The melanoma antigen (MAGE)-encoding genes are expressed in various tumor types, including lung, and are thought to be silent in all normal tissues except testis. In search of biomarkers for early lung cancer detection and cancer risk assessment, we investigated frequencies of expressional activation of MAGE-A1, -A3, and -B2 genes in non-small cell lung cancers (NSCLCs). Expression of these genes was evaluated by reverse transcription-PCR (RT-PCR) in 20 primary NSCLC samples and corresponding normal lung tissues as well as in 20 bronchial brush specimens from former smokers without lung cancer. mRNA in situ hybridization was done to confirm the gene expression pattern at the cellular level. Methylation-specific PCR was performed to evaluate the hypomethylation status of CpG sites in the promoter regions of these genes. Among the 20 primary NSCLC samples analyzed, 14 (70%) expressed MAGE-A1 and 17 (85%) each expressed MAGE-A3 and MAGE-B2. A substantial number of normal lung tissues adjacent to NSCLC also had a detectable level of MAGE expression (65, 75, and 80% for MAGE-A1, -A3, and -B2, respectively). We found that 7 (35%), 10 (50%), and 11 (55%) of the adjacent normal lung tissue samples exhibited promoter hypomethylation at MAGE-A1, -A3, and -B2, respectively, compared with 15 (75%), 16 (80%), and 16 (80%) of the NSCLC samples. Among the 20 bronchial epithelium samples from former smokers, 7 (35%), 10 (50%), and 12 (60%) had also detectable -A1, -A3, and -B2 expression, respectively. Activation of MAGE-A1, -A3, and -B2 genes is common not only in NSCLC but also in bronchial epithelium with severe carcinogen insult. These results suggest that MAGE genes may be activated very early in lung carcinogenesis and may be considered as targets for lung cancer prevention.

The human MAGE3 gene family consists of a large number of chromosome-X-linked genes originally identified because they encode products that can be recognized by autologous cytotoxic T cells (1, 2, 3, 4, 5). The function of MAGE proteins is not known, but because MAGE genes are not expressed in normal adult tissues except testis and are expressed in a large variety of neoplastic lesions, MAGE genes are considered tumor-specific antigens and ideal targets for cancer immunotherapy (6, 7). In search of biomarkers for early lung cancer detection and cancer risk assessment, we investigated frequencies of expressional activation of MAGE genes in primary NSCLCs. Three MAGE genes (MAGE-A1, -A3, and -B2), which have already been reported to be expressed in lung cancers, were analyzed (8, 9, 10). We found that in addition to NSCLC, normal lung tissues adjacent to cancers and bronchial epithelial cells from former smokers without lung cancer frequently expressed the MAGE genes, suggesting that activation of MAGE genes can occur at a very early stage of lung carcinogenesis.

Clinical Samples.

Surgical specimens were obtained from a total of 20 patients with early stages of lung cancer who underwent a surgical resection at the Department of Thoracic and Cardiovascular Surgery at The University of Texas M. D. Anderson Cancer Center (Table 1). Fresh-frozen primary lung cancer samples and adjacent normal lung were subjected to mRNA and genomic DNA extraction. Formalin-fixed, paraffin-embedded tissue blocks of lung cancers and adjacent histologically normal lung tissues were used for mRNA in situ hybridization. Bronchial brush samples from 20 former smokers committed in an ongoing chemoprevention clinical trial were used to isolate mRNA. Cell suspensions of bronchial brush samples were also cultured overnight in keratinocyte culture medium (Life Technologies, Inc., Grand Island, NY) on glass slides for mRNA in situ hybridization. Signed informed consent was obtained from every patient to allow the use of biological materials for biomarker studies. The study was reviewed and approved by the Institution’s Surveillance Committee.

Tissue Microdissection, mRNA Isolation, and Genomic DNA Extraction.

Fresh-frozen lung cancer samples and normal tissues were cut into 4-μm sections using a cryostat microtome at −20°C. The first and last sections were immediately stained with methylene blue and examined under a microscope to confirm histologically normal tissues without tumor cell infiltration and tumor tissues consisting of at least 80% tumor cells. Tissue microdissection was performed manually under a stereomicroscope using a 25-gauge needle. mRNA was isolated using a QIAamp mRNA extraction kit (Qiagen, Hilden, Germany), and genomic DNA was extracted by proteinase K digestion followed by phenol-chloroform extraction and precipitation.

RT-PCR and Direct Sequencing of PCR Products.

mRNA (100 ng) was reverse-transcribed using Superscript Reverse Transcriptase (Life Technologies), according to the manufacturer’s protocol, to generate cDNA. To test cDNA integrity, β-actin gene was amplified for each sample. A normal lung cDNA library, a lung cancer cell line expressing a high level of MAGE genes, and genomic DNA from each tissue were used as controls. To avoid amplification of genomic DNA of the MAGE genes, we designed primer sets flanking intron(s): MG1 forward (5′-TGTGGGCAGGAGCTGGGCAA-3′) and MG1 reverse (5′-GCCGAAGGAACCTGACCCAG-3′) for MAGE-A1; MG3 forward (5′-AAGCCGGCCCAGGCTCGGT-3′) and MG3 reverse (5′-GCTGGGCAATGGAGACCCAC-3′) for MAGE-A3; and MGB2 forward (5′-CAGCCAGGGGTGAATTCTCAG-3′) and MGB2 reverse (5′-TTCTCACGGGCACGGAGCTTA-3′) for MAGE-B2. PCR conditions for each MAGE gene were 95°C for 15 min; 35 cycles of 94°C for 30 s, 62°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 5 min in a volume of 12.5 μl with 0.5 units of Hotstar Taq Polymerase (Qiagen, Chatsworth, CA). PCR was also carried out using previously published conditions as well as previously published primers for MAGE-A1, -A3, and -B2(8). RT-PCR products were separated on 2.5% agarose gels. Representative DNA bands in agarose gels were eluted in 50 μl of sterile water using a QIAamp spin column (Qiagen) and directly sequenced using an AmpliCycle Sequencing kit (Perkin-Elmer, Foster City, CA) according to the manufacturer’s protocol.

Methylation-specific PCR.

Genomic DNA (1 μg) samples from 20 NSCLCs and adjacent normal tissues were treated with sodium bisulfite in a 50-μl reaction volume. We designed specific primer sets for methylated or unmethylated promoter sequences of the MAGE-A1, -A3, and -B2 genes: MG1M forward (5′-ATTTAGGTAGGATTCGGTTTTC-3′) and MG1M reverse (5′-AAACTAAAACGTCTTC-CCGCG-3′) for the methylated sequence in MAGE-A1; MG1U forward (5′-ATTTAGGTAGGATTTGGTTTTT-3′) and MG1U reverse (5′-AAACTAAAACATCTTCCCACA-3′) for the MAGE-A1 unmethylated sequence; MG3M forward (5′-CGTTTTGAGTAACGAGCGAC-3′) and MG3M reverse (5′-ACTAAAACGACGAAAATCGACG-3′) for the MAGE-A3 methylated sequence; MG3U forward (5′-TGTTTTGAGTAATGAGTGAT-3′) and MG3U reverse (5′-ACTAAAACAACAAAAATCAACA-3′) for the MAGE-A3 unmethylated sequence; MGB2M forward (5′-AACGTTAGAATAGTGACGTTC-3′) and MGB2M reverse (5′-AAAATAAACCACATCCGCTCG-3′) for the MAGE-B2 methylated sequence; and MGB2U forward (5′-AATGTTAGAATAGTGATGTTT-3′) and MGB2U reverse (5′-AAAATAAACCACATCCACTCA-3′) for the MAGE-B2 unmethylated sequence. These primer sets were not able to amplify untreated genomic DNA. The conditions for methylation specific PCR were 95°C for 15 min; 35 cycles of 94°C for 30 s, 54°C for 1 min, and 72°C 1 min; and a final extension at 72°C for 5 min in a volume of 12.5 μl with 0.5U Hotstar Taq Polymerase (Qiagen). Amplification products were separated on 2.5% agarose gels.

Generation of Riboprobes and RNA in Situ Hybridization.

Sequence-specific antisense and sense riboprobes were generated by in vitro transcription of TOPO TA cloning vector (pCRII-TOPO; Invitrogen, Carlsbad, CA) containing the RT-PCR products of each MAGE gene. HindIII- and XhoI-linearized plasmids were in vitro transcribed with T7 and SP6 RNA polymerase (Promega, Madison, WI) with a digoxigenin-UTP labeling mixture (DIG RNA labeling kit; Roche, Indianapolis, IN).

For mRNA in situ hybridization, paraffin-embedded tissue sections (4 μm) were mounted onto silane-coated slides (Sigma Chemical Co., St. Louis, MO). The sections were deparaffinized in xylene, and rehydrated in gradually decreasing concentrations of ethanol. Sections were then treated with proteinase K for 15 min at 37°C, washed three times with 1× PBS, and post-fixed in 4% paraformaldehyde for 5 min at room temperature. Sections were then acetylated in 0.25% acetic anhydride-0.1 m triethanolamine for 10 min, followed by dehydration in ethanol prior to hybridization. After being prehybridized in hybridization buffer (20× SSC, 50% deionized formamide, 2.5 mg of predenatured salmon sperm DNA, 1 g of dextran sulfate, 2% 100× Denhardt’s solution, 2% DTT, and 4 mg of yeast tRNA) for 1 h at 42°C, sections were hybridized at 42°C for 4 h in hybridization buffer containing a specific probe (400 ng/ml). The sections were then washed in 2× SSC, followed by treatment with RNase A (40 μg/ml) and RNase T1 (10 units/ml) in 10 mm Tris-HCl (pH 8.0), 1 mm EDTA, 0.5 m NaCl for 20 min at 37°C). Sections were then incubated with agitation for 2 h with 2× SSC containing 0.05% Triton X-100 and 2% normal sheep serum at room temperature. After sections were incubated overnight at 4°C with an alkaline phosphatase antidigoxigenin antibody (1:500), the staining signal was developed using 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium chloride and revealed under light microscope. For bronchial brush samples, signal amplification was achieved by a mouse antidigoxigenin antibody (Roche), detected by a biotinylated antimouse antibody (Vector Laboratories, Burlingame, CA). Further amplification was achieved by adding a preformed avidin and biotinylated alkaline phosphatase H macromolecular complex (Vector Laboratories). Sections incubated with digoxigenin-labeled sense probe in the same conditions as well as sections pretreated with RNase were used as negative controls.

We initially analyzed 20 tissue samples of primary NSCLC to determine mRNA expression of the three MAGE genes by RT-PCR. To distinguish cDNA from potential contaminating genomic DNA in the RT-PCR analysis, all primer sets were designed to flank at least one intron. We found that 14 (70%) of the 20 tumors expressed MAGE-A1 and 17 each (85%) expressed MAGE-A3 and -B2. Similar results were also observed when a panel of NSCLC cell lines was tested (data not shown). When using previously reported primers and under previously published conditions (8), we found in primary tumors a positive rate of 45% for MAGE-A1, 50% for MAGE-A2, and 35% for MAGE-B2. Expression of MAGE genes was not related to age, race, sex, tumor stage, or histology (Table 1). Surprisingly, these genes were transcriptionally activated not only in most of the primary NSCLC tissue samples, but also in adjacent normal lung tissues from patients with NSCLC (Fig. 1 a). Thirteen (65%) of the 20 adjacent normal-appearing lung tissues expressed MAGE-A1, 15 (75%) expressed MAGE-A3, and 16 (80%) expressed MAGE-B2, respectively. All RT-PCR products were directly sequenced and confirmed to be the expected MAGE gene transcripts (data not shown). When we used genomic DNA as a template, our primers failed to amplify a specific band, therefore excluding the presence of a pseudogene.

Interestingly, additional larger RT-PCR products were observed in some of specimens when MAGE-A3 and MAGE-B2 primers were used. Sequence analysis showed that all three MAGE genes analyzed in this study have at least one alternative spliced mRNA form in lung tissues. For MAGE-A1, an alternative splicing form that included five additional bp at the 3′ end of exon 1 was observed as a predominant form in some tissues (Fig. 1,b). Because of the subtle difference in the two MAGE-A1 mRNAs, it is difficult to distinguish them on agarose gels. Alternative splicing forms that include 64 bp at the 5′ end of exon 2 for MAGE-A3 and 43 bp for MAGE-B2 were also observed in 77 and 50% of lung tissues, respectively (Fig. 1, a and b). It was noticed that the patterns of MAGE-A3 and -B2 expression were different between paired normal lung and NSCLC tissues in some cases (Fig. 1 a). Expression of different alternatively spliced mRNA forms was found in 7 (35%) of the 20 tumor-normal pairs for MAGE-A3 and 8 (40%) of the 20 pairs for MAGE-B2.

Because of the striking finding that the three MAGE genes were expressed in adjacent normal lung tissues in patients with NSCLC, we wondered whether the expression is a consequence of the presence of tumors or is an early event in lung tumorigenesis. We further analyzed bronchial brush specimens from former smokers without evidence of lung cancer who enrolled in one of our ongoing clinical chemoprevention trials. Among the 20 specimens analyzed, 7 (35%), 10 (50%), and 12 (60%) had detectable MAGE-A1, -A3, or -B2 expression, respectively (Fig. 2). Again, RT-PCR fragments from each sample were recovered from gels and sequenced to confirm the identities to the corresponding mRNAs.

Because the mechanism of MAGE gene activation is promoter hypomethylation, we developed a methylation-specific PCR strategy to analyze hypomethylation status of CpG sites in the promoter regions of these genes. We found that 7 (35%), 10 (50%), and 11 (55%) of the adjacent normal tissues exhibited promoter hypomethylation at MAGE-A1, -A3, and -B2, respectively, compared with 15 (75%), 16 (80%), and 16 (80%) of the NSCLC samples. The hypomethylation status in these promoter regions was further confirmed by sequencing analysis of bisulfate-treated DNA fragments (data not shown).

To verify that expression of the MAGE genes was from lung epithelial cells, we conducted mRNA in situ hybridization using RNA probes specific to individual MAGE mRNA species. We first analyzed four pairs of NSCLC tissues and the corresponding adjacent normal lung tissues. We found that both tumor cells and adjacent normal-appearing bronchial epithelial cells expressed MAGE genes, but no signal was found in stromal cells. Fig. 3, A–F, shows examples of MAGE gene expression measured by mRNA in situ hybridization. In addition, we analyzed bronchial epithelial cells from bronchial brush specimens obtained from four former smokers without evidence of lung cancer. Results of the in situ hybridization were consistent with expression profiles measured by RT- PCR. Examples of these results are shown in Fig. 3, G–R.

Previous studies have shown expression of MAGE genes in lung cancers, with most reports showing MAGE gene expression in only 30–50% of lung tumor tissues (7, 8, 9, 10, 11). The higher rates of gene expression found in this study (70–85%) may be the result of using smaller amplicons or different primer designs that might have improved PCR amplification efficiency. When we used previously reported primer sets to test expression of the MAGE genes, we observed rates of expression in these tumors similar to those reported in the literature. Although mRNA expression may not necessarily indicate the expression of proteins, measuring mRNA was a common method to evaluate the expression patterns of MAGE genes in most of the previous studies because there are no commercially available antibodies.

All three genes studied here consist of small 5′-untranslated exon(s) and a large last exon containing a complete coding sequence (3, 4, 5, 6). Alternative splicing of the untranslated exon is known as a unique feature of MAGE-A4(12). Our finding that all three genes have at least one alternative slicing form suggests that alternative splicing may be a more general phenomenon of the MAGE gene family. Because the translation initiation sites of the genes are at exon 3 in MAGE-A1 and -A3 and at exon 2 in MAGE-B2, these alternative mRNA forms should not change the protein structures; however, they may impact translation efficiency. Nevertheless, additional work is needed to understand the biological significance of alternative splicing of the MAGE gene family.

The notion that MAGE genes are expressed only in tumor tissues or tumor cells but are silent in normal adult tissues, except testis, placenta, and skin during wound healing, has been widely accepted (11, 13, 14, 15). The observation of MAGE gene expression in normal-appearing lung tissues reported here is surprising and raises the intriguing possibility that MAGE gene activation may not be restricted to cancer cells. The finding that MAGE genes were expressed in 35–60% of bronchial epithelium samples from former smokers with no evidence of lung cancer supports the notion that activation of MAGE is a common phenomenon in carcinogen-exposed (transformed?) bronchial epithelium. The frequencies of expression of MAGE-A1 and -A3 are significantly greater in tumor sections than in bronchial brushes (P = 0.05 and 0.04, respectively, Fisher’s exact test). This is probably related to the fact that all bronchial brushes were obtained from former smokers without detectable lung cancer. In fact, we have analyzed a cDNA library derived from the lung of a 17-year-old female nonsmoker and did not detect expression of any of the three MAGE genes (data not shown). Taken together, these results suggest that activation of MAGE genes may be widespread in the carcinogen-damaged lungs and likely develops early during lung tumorigenesis at multiple sites. These results are consistent with our previous observation that multiple independent clones with genetic abnormalities exist widely in smoking-damaged lungs (16). However, and because smokers in general tend to have chronic bronchitis, it is also possible that the MAGE genes are activated merely by inflammatory signals to the bronchial epithelium.

Because the mechanism of MAGE gene activation is promoter hypomethylation, we developed a methylation-specific PCR strategy to analyze hypomethylation status of CpG sites in the promoter regions of these genes. We found an overall good correlation between mRNA expression of MAGE genes and hypomethylation of their promoters (P = 0.01 for MAGE-A1, P = 0.004 for MAGE-A3, and P = 0.04 for MAGE-B2, respectively, Fisher’s exact test). It is possible that the lower frequencies of hypomethylation compared with expression in the adjacent normal lung tissues may reflect a less extensive demethylation process in the promoters in nontumor cells. Furthermore, the primers used cover only a few CpG sites within promoter regions and may fail to locate all critical sites controlling expression of the MAGE genes. Nevertheless, these data indicate the presence of frequent promoter CpG hypomethylation in the MAGE genes and suggest that detection of such hypomethylation by methylation-specific PCR may be a potential marker for MAGE activation.

Because the tissues we used for our analyses may consist of various types of cells, we conducted RNA in situ hybridization to verify that expression of the MAGE genes did indeed arise from lung epithelium. We found that both tumor cells and adjacent normal-appearing bronchial epithelial cells expressed MAGE genes, but no signal was found in stromal cells. Isolated bronchial epithelial cells obtained from bronchial brushes performed in former smokers also expressed MAGE genes in some cases. These data demonstrate that the MAGE genes are expressed in carcinogen- exposed non-tumor-bearing lungs and suggest that expression profiles of these genes may be useful as molecular markers for risk assessment. However, additional carefully designed experiments are necessary to determine MAGE gene expression profiles in other histological tissues and their preneoplastic lesions. Analysis of MAGE gene expression in additional “normal” lung tissues as well in benign lung tissues (hamartomas, infectious lesions, and others) of nonsmokers is clearly warranted. If our findings are confirmed in further investigations, a vaccination strategy targeting these genes may have a significant impact not only in cancer treatment (17, 18, 19) but also in cancer prevention.

Fig. 1.

Expression and alternative splicing of three MAGE genes in paired tumor (T)/normal (N) tissues from patients with NSCLC. a, RT-PCR analysis of microdissected tissue samples. Alternative splicing of MAGE-A1 is not visible in this 2.5% agarose gel electrophoresis sample. For MAGE-A3 and -B2, 4 of 10 tissue pairs expressed different length of mRNA between normal and tumor tissues. Six of 10 tumors, in contrast to 2 normal tissues, expressed the long form of MAGE-A3 mRNA. Three of 10 tumors, in contrast to 1 normal tissue, expressed the long form of MAGE-B2 mRNA. b, RT-PCR amplicons were directly sequenced, confirming the specificity of the bands and demonstrating alternative splicing. For MAGE-A1, the alternative splicing form included five additional bp at the 3′ end of exon 1. Alternative splicing forms included 64 bp at the 5′ end of exon 2 for MAGE-A3 and 48 bp for MAGE-B2.

Fig. 1.

Expression and alternative splicing of three MAGE genes in paired tumor (T)/normal (N) tissues from patients with NSCLC. a, RT-PCR analysis of microdissected tissue samples. Alternative splicing of MAGE-A1 is not visible in this 2.5% agarose gel electrophoresis sample. For MAGE-A3 and -B2, 4 of 10 tissue pairs expressed different length of mRNA between normal and tumor tissues. Six of 10 tumors, in contrast to 2 normal tissues, expressed the long form of MAGE-A3 mRNA. Three of 10 tumors, in contrast to 1 normal tissue, expressed the long form of MAGE-B2 mRNA. b, RT-PCR amplicons were directly sequenced, confirming the specificity of the bands and demonstrating alternative splicing. For MAGE-A1, the alternative splicing form included five additional bp at the 3′ end of exon 1. Alternative splicing forms included 64 bp at the 5′ end of exon 2 for MAGE-A3 and 48 bp for MAGE-B2.

Close modal
Fig. 2.

Expression of three MAGE genes in bronchial brush samples from 20 former smokers (RT-PCR). NSCLC cell lines H460 (for MAGE-A1 and -A3) and H1648 (for MAGE-B2) were used as positive controls. The signal intensity of some samples appears to be as strong as that of tumor tissues. Alternative spliced mRNA of MAGE-A3 and -B2 was visible in some samples (indicated by arrows). The mRNA integrity was tested by RT-PCR of β-actin mRNA.

Fig. 2.

Expression of three MAGE genes in bronchial brush samples from 20 former smokers (RT-PCR). NSCLC cell lines H460 (for MAGE-A1 and -A3) and H1648 (for MAGE-B2) were used as positive controls. The signal intensity of some samples appears to be as strong as that of tumor tissues. Alternative spliced mRNA of MAGE-A3 and -B2 was visible in some samples (indicated by arrows). The mRNA integrity was tested by RT-PCR of β-actin mRNA.

Close modal
Fig. 3.

In situ hybridization of three MAGE genes in paired tumor-normal tissue samples (A–F) and cytological preparations of bronchial brush samples (G–R). A and B, RNA in situ hybridization for MAGE-A1 in squamous cell carcinoma (right) and adjacent normal-appearing bronchus (left). Strong staining was achieved when antisense probe was used (B) compared with control sense probe (A). C and D, RNA in situ hybridization for MAGE-A3 in squamous cell carcinoma (right) and adjacent normal-appearing bronchus (left). Strong staining was observed when antisense probe was used (D) compared with control sense probe (C). E and F, RNA in situ hybridization for MAGE-B2 in bronchoalveolar carcinoma and adjacent normal-appearing alveolar epithelial cells (inset). Strong staining was seen when antisense probe was used (F) compared with control sense probe (E). G–J, RNA in situ hybridization for MAGE-A1 in bronchial brush samples from former smokers. G, control sense probe. Clear cytoplasmic staining was identified in some cells of the bronchial epithelial clusters (H–J). K–N, RNA in situ hybridization for MAGE-A3 in bronchial brush samples from former smokers. Strong (L), weak (M), and negative (N) staining were identified. K, control sense probe. O–R, RNA in situ hybridization for MAGE-B2 in bronchial brush samples from former smokers. Strong (P and Q) and weak (R) staining was observed. O, control sense probe.

Fig. 3.

In situ hybridization of three MAGE genes in paired tumor-normal tissue samples (A–F) and cytological preparations of bronchial brush samples (G–R). A and B, RNA in situ hybridization for MAGE-A1 in squamous cell carcinoma (right) and adjacent normal-appearing bronchus (left). Strong staining was achieved when antisense probe was used (B) compared with control sense probe (A). C and D, RNA in situ hybridization for MAGE-A3 in squamous cell carcinoma (right) and adjacent normal-appearing bronchus (left). Strong staining was observed when antisense probe was used (D) compared with control sense probe (C). E and F, RNA in situ hybridization for MAGE-B2 in bronchoalveolar carcinoma and adjacent normal-appearing alveolar epithelial cells (inset). Strong staining was seen when antisense probe was used (F) compared with control sense probe (E). G–J, RNA in situ hybridization for MAGE-A1 in bronchial brush samples from former smokers. G, control sense probe. Clear cytoplasmic staining was identified in some cells of the bronchial epithelial clusters (H–J). K–N, RNA in situ hybridization for MAGE-A3 in bronchial brush samples from former smokers. Strong (L), weak (M), and negative (N) staining were identified. K, control sense probe. O–R, RNA in situ hybridization for MAGE-B2 in bronchial brush samples from former smokers. Strong (P and Q) and weak (R) staining was observed. O, control sense probe.

Close modal

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by American Cancer Society Grant RPG-98-054 (to L. M.); National Cancer Institute Grants PO1 CA 74173 and U19 CA 68437 (to W. K. H.); a Fondation de France, AP-HP and Lilly Foundation Grant (to J-C. S.); and the Tobacco Research Fund from the State of Texas (to M. D. Anderson Cancer Center). W. K. H. is an American Cancer Society Clinical Research Professor.

3

The abbreviations used are: MAGE, melanoma antigen; NSCLC, non-small cell lung cancer; RT-PCR, reverse transcription-PCR.

Table 1

MAGE gene status according to clinicopathological features of patients

PatientAge (years)RaceaGender (M/F)HistologybTNMStageMAGE-A1cMAGE-A3MAGE-B2
61 Ad T1N0M0 +N, −T +N, +T +N, +T 
48 SCC T1N0M0 −N, +T +N, +T +N, +T 
58 SCC T1N0M0 +N, +T +N, +T +N, +T 
52 SCC T2N0M0 −N, +T −N, +T +N, +T 
65 SCC T1N0M0 +N, +T +N, +T +N, +T 
66 Ad T1N0M0 +N, −T −N, −T +N, +T 
67 SCC T2N0M0 +N, +T −N, +T +N, +T 
54 Ad T1N0M0 +N, +T +N, +T +N, +T 
57 Ad T1N0M0 +N, −T +N, +T +N, +T 
10 45 SCC T2N0M0 +N, −T +N, +T +N, −T 
11 64 SCC T3N0M0 III −N, −T +N, +T −N, +T 
12 56 Ad T1N0M0 +N, +T +N, +T +N, +T 
13 60 SCC T1N0M0 −N, +T +N, +T +N, +T 
14 67 Ad T1N0M0 −N, +T −N, −T −N, +T 
15 58 SCC T2N0M0 +N, +T −N, −T +N, +T 
16 64 SCC T1N0M0 +N, +T +N, +T −N, −T 
17 69 Ad T1N0M0 −N, +T +N, +T +N, +T 
18 61 Ad T2N0M0 −N, −T +N, +T −N, −T 
19 56 Ad T1N1M0 II +N, +T +N, +T +N, +T 
20 50 BAC T1N0M0 +N, +T +N, +T +N, +T 
PatientAge (years)RaceaGender (M/F)HistologybTNMStageMAGE-A1cMAGE-A3MAGE-B2
61 Ad T1N0M0 +N, −T +N, +T +N, +T 
48 SCC T1N0M0 −N, +T +N, +T +N, +T 
58 SCC T1N0M0 +N, +T +N, +T +N, +T 
52 SCC T2N0M0 −N, +T −N, +T +N, +T 
65 SCC T1N0M0 +N, +T +N, +T +N, +T 
66 Ad T1N0M0 +N, −T −N, −T +N, +T 
67 SCC T2N0M0 +N, +T −N, +T +N, +T 
54 Ad T1N0M0 +N, +T +N, +T +N, +T 
57 Ad T1N0M0 +N, −T +N, +T +N, +T 
10 45 SCC T2N0M0 +N, −T +N, +T +N, −T 
11 64 SCC T3N0M0 III −N, −T +N, +T −N, +T 
12 56 Ad T1N0M0 +N, +T +N, +T +N, +T 
13 60 SCC T1N0M0 −N, +T +N, +T +N, +T 
14 67 Ad T1N0M0 −N, +T −N, −T −N, +T 
15 58 SCC T2N0M0 +N, +T −N, −T +N, +T 
16 64 SCC T1N0M0 +N, +T +N, +T −N, −T 
17 69 Ad T1N0M0 −N, +T +N, +T +N, +T 
18 61 Ad T2N0M0 −N, −T +N, +T −N, −T 
19 56 Ad T1N1M0 II +N, +T +N, +T +N, +T 
20 50 BAC T1N0M0 +N, +T +N, +T +N, +T 
a

W, white; B, black.

b

Ad, adenocarcinoma; SCC, squamous cell carcinoma; BAC, bronchoalveolar carcinoma.

c

N, normal lung tissue adjacent to tumor; T, tumor.

We thank Sandra Ideker for artwork, Julie Starr for editing the manuscript, and Diane D. Liu for statistical analysis.

1
van der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Van den Eynde B., Knuth A., Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
Science (Wash. DC)
,
254
:
1643
-1647,  
1991
.
2
De Plaen E., Arden K., Traversari C., Gaforio J. J., Szikora J. P., De Smet C., Brasseur F., van der Bruggen P., Lethe B., Lurquin C. Genome. Structure, chromosomal localization and expression of twelve genes of the MAGE family.
Immunogenetics
,
40
:
360
-369,  
1994
.
3
Rogner U. C., Wilke K., Steck E., Korn B., Poustka A. The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28.
Genomics
,
29
:
725
-731,  
1995
.
4
Muscatelli F., Walker A. P., De Plaen E., Stafford A. N., Monaco A. P. Isolation and characterization of a new MAGE gene family in the Xp21.3 region.
Proc. Natl. Acad. Sci. USA
,
92
:
4987
-4991,  
1995
.
5
Lucas S., De Plaen E., Boon T. MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members of the MAGE family with tumor-specific expression.
Int. J. Cancer
,
87
:
55
-60,  
2000
.
6
Lurquin C., De Smet C., Brasseur F., Muscatelli F., Martelange V., De Plaen E., Brasseur R., Monaco A. P., Boon T. Two members of the human MAGEB gene family located in Xp21.3 are expressed in tumors of various histological origins.
Genomics
,
46
:
397
-408,  
1997
.
7
Lucas S., De Smet C., Arden K. C., Viars C. S., Lethe B., Lurquin C., Boon T. Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.
Cancer Res.
,
58
:
743
-752,  
1998
.
8
Weynants P., Lethe B., Brasseur F., Marchand M., Boon T. Expression of MAGE genes by non-small cell lung carcinomas.
Int. J. Cancer
,
56
:
826
-829,  
1994
.
9
Yoshimatsu T., Yoshino I., Ohgami A., Takenoyama M., Hanagiri T., Nomoto K., Yasumoto K. Expression of melanoma antigen-encoding gene in human lung cancer.
J. Surg. Oncol.
,
67
:
126
-129,  
1998
.
10
Gotoh K., Yatabe Y., Sugiura T., Takagi K., Ogawa M., Takahashi T., Takahashi T., Mitsudomi T. Frequency of MAGE-3 gene expression in HLA-A2 positive patients with non-small cell lung cancer.
Lung Cancer
,
20
:
117
-125,  
1998
.
11
Van den Eynde B. J., van der Bruggen P. T cell defined tumor antigens.
Curr. Opin. Immunol.
,
9
:
684
-693,  
1997
.
12
De Plawn E., Naerhuyzen B., De Smet C., Szikora J. P., Boon T. Alternative promoters of gene MAGE4a.
Genomics
,
40
:
305
-313,  
1997
.
13
Becker J. C., Gillitzer R., Brocker E. B. A member of the melanoma antigen-encoding gene (MAGE) family is expressed in human skin during wound healing.
Int. J. Cancer
,
58
:
346
-348,  
1994
.
14
Takahashi K., Shichijo S., Noguchi M., Hirohata M., Itoh K. Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis.
Cancer Res.
,
55
:
3478
-3482,  
1995
.
15
Jungbluth A. A., Busam K. J., Kolb D., Iversen K., Coplan K., Chen Y. T., Spagnoli G. C., Old L. J. Expression of MAGE-antigens in normal tissues and cancer.
Int. J. Cancer
,
85
:
460
-465,  
2000
.
16
Mao L., Lee J. S., Kurie J. M., Fan Y. H., Lippman S. M., Lee J. J., Ro J. Y., Broxson A., Yu R., Morice R. C., Kemp B. L., Khuri F. R., Walsh G. L., Hittelman W. N., Hong W. K. Clonal genetic alterations in the lungs of current and former smokers.
J. Natl. Cancer Inst. (Bethesda)
,
89
:
857
-862,  
1997
.
17
Marchand M., Weynants P., Rankin E., Arienti F., Belli F., Parmiani G., Cascinelli N., Bourlond A., Vanwijck R., Humblet Y. Tumor regressions responses in melanoma patients treated with a peptide encoded by gene MAGE-3.
Int. J. Cancer
,
63
:
883
-885,  
1995
.
18
Thurner B., Haendle I., Roder C., Dieckmann D., Keikavoussi P., Jonuleit H., Bender A., Maczek C., Schreiner D., von den Driesch P., Brocker E. B., Steinman R. M., Enk A., Kampgen E., Schuler G. Vaccination with MAGE-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma.
J. Exp. Med.
,
190
:
1669
-1678,  
1999
.
19
Marchand M., van Baren N., Weynants P., Brichard V., Dreno B., Tessier M. H., Rankin E., Parmiani G., Arienti F., Humblet Y., Bourlond A., Vanwijck R., Lienard D., Beauduin M., Dietrich P. Y., Russo V., Kerger J., Masucci G., Jager E., De Greve J., Atzpodien J., Brasseur F., Coulie P. G., van der Bruggen P., Boon T. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1.
Int. J. Cancer
,
80
:
219
-230,  
1999
.