Selective cancer-germline gene expression in pediatric brain tumors

Fresh-frozen tumor samples were available at the Department of Pathology at the Radboud University Nijmegen Medical Centre. All samples were from pediatric patients (0–19-years-old) with a brain tumor diagnosed at the Department of Pediatric Hemato-Oncology. Sections of the frozen samples were stained with hematoxylin-eosin and reviewed by the pathologist to verify tumor histology and to evaluate the percentage of tumor cells. Samples were only considered for study if the contents of tumor cells was ≥80%.

Total RNA was isolated with TriZol reagent (Invitrogen, Carlsbad, CA) and samples were treated with Deoxyribonuclease I (Invitrogen) according to the manufacturer’s protocol. To generate cDNA, 1 μg DNase-treated RNA was reverse-transcribed with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo(dT) primer and 50 units SuperScript II, according to the manufacturer’s protocol. After first-strand synthesis, samples were diluted to a final volume of 100 μl with water.

Duplex PCR amplification of β-actin and GAPDH transcripts was carried out in a 25-μl reaction volume containing 2.5 μl of cDNA, 1× PCR Buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂), 100 μM each dNTP, 0.4 μM each primer, and 0.625 units Taq DNA polymerase (TaKaRa, Shiga, Japan). β-actin primers were as described [12]. GAPDH primers (originally available from Clontech, Palo Alto, CA; kindly provided by Dr. B. Lethé) were 5′-TgAAggTCggAgTCAACggATTTggT-3′ (sense) and 5′-CATgTgggCCATgAggTCCACCAC-3′ (antisense). Cycling was performed in a TRIO-Thermoblock thermocycler (Biometra, Göttingen, Germany) as follows: 94°C for 4 min, followed by 22 cycles of 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. Cycling was concluded with a final extension step at 72°C for 15 min. PCR products were fractionated in 1.3% agarose gel and visualized by ethidium bromide fluorescence (β-actin, 626 bp; GAPDH, 983 bp). PCR amplification of MAGE-A transcripts was carried out with the primer pair designed by Zammatteo et al. [13]. These primers derive from a consensus nucleotide sequence for the last exon of the 12 MAGE-A genes and give amplicons of ~539 bp. PCR conditions were as described above, except that PCR was performed for 30 cycles.

Expression of CGGs and of the reference gene β-actin, was measured by quantitative PCR, based on TaqMan methodology, using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). PCR reactions were prepared with the qPCR Core Kit w/o dUTP reagents (Eurogentec, Seraing, Belgium). Each reaction (25 μl) contained 2.5 μl of cDNA, 1× PCR buffer containing the passive reference dye ROX, 5 mM MgCl₂, 200 μM each dNTP, 200 nM each primer, 100 nM probe, and 0.625 units DNA polymerase. Primers, probes and thermal cycling conditions are given in Table 1 [14, 15]. Probes with 6FAM and TAMRA labels were from Eurogentec. Probes with 6FAM and MGB-NFQ labels (for MAGE-A3, MAGE-A6 and MAGE-A12) were from Applied Biosystems. Quantification of the samples was achieved by extrapolation from a standard curve of serial dilution points of cDNA of the relevant gene (Supplementary Figure 1). Samples and standard dilution points were assayed in duplicate or triplicate. Standard calibration curves for β-actin and all CGGs were linear over 4 (CGGs) or 5 (ß-actin) orders of magnitude and had similar PCR efficiencies (slope from −3.45 to −3.77). Differences in sensitivity between the assays for the various genes (y-intercept from 38.7 to 41.6) were due in part to differences in the actual cDNA copy number in the standard dilutions. cDNA copy numbers in the standards were verified by testing minimally 12 replicates of the 1-copy dilution in each qPCR run. If needed, copy numbers of the test samples were corrected by a factor calculated on the basis of the results for the 1-copy dilution. Normalization of samples was achieved by dividing the copy number of CGG by that of the reference gene, β-actin.

Immunochemistry was performed on 4 μm tissue sections of formalin-fixed paraffin-embedded tissue blocks. Sections were heated for 20 min in citrate buffer (10 mM, pH 6.0) for antigen retrieval. The following mouse IgG1 monoclonal antibodies (mAb) were used: E978 (anti-NY-ESO-1) [16] (Zymed, San Francisco, CA), MA454 (anti-MAGE-A1) [17] (Zymed, San Francisco, CA), and 57B (anti-MAGE-A4) [18, 19] (kindly provided by Dr. G.C. Spagnoli, University Hospital Basel, Switzerland). Testis tissue with intact spermatogenesis was used as positive control. Tissue sections were incubated with mAb diluted in PBA: E978 (2.5 μg/ml), MA454 (1 μg/ml), or 57B (5 μg/ml), or with IgG1 negative control antibody, at room temperature for 1 h. Binding sites of primary antibodies were then detected by a biotinylated horse-antimouse secondary reagent (Vector Laboratories, Burlingame, CA) followed by an avidin–biotin complex system (ABC Elite, Vector Laboratories). Diaminobenzidine tetrachloride served as a chromogen. Immunoreactivity was assessed blindly with respect to the mAb used.

Normalized CGG values are presented as means ± standard deviation (SD). The SD of the normalized CGG values was calculated from the SD of the CGG and the β-actin values using the following formula: CV = SQRT [CV² _β-actin + CV _CGG ² ], where CV = SD/mean value (as described in the Sequence Detection System User Bulletin 2, 1997, Applied Biosystems). Differences in mRNA expression levels between pediatric and adult glioblastomas are calculated with the Spearman rank correlation. All statistical tests were two-sided, significance was determined as P < 0.05.