XAGE-1 Expression in Non–Small Cell Lung Cancer and Antibody Response in Patients Free

Cancer/testis (CT) antigens are expressed in various types of cancer and male germ cells in the testis but not in adult somatic tissues (1, 2). Forty-four CT antigen genes or gene families have been identified to date (3). The expression of CT antigens such as NY-ESO-1 and SSX 2 in cancer has been shown to elicit humoral and cellular immune responses in patients simultaneously (4, 5). Because of their broad expression in cancer and restricted expression in normal tissues and high immunogenicity, CT antigens are the most attractive targets for cancer vaccine.

XAGE-1 was originally identified by the search for PAGE/GAGE-related genes using expression sequence tag database (6) and was shown to exhibit characteristics of CT-like antigen (7). Four transcript variants XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d have been identified and were shown to be expressed in metastatic melanoma, Ewing sarcoma, and various epithelial tumors such as breast, lung, and prostate cancers (8–10). In the serologic search for antigens using recombinant expression cloning (SEREX), we recently identified XAGE-1b as a dominant antigen recognized by serum from a lung adenocarcinoma patient using an autologous tumor cell line established from malignant pleural effusion as a source of a cDNA library (11). We also observed serum reactivity against XAGE-1b in 8 of 32 sera from lung cancer patients, indicating that XAGE-1 is immunogenic (11).

In this study, we investigated the mRNA expression of four XAGE-1 transcript variants by conventional 30-cycle and quantitative real-time reverse transcription-PCR (RT-PCR) in 49 non–small cell lung cancer (NSCLC) specimens. XAGE-1b mRNA expression was observed most frequently and abundantly in tumor tissues, indicating that XAGE-1b was the major transcript variant. In immunohistochemistry using a XAGE-1 monoclonal antibody (mAb), positive nuclear staining was observed in most of the XAGE-1b mRNA-positive specimens. Humoral immune responses to XAGE-1b were detected in lung cancer patients by ELISA and Western blotting.

Patients and specimens. Forty-nine NSCLC and adjacent normal lung tissue specimens were surgically obtained from patients at the Okayama University Hospital. Sera were obtained from 74 lung cancer patients and 40 healthy donors. Written informed consent was obtained from each patient for the use of specimens and sera in this study. This study was approved by the Ethics Committee of Okayama University Graduate School of Medicine and Dentistry.

Total RNA isolation and cDNA synthesis. Total RNA was isolated from frozen tumor and normal lung specimens using RNeasy Mini Kit (Qiagen, Hilden, Germany), and 2 μg were reverse transcribed into single-strand cDNA using Molony murine leukemia virus reverse transcriptase (Ready-To-Go You-Prime First-strand Beads, Amersham Biosciences, Piscataway, NJ) and oligo (dT)₁₅ as a primer. cDNAs were tested for integrity by amplification of the G3PDH gene. cDNA from normal tissues was purchased from BD Biosciences Clontech (Palo Alto, CA).

Preparation of plasmid vectors. To generate the expression vector, XAGE-1b cDNA spanning nucleotides 31 to 395 was amplified by PCR using human testis cDNA. Primers were designed to create an EcoRI site at the 5′ end, and a XhoI site at the 3′ end of the PCR product. The amplified cDNA was ligated into pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA). Recombinant vector, pcDNA3.1/XAGE-1b, was introduced into TOP10 Escherichia coli cells. Insertion of the cDNA of the cloned transformant was confirmed by DNA sequencing. Plasmid DNA was purified using the Endfree Plasmid MegaKit (Qiagen).

Production of recombinant XAGE-1b protein. The XAGE-1b cDNA spanning nucleotides 128 to 367 was amplified by PCR using human testis cDNA. Primers were designed to create an EcoRI site at the 5′ end, and 6 His-tag and a SalI site at the 3′ end of the PCR product. The amplified DNA was ligated into the glutathione S-transferase (GST)–containing vector pGEX-6P-1 (Amersham Biosciences). C-His-tagged GST-XAGE-1b fusion protein was expressed in BL21 E. coli and purified by nickel ion affinity chromatography under native condition (HiTrap Chelating, Amersham Biosciences).

Production of XAGE-1 monoclonal antibody, USO9-13. BALB/c mice were i.m. injected with pcDNA3.1/XAGE-1b (100 μg) into the anterior tibial muscle and pulsed with an electric pulse generator (CUY-21, BEX, Tokyo, Japan) using a 1.0-cm-diameter round plate electrode twice at a 2-week interval. The mouse which had the highest antibody titer was i.p. boosted with C-His-tagged GST-XAGE-1b protein (100 μg) 3 days before fusion. Fused spleen cells with NS-1 were cultured in HY soft agar with hypoxanthine-aminopterin-thymidine medium. Visible hybridoma colonies were selected and screened for production of XAGE-1 mAb by ELISA using C-His-tagged GST-XAGE1b protein as an antigen. A hybridoma clone USO9-13 was obtained by limiting dilution.

Reverse transcription-PCR. A schematic representation of the structure of the XAGE-1 gene and transcripts including locations of primers used in this study is shown in Fig. 1. Sequences for primer pairs used for RT-PCR are listed in Table 1. The amplification program for XAGE-1 transcript variants was 1 minute at 94°C, 1 minute at 60°C, and 1.5 minutes at 72°C for 30 cycles after 10 minutes at 94°C. The amplification program for G3PDH was 1 minute at 94°C, 1 minute at 66°C, and 1.5 minutes at 72°C for 30 cycles after 10 minutes at 94°C. These cycles were followed by a 10-minute elongation step at 72°C. PCR products were analyzed on 0.8% or 2% agarose gel.

Real-time reverse transcription-PCR. Sequences of gene-specific primers and Taqman probes for XAGE-1b and XAGE-1d are shown in Table 1. Real-time RT-PCR was run on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Fifty two-step cycles amplification was done with 1 μL cDNA (corresponding to 60 ng total RNA) solution extracted from tumor specimens or plasmid solution with 25 μL Taqman Universal PCR Master Mix (Applied Biosystems), the primer pair, and 200 nmol/L Taqman probe in a total volume of 50 μL after a 10-minute activation of AmpliTaq GOLD DNA polymerase at 95°C. The amplification conditions were as follows: XAGE-1b, 95°C for 15 seconds and 60°C for 60 seconds with 500 nmol/L of the forward (R-1bS) and reverse (R-1bAS) primers; XAGE-1d, 95°C for 15 seconds and 60°C for 60 seconds with 100 nmol/L of the forward (X-6) and reverse (X-2) primers. Amplification was not observed using human genomic DNA as templates (BD Biosciences Clontech).

Immunohistochemistry. Tumor specimens were fixed with buffered formalin and embedded in paraffin. Five-micrometer sections were placed on glass slides, heated at 60°C overnight, and deparaffinized with xylene and ethanol. For antigen retrieval, tumor specimens mounted on glass slides were microwave heated in an antigen retrieval buffer [10 nmol/L citrate buffer (pH 6.0)] with a pressure cooker for 10 minutes. After the inactivation of endogenous peroxidase with 0.3% H₂O₂ for 5 minutes, specimens were preincubated with serum-free blocking solution (DakoCytomation, Kyoto, Japan). XAGE-1 mAb, USO 9-13, was added at a concentration of 2 μg/mL and incubated at 4°C overnight. After washing, DAKO EnVision⁺ horseradish peroxidase–conjugated goat anti-mouse IgG (DakoCytomation) was applied and incubated for 30 minutes at room temperature. Specimens were visualized with 3,3′-diaminobenzidine in H₂O₂ and counterstained with hematoxylin solution. As a negative control, 2 μg/mL of isotype-matched myeloma protein (mouse IgG2a κ, UPC-10; Sigma, St. Louis, MO) were used.

ELISA. Recombinant GST-XAGE-1b protein (2 μg/mL) in 0.05 mol/L carbonate buffer (pH 9.6) was adsorbed onto 96-well plates (Nunc, Rochester, NY) at 4°C overnight. GST protein was used as a negative control. ELISA was done as described elsewhere (12). A positive reaction was defined as an absorbance value for 1:100 and 1:400 diluted sera that exceeded the mean absorbance value of sera from 40 healthy donors by 4 SDs.

Western blot analysis. 293 T cells were transfected with pcDNA3.1 alone or pcDNA3.1/XAGE-1b using LipofectAMINE 2000 (Invitrogen). The transfectants were incubated at 37°C for 48 hours and lysed with extraction buffer. Twenty micrograms of cell lysates were diluted in SDS and run on a 15% SDS gel. The protein was then transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Biosciences). The blotted membrane was blocked with 5% skim milk/Tween 20/TBS and incubated with patients' sera at a 1:1,000 dilution or with 1 μg/mL XAGE-1 mAb as a positive control for 2 hours at room temperature. Peroxidase-conjugated anti-human or anti-mouse IgG (Amersham Biosciences) was added and incubated for 2 hours at room temperature. The ECLplus Western Blotting Detection System (Amersham Biosciences) was used as the detection reagent.

Statistical analysis. Statistical analysis was done with the χ² test and Fisher's exact probability test. P < 0.05 was considered significant.

XAGE-1 mRNA expression in non–small cell lung cancer. Four XAGE-1 transcript variants, XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d, have been identified (8–10). We investigated the expression of these transcript variants in 49 NSCLC and adjacent normal lung tissues by conventional 30-cycle RT-PCR using specific primer pairs (see ref. 11; Fig. 1; Table 1). The PCR product was analyzed in agarose gel. Figure 2 shows a representative RT-PCR and Table 2 summarizes the results. The expression of XAGE-1b and XAGE-1d but not XAGE-1a nor XAGE-1c mRNA was observed. Histologically, XAGE-1b mRNA expression was observed in 14 of 31 (45%) adenocarcinoma and 1 of 15 (7%) squamous cell carcinoma specimens. No positive expression was observed in two large cell carcinoma specimens and a carcinoid. On the other hand, XAGE-1d mRNA expression was observed in six (19%) adenocarcinoma specimens but not in the others. XAGE-1d mRNA expression was associated with XAGE-1b mRNA expression. No XAGE-1 mRNA expression was observed in 49 adjacent normal lung tissues. The expression of XAGE-1b and XAGE-1d but not XAGE-1a nor XAGE-1c mRNA was similarly observed in the testis.

Quantitative real-time reverse transcription-PCR analysis of XAGE-1b and XAGE-1d mRNA expression in non–small cell lung cancer, adjacent normal lung tissues, and a panel of normal tissues. We analyzed XAGE-1b and XAGE-1d mRNA expression quantitatively by real-time RT-PCR using Taqman probes shown in Fig. 1 and Table 1 in 49 NSCLC and adjacent normal lung tissues. As shown in Fig. 3A, the XAGE-1b mRNA copy number ranged from 3 × 10³ to 7 × 10⁵ per 60 ng total RNA in 15 specimens that were shown to be positive for XAGE-1b mRNA expression by conventional RT-PCR. The XAGE-1d mRNA copy number ranged from 10³ to 10⁴ in six specimens that were shown to be positive for XAGE-1d mRNA expression by conventional RT-PCR. XAGE-1b and XAGE-1d mRNA copy numbers were <10³ in 49 adjacent normal lung tissues.

We then investigated XAGE-1b and XAGE-1d mRNA expression in various normal tissues including lung using the Clontech normal tissue panel. Figure 3B shows amplification curves and copy numbers for XAGE-1b and XAGE-1d mRNA. The XAGE-1b mRNA copy number was 1.8 × 10⁵/60 ng total RNA in testis and <10³ in other normal tissues. The XAGE-1d mRNA copy number was <10³ per 60 ng total RNA in normal tissues including testis.

We examined the correlation between XAGE-1b mRNA expression and clinicopathologic features (Table 3). Significantly higher expression of XAGE-1b mRNA expression was observed more frequently in adenocarcinoma as shown above. No correlation was observed between XAGE-1b mRNA expression and age or sex of patients, disease stage, or histologic grade.

XAGE-1b protein expression in non–small cell lung cancer by immunohistochemistry. XAGE-1 protein expression was examined in 49 NSCLC specimens by immunohistochemistry using a XAGE-1 mAb, USO 9-13. The mAb reacted against the recombinant XAGE-1b-GST fusion protein but not control GST or irrelevant NY-ESO-1 protein by ELISA and Western blot. Because the epitope of USO 9-13 mAb is located at the XAGE-1 COOH terminus, residues 65 to 81 (data not shown), the mAb is potentially reactive with XAGE-1a, XAGE-1b, and XAGE-1c. However, Western blot analysis using XAGE-1a- or XAGE-1b-transfected 293 T cell lysates revealed that USO 9-13 mAb detected only a 9-kDa band equivalent to the molecular size of XAGE-1b (data not shown), which was consistent with the previous report showing that translation of XAGE-1 starts with the second initiation codon located at exon 2 (see Fig. 1 and ref. 10). No specific band was observed using XAGE-1c-transfected 293 T cell lysates. Thus, USO 9-13 detects XAGE-1b. Positive staining was observed in 14 and 3 of 15 and 34 XAGE-1b mRNA-positive and mRNA-negative specimens, respectively (Table 4). The former 14 positive specimens include 13 adenocarcinomas and a squamous cell carcinoma and the latter three positive specimens include three adenocarcinomas. As shown in Fig. 4, staining was observed in the nuclei of tumor cells, predominantly in the nucleoli. The staining pattern was focal (5-10% of cancer cells were stained; see Fig. 4B and C), intermediate (10-50% were stained), or diffuse (>50% were stained; see Fig. 4E and F). Focal, intermediate, and diffuse stainings were observed in four, six, and seven specimens, respectively. No positive staining was observed in the noncancerous lung tissues examined.

In normal tissues, staining was observed only in the nucleus of spermatogonia and spermatocytes in testis (Fig. 4G and H). The isotype-matched antibody UPC-10 showed no reactivity.

Correlation between XAGE-1b protein expression and histologic type for dominant expression in adenocarcinoma was observed (Table 3).

XAGE-1b antibody response in cancer patients. Sera from 74 NSCLC patients and 40 healthy donors were analyzed for XAGE-1b antibody by ELISA using recombinant GST-XAGE-1b fusion protein. Figure 5 shows typical titration curves for positive and negative sera, and Tables 4 and 5 summarize the results. We observed antibody production in 5 of 56 adenocarcinoma patients but none of 18 patients with other histologic types. XAGE-1 antibody was IgG1 isotype in all seropositive patients (data not shown). No antibody was detected in 40 healthy donors. No antibody against GST recombinant protein was detected in sera from five seropositive patients. Western blot analysis showed that five ELISA-positive sera reacted against XAGE-1b recombinant protein (data not shown) and XAGE-1b protein expressed in 293 T cells transfected with pcDNA3.1/XAGE-1b (Fig. 6).

XAGE-1 was identified originally by database mining of expressed sequence tags and shown to be expressed in testis in normal tissues and Ewing sarcoma (6, 7). Recently, the transcript variants, XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d, have been identified (8–10). Expression analysis showed that they belong to CT-like antigens. In the present study, we showed that XAGE-1b mRNA was expressed at high frequency and strongly in lung adenocarcinoma, and XAGE-1d mRNA at lower frequency and less strongly. No expression of XAGE-1a or XAGE-1c was observed. Analysis with conventional 30-cycle and quantitative real-time RT-PCR revealed that XAGE-1b and XAGE-1d mRNA was expressed in 15 and 6 of 49 NSCLC, respectively. Fourteen of 15 XAGE-1b mRNA-positive and six of six XAGE-1d mRNA-positive tumors were adenocarcinoma. XAGE-1d mRNA expression was associated with XAGE-1b mRNA expression. Thus, XAGE-1b was the transcript variant dominantly expressed in adenocarcinoma. A similar expression profile of XAGE-1 transcript variants was reported in metastatic melanoma (9).

Quantitative real-time RT-PCR revealed that the XAGE-1b mRNA copy number ranged from 3 × 10³ to 7 × 10⁵ per 60 ng total RNA in specimens that were shown to be positive by conventional 30-cycle RT-PCR and was <10³ in normal tissues except testis in which 1.8 × 10⁵ copies were detected. On the other hand, the XAGE-1d mRNA copy number was in the range of 10³ to 10⁴ in six specimens that were shown to be positive by conventional 30-cycle RT-PCR and was <10³ in normal tissues including testis. Thus, positive mRNA expression detected by conventional 30-cycle RT-PCR was observed in specimens that express >10³ copy number with both XAGE-1b and XAGE-1d mRNA.

To analyze XAGE-1b protein expression in tissues, we produced a XAGE-1 mAb, USO 9-13. Antibody specificity was confirmed by ELISA and Western blotting using the recombinant proteins or cell lysates from 293 T cells transfected with cDNA of XAGE-1 transcript variants. Immunohistochemistry using USO 9-13 mAb showed that positive staining was observed in testis among normal tissues. Staining was observed in the nuclei of spermatogonia and spermatocytes. In NSCLC, positive staining was observed at extremely high frequency. Fourteen of 15 mRNA-positive specimens were stained positively. Three of 34 mRNA-negative specimens were also stained positively. This discrepancy may derive from the difference of tumor samples in the same tumor specimens for RT-PCR and immunohistochemistry, because the tumor specimens kept frozen for analysis were quite large (3-5 cm). Staining was localized in the nuclei of cancer cells. Nuclear localization was also seen in a XAGE-1b mRNA-positive melanoma cell line, SK-MEL-37 and 293 T cells transfected with pcDNA3.1/XAGE-1b, which was consistent with the previous report showing that GFP-XAGE-1b fusion protein was visible in the nuclei of COS-1 cells transfected with XAGE-1b/GFP (9). XAGE-1b contains the nuclear localization signal with two basic amino acid domains separated by 10 spacer amino acids (13).

XAGE-1b expression was observed mostly in adenocarcinoma among the cases of NSCLC. On the other hand, we detected XAGE-1b mRNA expression at high frequency in liver cancer (11 of 19, 58%), at intermediate frequency in prostate cancer (14 of 54, 24%), but at low frequency in breast cancer (1 of 20, 5%) by RT-PCR, suggesting that XAGE-1b could be the target for cancer vaccine in some types of cancer.

Although CT antigens such as MAGE and SSX were expressed at high frequency in lung cancer (2, 3), humoral immune responses were rarely observed in patients (14). We observed antibody production against XAGE-1b in 5 of 56 patients with adenocarcinoma but not in 18 patients with other histologic types or 40 healthy volunteers. In our SEREX analysis, we observed XAGE-1b antibody in sera from 8 of 18 patients with adenocarcinoma (11). These findings indicated that XAGE-1b expressed in lung adenocarcinoma was immunogenic in patients. It should be determined whether CD8 T-cell responses specific to XAGE-1b can be evoked in seropositive patients. If so, this will provide a solid rationale for establishing immunotherapy targeting XAGE-1b and will prompt the identification of CD8 T-cell epitopes from XAGE-1b.

We thank M. Isobe and T. Akimoto for their excellent technical assistance and J. Mizuuchi for preparation of the article.