| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Oncology, Markers, Clinical Correlates |
Departments of Haematology and Oncology [D. L. S., C. E., D. M. A.] and Anatomical Pathology [C. W. C.], Royal Children’s Hospital, and Departments of Anatomical Pathology [M. F. G.] and Surgery [D. M. A.], Royal Melbourne Hospital, Parkville 3052, Melbourne, Victoria, Australia; and Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Université Catholique de Louvain, B-1200 Brussels, Belgium [F. B.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several approaches to cancer immunotherapy are under investigation in brain tumors. One such approach is the use of active immunotherapy. Previous studies (1 , 2) using animal models of brain tumors have demonstrated that vaccinations of autologous tumor cells that have been genetically modified are able to protect mice against intracranial tumor challenge and prolong survival in mice with established intracranial tumors.
However, there may be several barriers to these approaches in human patients. These include the technical difficulties in preparing vaccines from tumor specimens and the risk of causing experimental allergic encephalomyelitis because it is often difficult to separate normal brain from tumor tissue (3 , 4) . It has been shown that nonhuman primates and guinea pigs immunized with human GBM3 tissue developed experimental allergic encephalomyelitis that was lethal (5) . Therefore, the targeting of a tumor-associated antigen may represent a reasonable initial approach to human active immunotherapy for high-grade brain tumors.
Several tumor-associated antigens have now been identified, and these are coded by the MAGE (6 , 7) , BAGE (8) , and GAGE (9) 4 families of genes. Although these antigens were originally identified in melanoma, they have since been found to be present in a variety of tumor types, including lung and bladder carcinomas, sarcomas, and head and neck tumors (e.g., Refs. 8 , 9 , and 10) . Lack of expression of MAGE and GAGE in a large panel of normal adult tissues, including stomach, lung, breast, colon, skin and uterus, fetal liver, and brain has been demonstrated previously (7 , 9) . The only normal tissues in which expression of these genes has been observed was testis (9) and placenta for MAGE-3 and -4 only (7) .
Because melanocytes and glial cells are both derived from the neuroectoderm, it has been hypothesized that glial tumors also express one or more of these genes. However, studies examining the expression of these genes in glioma are limited. Chi et al. (11) examined the expression of MAGE-1 and -3 in 21 GBM specimens by RT-PCR and Southern blotting and demonstrated frequencies of expression of 38 and 33%, respectively.
We have analyzed the expression of MAGE-1, -2, -3 and -6 and the GAGE family of genes in a panel of GBMs from adult and pediatric patients as well as medulloblastomas, ependymomas, and two other high-grade brain tumors using the sensitive method of RT-PCR and Southern blotting. A panel of normal brain specimens was also included for analysis of GAGE expression.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Tumor Specimens.
Brain tumor specimens from adult patients were snap-frozen in liquid nitrogen after surgical resection, and those from pediatric patients were embedded and frozen in OCT. All specimens were stored at -80°C. Classification of tumor type and grade was made by two independent pathologists in a blinded fashion in accordance with the WHO histological typing of central nervous system tumors (12)
.
RNA Isolation and cDNA Synthesis.
Total RNA was isolated from brain tumor specimens and cell lines using the acid guanidium thiocyanate-phenol-chloroform method (13)
, quantitated by UV spectrophotometry and stored at -80°C in 100% ethanol and 75 mM sodium acetate pH6. An aliquot containing 1 µ of RNA was removed and centrifuged, and the dried pellet was resuspended in 20 µl of the reverse transcription mix, which contained the following: 1x reaction buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT], 1 mM each dNTP (Promega, Madison, WI), 1 nmol pd(N)6 random primer (Pharmacia Biotech, Uppsala, Sweden), 9.2 units of RNasin (Promega), and 200 units of Moloney murine leukemia virus reverse transcriptase RNase H minus (Promega). The reaction was incubated at 37°C for 60 min and then stopped and stored at -20°C.
For normal brain specimens, total RNA was extracted using the guanidinium isothiocyanate/cesium chloride method (14) , and then 2 µg were reverse-transcribed as described previously (15) .
PCR.
Sequences of oligonucleotide primers for PCR amplification and expected product sizes are listed in Table 1
. ß2-microglobulin was used to assess the integrity of the mRNA.
|
Southern Blotting.
The entire PCR was electrophoresed on a 1.5 or 2% agarose gel and transferred onto a Hybond-N+ membrane (Amersham Life Sciences, Cleveland, OH) in 0.4 M NaOH. The DNA was fixed to the membrane in 0.4 M NaOH and then washed in 5x SSC. Membranes were prehybridized in 0.5 M sodium phosphate (pH 7.2), 7% SDS, and 1.5 mM EDTA for 45 min. Sequences for probes used for the mRNA detection of all antigens and ß2-microglobulin are listed in Table 1
. GAGE-1, -2, and -7 and GAGE-3–6 and -8 PCR products were probed with the GAGE-1–8 probe and GAGE-1 PCR products with the GAGE-1-specific probe. All probes were labeled at their 5' end with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [
-32P]ATP (DuPont, Wilmington, DE) and added directly to the prehybridization solution, and membranes were hybridized overnight at 37°C. Membranes were washed in 2x SSC at room temperature for 5 min and then two washes in 0.2x SSC-0.1% SDS at 37°C for 15 min each.
Sequencing.
To verify MAGE- and GAGE-specific sequences of amplified products, sequencing was performed on two to three specimens from each tumor type and on positive control cell lines for each gene amplified. PCR was performed as described above except that reaction volume was increased to 100 µl, and amplification was increased to 40 cycles to increase the amount of amplicon. PCR products were then purified in one of two ways: (a) agarose gel electrophoresis, isolation of bands of interest, and DNA purification with the BRESAclean DNA purification kit (Bresatec, Adelaide, South Australia, Australia); or (b) direct purification with the PCR product presequencing kit (Amersham). Sequencing was performed by the dideoxy-chain termination method using the T7 Sequenase version 2.0 kit (Amersham). Samples were sequenced with the sense or antisense primer used in the PCR. Sequences were entered into the BLAST sequence program (www.ncbi.nlm.nih.gov/BLAST/) to search for sequence homology.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and 3
, and representative autoradiographs are shown in Figs. 1
and 2
. Unless otherwise indicated, all data for analysis of MAGE-3&6 expression were obtained using the MAGE-3/6F/BLE-5 primer pair for simultaneous gene amplification.
|
|
|
|
; Table 2)
. However, when GAGE-1 only was amplified, none of the specimens were positive for this gene, thus indicating that GAGE-2 and/or -7 only are expressed in normal brain. No expression of GAGE-3–6 or -8 was observed. The normal brain specimens were analyzed for GAGE expression only, as absence of MAGE-1, -2, and -3 expression in normal brain has been reported previously using RT-PCR and Southern blotting (6)
.
Molecular Detection of MAGE and GAGE Expression in Brain Tumor Specimens.
Because no GAGE-1, -3–6, or -8 or MAGE was detected in normal brains, we examined tumor specimens for expression of these genes. In adult GBM, GAGE was the predominantly expressed gene, with 65% of specimens expressing GAGE-1 and 75% expressing GAGE-3–6 and -8. Little or no expression of the four MAGE genes was observed, with only one specimen expressing MAGE-2. The antigen mRNA expression profile in pediatric GBM was similar to that for adult GBM with respect to MAGE and GAGE-3–6 and -8 expression. However, only 11% of pediatric GBM specimens expressed GAGE-1. GBM025 did not express any of the genes.
The MAGE-3 and -6 expression data presented here for adult GBM specimens was initially determined using two pairs of PCR primers: AB1197/BLE5 and AB1197/MAGE-6R for individual amplification of MAGE-3 and -6, respectively (10) . Southern blot results from this analysis showed amplification of a 805-bp genomic DNA band, as expected. However, the cDNA band amplified in the tumor samples appeared slightly larger than expected. The cDNA band in the positive control, D566, was 725/727-bp, as predicted. We, therefore, sequenced the genomic DNA and cDNA bands from two GBM specimens and the cDNA band from the positive control for both MAGE-3 and -6. As expected, in all cases, the sequence of the 805-bp genomic DNA band was 100% homologous to published MAGE-3 and -6 sequences (7 , 16) ; however, the sequence of the cDNA band from the tumor specimens was not homologous to any human sequence. The sequence of the 725-bp MAGE-3 or 727-bp MAGE-6 cDNA bands from D566 was 100% homologous with published sequences. We then screened the specimens again with a third pair of PCR primers for simultaneous amplification of both MAGE-3 and -6 (MAGE-3/6F/BLE5). Using these primers, no expression of these genes was observed in tumor specimens; however, good amplification was noted in positive controls. In summary, in contrast to a previous report (11) , these data confirmed that MAGE-3 and -6 mRNA is not detectable in GBM specimens.
For medulloblastomas and ependymomas, GAGE-3–6 and -8 were more frequently expressed than GAGE-1: a similar profile to that for both adult and pediatric GBM. MAGE-1, -3, and/or -6 were expressed by medulloblastomas only—the only group of tumors examined that expressed any of these three genes. MAGE-2 was expressed at a high frequency by both medulloblastomas (60%) and ependymomas (57%). Of the two other glial tumors examined, both had the same expression profile, in that GAGE-3–6 and -8 only were expressed (Table 3)
.
Confirmation of Results.
In addition to sequencing MAGE-3 and -6 PCR products amplified from adult GBM specimens, we also sequenced two to three specimens from each tissue group (i.e., normal brain and tumors) for each gene amplified to confirm our Southern blot results. In normal brain, insufficient GAGE-1, -2 and -7 PCR product was obtained for sequencing due to the low expression levels of these genes. In all tumor specimens, however, sequence of all GAGE, MAGE-1, -2, and -3 and -6 (simultaneous gene amplification) PCR products (where amplified) was 100% homologous with published sequences (6
, 7
, 9
, 16
, 17)
, as were sequences of positive control PCR products.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although the lack of expression of MAGE in normal brain had been demonstrated by RT-PCR and Southern blotting (6) , a similar analysis had not been performed for GAGE. The data presented here using RT-PCR and Southern blotting suggest that two of the eight GAGE genes, GAGE-2 and/or -7, are expressed in normal brain, albeit at very low levels. This observation will need further investigation because we were unable to sequence the PCR products for GAGE-1, -2, and -7.
Of the MAGE genes we have analyzed, MAGE-2 showed the highest frequency of expression, with 5, 11, 60, and 57% of adult GBMs, pediatric GBMs, medulloblastomas and ependymomas, respectively, being positive for this gene. MAGE-1, -3, and/or -6 were expressed by medulloblastomas only. These results differ from those of Chi et al. (11) , who showed 38 and 33% expression of MAGE-1 and -3, respectively, in GBM and expression of MAGE-3 in one of two ependymomas but no expression of MAGE-1. We believe that this discrepancy may be explained by nonspecific hybridization of internal probes to non-MAGE amplification products. In this study, we encountered cross-hybridization of the MAGE-3/6 probe and so performed sequencing of PCR products amplified from adult GBM specimens to confirm our Southern blot results. This sequencing of MAGE-3 and -6 PCR products (from individual gene amplification) showed that there was nonspecific amplification and hybridization of MAGE-3 and -6 primers and probes indicating that there was no expression of either of these genes in GBM. This was further confirmed using primers which simultaneously amplified both MAGE-3 and -6.
The high level of expression of MAGE-2 in medulloblastomas and ependymomas, in contrast to GBM, is of interest. These differences in expression patterns may be exploited to differentiate these tumor types where diagnosis is proving difficult.
In contrast to the expression patterns of the four MAGE genes examined, GAGE was expressed in all tumor types with 11–65% expressing GAGE-1 and 47–93% expressing GAGE-3–6 and -8. This high frequency of expression is comparable only to that in neuroblastomas, in which 82% of this group of tumors express one or more GAGE genes (18) .
Although the expression of the antigen mRNA was heterogeneous within each tumor group, all genes were expressed in the GBM cell line, D566. Previous studies have demonstrated that transcriptional activation of MAGE-1 and the GAGE family of genes is dependent upon the methylation status of the promoter (19) .4
There is high sequence homology between GAGE-2 and -7, the 5' end of GAGE-1 and GAGE-3–6 and -8. GAGE-1 differs from GAGE-2 to -8 in that it has an additional 143-bp exon that encodes 22 COOH-terminal amino acids in the putative GAGE-1 protein. The unique sequence in GAGE-1 may represent a more specific target for immunotherapy for two reasons. First, immunizing against GAGE-3–6 or -8, which are expressed in a larger proportion of tumors, as opposed to GAGE-1, may be problematic as these genes share a high sequence homology with GAGE-2 and -7, which are expressed in normal brain. Second, the additional 22 COOH-terminal amino acids unique to GAGE-1 contains several potential immunoreactive epitopes.
Lethé et al. (15) demonstrated that CTL lysis of melanoma and other tumor cell lines occurs only if MAGE-1 expression exceeds 10% of that found in a control cell line. The feasibility of using GAGE as a target for specific immunotherapy must include a consideration of the level of expression of this gene in tumors examined. When compared with D566, a GBM cell line, the expression level of GAGE in tumors was <1% of that in D566 (data not shown). Thus, further investigation is required to determine whether GAGE would be a useful target for immunotherapy as the level of expression is low, requiring 30 cycles of PCR for detection, and a homogeneous expression within a tumor type has not been ascertained.
The specific diagnosis and grading of astrocytomas is often difficult. Significant inconsistencies in the pathological classification of astrocytomas can occur, often compromising patient management and jeopardizing the interpretations of clinical studies (20 , 21) . Molecular tools for diagnosis are urgently required. Thus, the pattern of MAGE and GAGE expression may also be potentially useful as a diagnostic marker for high-grade brain tumors. Studies examining the expression of MAGE and GAGE in WHO grades I, II, and III gliomas are currently underway.
Our results show that expression of MAGE-1, -3, and -6 in high grade brain tumors is infrequent. A large percentage of medulloblastomas and ependymomas express MAGE-2. GAGE was expressed by all four tumor types. The absence of GAGE-1 in normal brain, its relatively high frequency of expression in high-grade brain tumors, and its unique 3' sequence make it attractive as a potential target for specific immunotherapy. The detection method we used here may be used for rapid screening of biopsy specimens both for diagnostic purposes and to determine a patient’s eligibility for such therapy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by the Department of Haematology and Oncology, Royal Children’s Hospital (Melbourne, Australia). D. M. A. is a recipient of a Neil Hamilton-Fairley fellowship from the National Health and Medical Research Council, Australia. 
2 To whom requests for reprints should be addressed, at Department of Haematology and Oncology, Royal Children’s Hospital, Flemington Road, Parkville 3052, Melbourne, Victoria, Australia. Phone: 61 3 9345 5652; Fax: 61 3 9345 6524; E-mail: ashleyd{at}cryptic.rch.unimelb.edu.au
3 The abbreviations used are: GBM, glioblastoma multiforme; RT-PCR, reverse transcription-PCR.
4 O. De Backer, K. C. Arden, M. Boretti, C. De Smet, V. Vantomme, S. Czekay, C. S. Viars, E. De Plaen, P. van der Bruggen, F. Brasseur, P. Chomex, B. Van den Eynde, and T. Boon. Structure, chromosomal location and expression of the GAGE genes, submitted for publication.
Received 8/14/98; revised 11/16/98; accepted 11/24/98.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  N. Ludwig, A. Keller, N. Comtesse, S. Rheinheimer, C. Pallasch, U. Fischer, K. Fassbender, W. I. Steudel, H.-P. Lenhof, and E. Meese Pattern of Serum Autoantibodies Allows Accurate Distinction between a Tumor and Pathologies of the Same Organ Clin. Cancer Res., August 1, 2008; 14(15): 4767 - 4774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Zhang, J. Eguchi, C. A. Kruse, G. G. Gomez, H. Fakhrai, S. Schroter, W. Ma, N. Hoa, B. Minev, C. Delgado, et al. Antigenic Profiling of Glioma Cells to Generate Allogeneic Vaccines or Dendritic Cell-Based Therapeutics Clin. Cancer Res., January 15, 2007; 13(2): 566 - 575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Cadieux, T.-T. Ching, S. R. VandenBerg, and J. F. Costello Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation. Cancer Res., September 1, 2006; 66(17): 8469 - 8476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Soling, M. Sackewitz, M. Volkmar, D. Schaarschmidt, R. Jacob, H.-J. Holzhausen, and N. G. Rainov Minichromosome Maintenance Protein 3 Elicits a Cancer-Restricted Immune Response in Patients with Brain Malignancies and Is a Strong Independent Predictor of Survival in Patients with Anaplastic Astrocytoma Clin. Cancer Res., January 1, 2005; 11(1): 249 - 258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Liu, H. Ying, G. Zeng, C. J. Wheeler, K. L. Black, and J. S. Yu HER-2, gp100, and MAGE-1 Are Expressed in Human Glioblastoma and Recognized by Cytotoxic T Cells Cancer Res., July 15, 2004; 64(14): 4980 - 4986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Sienel, C. Varwerk, A. Linder, D. Kaiser, M. Teschner, M. Delire, G. Stamatis, and B. Passlick Melanoma associated antigen (MAGE)-A3 expression in Stages I and II non-small cell lung cancer: results of a multi-center study Eur. J. Cardiothorac. Surg., January 1, 2004; 25(1): 131 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sugita, M. Geraci, B. Gao, R. L. Powell, F. R. Hirsch, G. Johnson, R. Lapadat, E. Gabrielson, R. Bremnes, P. A. Bunn, et al. Combined Use of Oligonucleotide and Tissue Microarrays Identifies Cancer/Testis Antigens as Biomarkers in Lung Carcinoma Cancer Res., July 15, 2002; 62(14): 3971 - 3979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Sahin, M. Koslowski, O. Türeci, T. Eberle, C. Zwick, B. Romeike, J.-R. Moringlane, K. Schwechheimer, W. Feiden, and M. Pfreundschuh Expression of Cancer Testis Genes in Human Brain Tumors Clin. Cancer Res., October 1, 2000; 6(10): 3916 - 3922. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
