were collected intraoperatively between March and November 2001. Subsequent to tumor resection, 1 cm³ samples of the primary tumors were excised and stored in a RNA-stabilizing solution at -20°C. Subsequently, the specimens were shipped to the central laboratory in Belgium for analysis of MAGE-A3 expression. The tumors were classified according to the international union against cancer's TNM-classification [2]. The preoperative staging of all patients had resulted in T1–T3 tumors without evident distant metastasis (M0) and without ipsilateral mediastinal or contralateral mediastinal or supraclavicular lymph node involvement (N0–N1). In general, a lobectomy or pneumonectomy with systematic mediastinal lymphadenectomy was performed. Postoperative pathological tumor stages were determined by the local pathologist in each of the participating institutions. Five patients had to be excluded because of incomplete clinical data and four patients were postoperatively classified as Stage III resulting in 105 eligible Stage I patients and 99 eligible Stage II patients.

2.2. Analysis of MAGE-A3 expression
Primary tumor samples were stored and shipped in RNA-stabilizing solution (RNA-later, Ambion, Cambridgeshire, UK). Subsequently, total cellular RNA was extracted from about 100 mg of primary tumor using a TRI-Reagent [14] (Tripure, Roche, Mannheim, Germany) according to the manufacture's instructions except that the isopropanol precipitation was replaced by RNeasy purification (Qiagen, Hilden, Germany). RNA concentration was determined from the optical density value at 260 nm. cDNA synthesis from 2 µg of total RNA was performed in a 20 µl mixture containing 0.5 mM of each dNTP, 10 mM of dithiotreitol, 20 U of rRNase inhibitor (Promega, Mannheim, Germany), 2 µM of oligo (dT) primers and 200 U of moloney murine leukemia virus reverse transcriptase (Invitrogen – Life Technologies, Karlsruhe, Germany) for 1 h 30 at 42°C. cDNA corresponding to 50 ng of total RNA was amplified by polymerase chain reaction (PCR) with the sense primer AB1197: 5'-TGGAGGACCAGAGGCCCCC-3' and the antisense primer BLE-5: 5'-GGACGATTATCAGGAGGCCTGC-3' as described previously [15]. Ten µl aliquots of the PCR products were analyzed using agarose gel electrophoresis and visualization of DNA bands by ethidium bromide fluorescence. RNA amplicons are expected at a size of 725 bp and are clearly distinguishable from genomic DNA amplicons which are expected at a size of 805 bp. A tumor sample was considered MAGE-A3 positive when the amount of 725 bp amplicons was equal or greater than the amount of amplicons obtained using 0.5 ng total cellular RNA of MZ-2-3.0 cell culture cells, which overexpress MAGE-A3. Two further positive controls were included: MAGE-A3 genomic DNA cloned into a cosmid vector and a 10-fold dilution of MZ-2-3.0 cDNA. RNA-free water submitted to the RNA extraction, RNA-free water submitted to the reverse transcription and water submitted to the PCR served as negative controls.

2.3. Statistical analysis
Statistical analysis was performed using the SPSS software package, version 11.0 (SPSS, Inc, Chicago, USA). Two-tailed Pearson's Chi-square test was used to analyze the association between MAGE-A3 expression and the postoperative pathological Stage. The threshold for statistical significance was chosen at P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
Table 1 summarizes the results of the MAGE-A3 expression analysis in the eligible lung cancer patients. Reverse-transcriptase (RT)-PCR analysis of the primary tumor samples revealed a MAGE-A3 expression in 80 out of the 204 (39.2%) examined Stages I–II primary tumors. Stratification into UICC-Stages showed that 31 out of 105 (29.5%) patients with Stage I non-small cell lung cancer were MAGE-A3-positive. In Stage II, MAGE-A3 was expressed in 49 out 99 (49.5%) primary tumors. In comparison to Stage I, the rate of MAGE-A3 expression was significantly increased in Stage II (P=0.004; Chi-square test). Four additional patients who preoperatively showed clinical Stages I–II tumors were postoperatively classified as Stage III. However Stage III tumors were not eligible because tumor biologic considerations suggest that immunotherapy is more effective in patients with low tumor burden. Two (50%) of the four Stage III-patients exhibited a positive MAGE-A3 expression.

	4. Discussion

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
Although antibodies for the detection of MAGE-A expression have recently become available [16,17], they are not specific for individual MAGE-A proteins. Therefore, for the analysis of the MAGE-A3 expression in operable NSCLC, a RT-PCR assay was applied. It revealed that 31 out of 105 (29.5%) patients with Stage I NSCLC and 49 out of 99 (49.5%) patients with Stage II NSCLC expressed MAGE-A3 in their primary tumors. The rate of MAGE-A3 expression was significantly increased in Stage II (P=0.004; Chi-square test), suggesting that MAGE-A3 expression might be associated with a progression towards higher stages. Recent studies have reported a frequent MAGE-A expression in cancer progression [18,19] leading to the hypothesis of a tumor promoting effect of MAGE-A. This hypothesis goes conform with our observation of a higher rate of MAGE-A3 positive tumors in Stage II than in Stage I. However, it recently turned out that a higher frequency of MAGE-A expression is linked to a random demethylation of the MAGE-A genes [20]. Because demethylation is a normal process during cancer progression, the latter report argues against the hypothesis of a tumor promoting effect of MAGE-A.

Immunotherapy against a tumor target that is exclusively presented on the surface of tumor cells seems promising. MAGE-A3 is the most silent gene in germ line cells within the MAGE-A family and is therefore particularly suited as target of immunotherapy [11]. MAGE-A3 is only recognized by cytolytic T cells if it is presented on the HLA-A1 molecule [11]. Therefore, to be eligible for an adjuvant vaccination with MAGE-A3, candidates must both carry the HLA-A1 allele and express MAGE-A3 in their primary tumors. In Europe, about 26% of operable NSCLC patients carry the HLA-A1 allele and are therefore potential candidates for an anti-MAGE-A3 immunotherapy [8].

The second prerequisite for adjuvant anti-MAGE-A3 immunotherapy is the expression of MAGE-A3 in NSCLC cells. Previous studies investigating the rate of MAGE-A3 expression in NSCLC were only performed in a few patients [13] with early-stage NSCLC, and thus were of limited value for planning an anti-MAGE-A3 immunotherapy trial. Since the aim of the present study was restricted to examination of the rate of MAGE-A3 expression in tumors of potential candidates for an adjuvant immunotherapy trial in early-stage NSCLC, correlation with tumor parameters such as pT-status, pN-status and tumor histology was not intended. The present study revealed that about 30% Stage I NSCLC and about 50% Stage II NSCLC express MAGE-A3. Previously, MAGE-A3 expression was detected in nine out of 16 (56%) NSCLC [17] using immunohistochemical staining of primary tumors, however the applied antibody has been reported to react weakly with MAGE-A1, -A4, -A6 and -A12 antigens as well [17]. This antibody cross reactivity is one possible reason for the high expression rate of 56% contrasting the observed expression rate of 30–50% in the present study. Another explanation might be, that Jungbluth et al. examined higher stages, however, postoperative staging was not reported in his study. Recently, also RT-PCR approaches have been applied for the investigation of MAGE-A3 expression, however in a limited number of NSCLC [13]. They revealed a MAGE-A3 expression in five out of 12 (41.7%) NSCLC [12]. The reported RT-PCR method was performed reliably and the observed rate of MAGE-A3 expression was comparable to the rate of 30–50% in the present study.

This study showed that that about 30% Stage I patients and about 50% Stage II patients express MAGE-A3 in their primary NSCLC and are therefore possible candidates for trials investigating adjuvant therapy with active MAGE-A3 immunization if they carry the HLA-A1 allele. Recently, a clinical trial of anti-MAGE-A3 immunotherapy in malignant melanoma demonstrated, that vaccination with a MAGE-A3 peptide can induce tumor regression [8]. Currently, a multi-center phase two trial of adjuvant MAGE-A3 vaccination in operable NSCLC is in progress in the participating institutions of the present study.

	Acknowledgments

 
We thank all participating surgeons, pathologists and co-workers for their contribution to this work.

	Footnotes

 
Presented at the 10th Annual Meeting of the European Society of Thoracic Surgeons, Istanbul, Turkey, October 26–28, 2002.

	Appendix A. Participating institutions (in alphabetical order)

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 
S. Eggeling, Oskar-Ziethen Krankenhaus, Berlin, Germany.

C. Engelmann, Fachkrankenhaus für Lungenheilkunde und Thoraxchirurgie, Berlin-Buch, Germany.

A. Fisseler-Eckhoff, Institut für Pathologie, Horst-Schmidt-Kliniken Wiesbaden, Wiesbaden, Germany.

G. Friedel, Klinik Schillerhöhe, Stuttgart-Gerlingen, Germany.

H. Hoffmann, Thoraxklinik Heidelberg, Heidelberg, Germany.

A. Holzgreve, Krankenhaus Neukölln, Berlin, Germany.

U. Keilholz, Universitätsklinikum Benjamin-Franklin, Freie Universität Berlin, Germany.

D. Kaiser, Abteilung für Thoraxchirurgie, Zentralklinik Emil von Behring, Berlin, Germany.

A. Linder, Abteilung für Thoraxchirurgie, Lungenklinik Hemer, Hemer, Germany.

B. Passlick, Klinik für Thoraxchirurgie, Asklepios Fachkliniken München-Gauting, Gauting, Germany.

A. Pluzanska, Hospital M. Kopernik, Poland.

G. Stamatis, Zentrum für Thoraxchirurgie, Ruhrlandklinik, Essen, Germany.

C. Steppert, Klinik für Erkrankungen der Atmungsorgane, Bezirksklinikum Obermain-Kutzenberg, Ebensfeld, Germany.

E. Stoelben, Abteilung für Lungenchirurgie, Chirurgische Universitätsklinik Freiburg, Freiburg, Germany.

L. Strens, UZ-Gasthuisberg, Gasthuisberg, Belgium.

M. Teschner, Klinik für Thoraxchirurgie, Zentralkrankenhaus Bremen Ost, Bremen, Germany.

	References

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion Appendix A. Participating... References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sienel, W. Articles by Passlick, B. Search for Related Content PubMed PubMed Citation Articles by Sienel, W. Articles by Passlick, B. Related Collections Lung - cancer