Detection ofMAGE-ATranscripts in BoneMarrow Is an Independent Prognostic Factor in Operable Non ^ Small-Cell Lung Cancer

Purpose:MAGE-A gene expression in humans is mostly restricted to tumor cells, and the role of MAGE-A transcripts andpeptides as diagnosticmarkers and therapeutic targets is currently under investigation. Thus far, the clinical relevance of MAGE-A transcripts as marker for disseminated tumor cells in bonemarrowof patients with operable lung cancer without overt metastases is still unclear. Experimental Design: Preoperative bone marrow aspirates from 50 consecutive patients with operable non ^ small-cell lung cancer free of distant metastases (i.e., pT1-4 pN0-2 M0 R0) were admitted to the study. Each bone marrow sample was divided and examined using multimarker MAGE-A reverse transcription-PCR (RT-PCR) and immunocytochemical staining with the antipancytokeratin antibodyA45-B/B3. Multimarker MAGE-A RT-PCR consisted of multiple subtype-specific nested RT-PCRs with primers for MAGE-A1, MAGE-A2, MAGE-A3/6, MAGE-A4, andMAGE-A12.The median follow-up duration was 92 months (range,18-110 months). Results:Twenty-six (52%) lung cancer patients harboredMAGE-A transcripts in their bonemarrow, as opposed to none of the 30 healthy controls tested. In all 7 patients with immunocytochemically positive bone marrow, MAGE-A transcripts were also detected. All different MAGE-A subtypes (MAGE-A1, MAGE-A2, MAGE-A3/6, MAGE-A4, and MAGE-A12) were observed. Sixty-five percent of patients with MAGE-A transcripts in bone marrow exhibited only one subtype. Univariate (P = 0.03, log-rank-test) andmultivariate survival analysis showed thatMAGE-A transcripts in bonemarrowwere associatedwithpoor outcome inpN0 patients (P =0.02; relative risk, 7.6). Conclusions: Detection of MAGE-A transcripts in bone marrow predicts an unfavorable outcome in patients with early-stage operable lung cancer.This finding indicates that MAGE-A transcripts are clinically relevant markers of micrometastatic spread in lung cancer and supports further investigation of MAGE-A as potential future therapeutic target. The failure to reduce mortality of operable solid tumors most likely results from early dissemination of cancer cells, which is usually missed by conventional staging procedures at the time of surgery. Because adjuvant therapy aims to eradicate occult disseminated tumor cells (DTC) before metastatic disease becomes clinically evident, the early detection of tumor cell dissemination could identify patients who might benefit from such treatment (1). Over the past 10 years, various assays have been developed to detect micrometastatic spread at the single cell level in patients with solid tumors including non–smallcell lung cancer (NSCLC; refs. 2–6). However, the specificity and clinical significance of these assays are still under investigation. Moreover, markers that also represent potential therapeutic targets might be of broader clinical interest to facilitate not only the detection of micrometastatic spread but also its eradication to prevent metastatic relapse. Since the identification of the first MAGE gene in 1991 (7), the MAGE family has expanded to more than 25 genes that have been identified in humans (8). The functional role of most family members remains uncharacterized although they may play a role in embryonal development and tumor transformation or in certain aspects of tumor progression (9–14). Despite their normally restricted expression, many of the MAGE genes are expressed in a wide variety of histologically distinct tumors, likely because of transformation-dependent loss of the DNA methylation that normally silences these genes (15, 16). Thus, MAGE transcripts seem to be ideal markers for occult tumor cells that could be detected by a reverse transcription-PCR (RT-PCR) assay. Imaging, Diagnosis, Prognosis Authors’Affiliations: Department ofThoracic Surgery, Albert-LudwigsUniversity Freiburg, Freiburg, Germany; Institute for Immunology, Ludwig-Maximilians University Munich, Munich, Germany; and Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Received10/16/06; revised 2/28/07; accepted 4/18/07. Grant support: Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, and Sander Stiftung, Germany. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note:W. Sienel and I. Mecklenburg contributed equally to this work and share the first authorship. Requests for reprints:Wulf Sienel, Department of Thoracic Surgery, University Hospital Freiburg, Albert-Ludwigs-University Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. Phone: 49-761-270-2401; Fax: 49-761-270-2499; E-mail: wulf.sienel@uniklinik-freiburg.de. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-2507 www.aacrjournals. org Clin Cancer Res 2007;13(13) July1, 2007 3840 Research. on August 4, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals. org Downloaded from Among the MAGE families, the cluster MAGE-A, which has 12 subtypes (MAGE-A1-A12), has been observed most frequently in malignant tumors and has been characterized the best (5, 8, 11). Individual MAGE-A subtypes are expressed too sporadically and too weakly to be suitable for the detection of occult tumor cells (6); however, most carcinomas express at least one MAGE-A subtype (5, 6). Therefore, recent studies for the detection of occult tumor cells used either PCR primers that can simultaneously detect multiple MAGE-A subtypes in one reaction (5, 17) or multimarker RT-PCR settings with different MAGE-A primer pairs (6, 18). Using these MAGE-A RT-PCR assays, DTCs in bone marrow have been detected in 11 of 33 (33%) examined lung cancer patients without overt metastases (cM0; ref. 6), in 22 of 136 (16%) examined breast cancer patients without overt metastases (cM0; ref. 5), and in 18 of 30 (60%) of prostate cancer patients without distant metastases (cM0; ref. 6). However, the key question on whether DTCs detected by MAGE-A RT-PCR are associated with a poor clinical outcome has not been investigated thus far. Therefore, we analyzed the survival in operable NSCLC, which is the most aggressive entity of the cancers mentioned above, and compared the detection of MAGE-A transcripts in bone marrow to the observed cancer-related survival. Bone marrow is known to be a common homing organ of disseminated epithelial tumor cells (1) and a frequent site of metastasis in lung cancer. We focused on operable patients treated by complete resection without neoadjuvant therapy to minimize treatment bias on survival. Because of the severe prognostic effect of manifest distant or lymph node metastases in lung cancer (19), we focused on operable pN0 cM0 lung cancer patients. Here, we report for the first time that the detection of MAGE-A transcripts in bone marrow is an independent prognostic factor in earlystage operable NSCLC.