The clinical significance of MAGEA3 expression in pancreatic cancer

Authors

  • Joseph Kim,

    1. Gastrointestinal Research Section, Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA,USA
    2. Division of Surgical Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA, USA
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  • Howard A. Reber,

    1. Section of Gastrointestinal Surgery, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Oscar J. Hines,

    1. Section of Gastrointestinal Surgery, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Kevork K. Kazanjian,

    1. Section of Gastrointestinal Surgery, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Andy Tran,

    1. Gastrointestinal Research Section, Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA,USA
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  • Xing Ye,

    1. Division of Biostatistics, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA, USA
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  • Farin F. Amersi,

    1. Gastrointestinal Research Section, Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA,USA
    2. Division of Surgical Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA, USA
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  • Steve R. Martinez,

    1. Gastrointestinal Research Section, Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA,USA
    2. Division of Surgical Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA, USA
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  • Sarah M. Dry,

    1. Department of Pathology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Anton J. Bilchik,

    1. Division of Surgical Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA, USA
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  • Dave S.B. Hoon

    Corresponding author
    1. Gastrointestinal Research Section, Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA,USA
    • Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA, 90404, USA
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    • Fax: +1-310-449-5282, +1-310-449-5261.


Abstract

The MAGEA gene family that encodes cancer testis antigens is differentially expressed in many cancers. Though MAGEA3 expression has been detected in gastrointestinal malignancies, its role in pancreatic ductal adenocarcinoma (PDAC) has not been well established. We assessed 57 patients who underwent intent-to-cure surgery for PDAC. Total RNA from paraffin-embedded pancreatic tumors was extracted and assessed for MAGEA3 gene expression by an optimized probe-based quantitative real-time RT-PCR (qRT) assay. MAGEA3 gene expression was detected by qRT in 25 (44%) patients. For the entire cohort, detection of MAGEA3 expression was associated with significantly decreased overall survival (median, 16 vs 33 months; log-rank, p = 0.032). When clinicopathologic factors, including age, gender, stage, tumor extent, lymph node metastasis, tumor grade, perineural invasion and lymphovascular invasion were assessed by univariate analysis, MAGEA3 gene expression and tumor grade were significant prognostic factors for poor survival (HR 2.1, 95% CI: 1.0–4.4, p = 0.041; and HR 3.7, 95% CI: 1.8–7.6, p = 0.0004, respectively). Immunohistochemistry (IHC) was performed and confirmed MAGEA3 protein in PDAC specimens. In conclusion, MAGEA3 is differentially expressed in patients with PDAC; its expression correlates with significantly worse survival. Molecular assessment for MAGEA3 should be considered to improve prognostic evaluation and to identify eligible patients for potential immune-based therapy. © 2005 Wiley-Liss, Inc.

Pancreatic ductal adenocarcinoma (PDAC) has been a leading cause of cancer-related mortality, in part, due to ineffective treatment options. Although efforts to characterize the molecular features of PDAC have identified dysregulated signaling and transcriptional pathways (e.g., KRAS and DPC4/SMAD4 mutations, respectively)1, 2 that promote aggressive properties in pancreatic cancer, therapies to abrogate or block such pathways have been disappointingly ineffective. This may reflect nonspecific targeting of regulated, benign cells that share identical signaling pathways with cancer cells. Such may be a limitation for recently developed tyrosine kinase inhibitors.3 One potential method to bypass these constraints is to identify pancreatic tumor antigens that are not expressed on benign cells.

The MAGE family of cancer testis antigens (MAGEA, MAGEB and MAGEC) are unique tumor markers that are expressed normally only in the placenta and male germ cells.4, 5, 6 The remaining members of the human MAGE family, MAGED and necdin, are ubiquitously expressed.7 The MAGE antigens arise from transcription of genes that belong to a family of more than 20 closely related genes located on chromosome X.8, 9, 10, 11, 12, 13, 14, 15 Although originally identified in melanoma, the MAGE gene is commonly expressed in various tumors of epithelial origin, including breast, lung and colorectal carcinomas.16, 17, 18, 19 The MAGEA subfamily, which is comprised of 12 genes, has had one or more of its antigens detected in various gastrointestinal malignancies, including pancreatic cancer.20, 21, 22, 23, 24, 25, 26

We have previously assessed MAGEA3 gene expression in melanoma and other cancers and have found it to be a specific molecular biomarker for cancer cells.27, 28, 29, 30 However, DNA sequences of MAGEA3 and MAGEA6 are almost identical and, therefore, RT-PCR assays may not discriminate between the 2 MAGEA variants.31 We hypothesized that, as an antigen unique primarily to cancer cells and expressed in gastrointestinal malignancies, MAGEA3 may be expressed in pancreatic cancer. A recent report examined MAGEA3 expression in pancreatic cancer, albeit by gel-based RT-PCR methods.26 Here, we assessed PDAC specimens from patients who underwent surgical resection and sought to determine patterns of MAGEA3 gene expression by quantitative real-time RT-PCR (qRT) and whether differential patterns of expression correlated with clinical outcomes.

Material and Methods

Patients and resources

A multi-institutional cohort of patients (n = 57) was evaluated for this study. Patients were accrued from John Wayne Cancer Institute (JWCI), UCLA School of Medicine and the Cooperative Human Tissue Network (CHTN). All patients underwent intent-to-cure surgery for PDAC between the years 1996 and 2004. Benign and matched normal pancreas specimens (n = 15) were also obtained from all 3 sites. During this time interval PDAC patients were offered various adjuvant chemotherapy regimens consisting of 5-fluorouracil, leucovorin, mitomycin C, dipyridamole or gemcitabine. Adjuvant radiation therapy was offered to patients at the discretion of their treating physicians. A Human Subjects Institutional Review Board (IRB) approval was obtained for the purposes of this study at the participating institutions. Informed consent waivers were obtained for all patients in this study to allow collection of retrospective clinical data and to conduct an analysis of archived paraffin-embedded specimens.

RNA isolation

Eight established pancreatic cancer cell lines (MIA PaCa-2, PANC-1, Capan-1, BxPC-3, COLO357, CFPAC-1, AsPC1 and Hs 766T) were obtained from the American Type Culture Collection (American Type Culture Collection, ATCC, Manassas, VA) and maintained as recommended. The immortalized normal human pancreas ductal epithelial (HPDE) cell line32, 33 was kindly provided by Dr. Tsao (University of Toronto, Toronto, Ontario, Canada) and was used as a negative cell line control for MAGE-A3 gene expression. All cells were incubated at 37°C with 5% CO2. Total RNA from cell lines was extracted, isolated and purified using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as previously described.28, 29

Paraffin-embedded archival tissue (PEAT) blocks of PDAC specimens were reviewed by a surgical pathologist to confirm the diagnosis of PDAC and ensure the presence of tumor in the tissue blocks. Additional paraffin blocks of tumor from the same patient were unavailable due to procedural restrictions of tumor procurement. Therefore, potential tumor heterogeneity was not assessed. Also, tumor blocks were not microdissected because MAGEA3 is not expressed in normal tissues other than placenta or male germ cells.4, 5, 6

The paraffin blocks were sectioned (20 μm) under RNAse-free conditions and placed in sterile microcentrifuge tubes (Eppendorf, Westbury, NY). After deparaffinization with xylene and washings with 100% ethanol, the specimens were treated with a proteinase K digestion buffer for 3 hr. Total RNA was extracted, isolated and purified using a modification of the RNAWiz (Ambion, Austin, TX) phenol–chloroform extraction method as previously described.34 RNA was quantified and assessed for purity by ultraviolet spectrophotometry and RIBOGreen detection assay as previously described (Molecular Probes, Eugene, OR).30

Primers and RT-PCR

Primer and probe sequences were designed as previously described.35 Specific primers were designed to amplify sequential exon–exon regions to avoid potential amplification of contaminating genomic DNA and to produce amplicon sizes less than 150 bp to optimally amplify PEAT RNA. The primers and FRET probe sequences used were: MAGEA3: 5′-AGGAGAAGATCTGCCAGTGG-3′ (forward); 5′-AGTGCTGACTCCTCTGCTCA-3′ (reverse); and 5′-FAM-AGCTCCTGCCCACACTCCCGCCTGT-BHQ-1-3′ (FRET probe). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′-GGGTGTGAACCATGAGAAGT-3′ (forward); 5′-GACTGTGGTCATGAGTCCT-3′ (reverse); and 5′-FAM-CAGCAATGCC TCCTGCACCACCAA-BHQ-1-3′ (FRET probe). PCR products were assessed by gel electrophoresis to confirm amplicon sizes.

Reverse transcription of total RNA was performed using Moloney Murine Leukemia Virus RT (Promega, Madison, WI). To ensure more robust cDNA production, Oligo dT (Gene Link, Hawthorne, NY) and random hexamers (Roche, Indianapolis, IN) were added to the PEAT reaction mixtures as previously described.30 The qRT assay was performed with the iCycler iQ RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using 250 ng of total RNA for each reaction.

Each PCR reaction was performed with 1 μM of each primer, 200 μM each deoxynucleotide triphosphate, 4.0 mM MgCl2, 10× AmpliTaq Buffer and 1 U of AmpliTaq Gold Polymerase (Applied Biosystems, Branchburg, NJ) and was subjected to 45 cycles at 95°C for 60 sec, 58°C for 60 sec and 72°C for 60 sec for MAGEA3; 45 cycles at 95°C for 60 sec, 55°C for 60 sec and 72°C for 60 sec for housekeeping gene, GAPDH. Each sample was assayed in triplicate by qRT and cDNA derived from cancer cell lines and volunteer lymphocytes served as positive and negative controls, respectively, for the MAGEA3 qRT assay. The expression of the housekeeping gene GAPDH was assessed in all pancreatic cancer specimens to verify mRNA integrity. Only specimens with adequate RNA (positive MAGEA3 expression or adequate GAPDH gene expression, i.e., copy numbers ≥1,000) were included in the study.

Immunohistochemistry

Immunohistochemistry (IHC) was performed to confirm the translation of MAGEA3 mRNA to protein in PDAC specimens. Tumor blocks were sectioned (5 μm), dried overnight at 37°C and then deparaffinized with xylene. The sections were treated with an antigen retrieval solution (Target Retrieval, DakoCytomation, Carpinteria, CA) at 95°C for 15 min, cooled to room temperature and then treated with dilute hydrogen peroxide to block endogenous peroxidase as previously described.35 Nonspecific antibody binding was diminished with 5% milk. The sections were incubated overnight at 4°C with a polyclonal rabbit anti-human MAGEA3 antibody (Abgent Inc., San Diego, CA) at a dilution of 1:100. We note that this anti-MAGEA3 antibody may cross-react with MAGEA6 because of the protein sequence homology between the 2 variants. The next day, sections were labeled with a secondary Link-Streptavidin HRP solution (Dako), developed with diaminobenzidine (DAB), counterstained with hematoxylin and examined at 200× and 400× magnifications.

Statistical analysis

Patient characteristics and MAGEA3 expression were summarized using mean, median and frequency. Clinicopathologic factors of MAGEA3 negative and positive patients were compared by Student's t-test and Fisher's Exact test. Overall survival curves with respect to MAGEA3 expression were compared using Kaplan–Meier's method. The log-rank test was used to compare the equality of the 2 curves. Univariate analysis of prognostic factors including age, gender, stage, tumor extent, lymph node metastasis, grade, size, perineural invasion and lymphovascular invasion was assessed. A multivariate analysis using the Cox proportional hazard regression model was also performed to evaluate the prognostic significance of MAGEA3 expression when clinical prognostic factors were adjusted. A stepwise method was chosen for covariate selection. The relationship of MAGEA3 expression with tumor grade was determined by Chi square analysis. All analyses were performed using SAS (SAS /STAT User's Guide, version 8; SAS Institute Inc, Cary, NC) and tests were 2-sided with significance level of p < 0.05.

Results

MAGEA3 gene expression in control specimens

In 8 of 8 established pancreas cancer cell lines (MIA PaCa-2, PANC-1, Capan-1, BxPC-3, COLO357, CFPAC-1, AsPC1 and Hs 766T), MAGEA3 gene expression was detected by qRT. All but 2 of the lines (PANC-1 and BxPC-3) were derived from metastatic pancreatic cancers and only one (PANC-1) was derived from the exocrine ducts. MAGEA3 expression was undetectable by qRT in the normal control line HDPE (Fig. 1). BxPC3, COLO357 and Hs 766T had the highest levels of MAGEA3 gene expression relative to GAPDH expression. Additionally, mRNA from the peripheral lymphocytes of healthy volunteers (n = 5) were negative for MAGEA3 expression by qRT. Matched benign pancreas specimens (n = 10) and pancreas tissue with chronic pancreatitis (n = 5) were obtained and analyzed. In 14 of 15 (94%) specimens, MAGEA3 expression was not detected. One pancreas specimen from a patient with chronic pancreatitis was positive for MAGEA3 gene expression.

Figure 1.

MAGEA3 gene expression in 8 pancreatic cancer cell lines and one normal pancreas ductal epithelial cell line. MAGEA3 expression is plotted as a ratio to the expression of the housekeeping gene GAPDH.

MAGE-A3 expression in patients with pancreatic cancer

Fifty-seven patients were assessed for MAGEA3 gene expression by qRT; the patient demographics are listed in Table I. Because of our use of paraffin-embedded samples, we assessed for MAGEA3 expression and stratified specimens into a group with (+) expression of any level and a group with (−) expression. We utilized zero copy number as the cut-off between (+/−) expression. This cut-off was determined by generating standard PCR curves using serially diluted cDNA standard templates for MAGEA3 and using the threshold cycle (Ct) of templates with known numbers of copies. We utilized the optimized threshold cycle number that would amplify at least 1 mRNA copy number of MAGEA3. At 45 cycles, our MAGEA3 positive controls (pancreas cancer cell lines) were PCR (+) and our negative controls (normal pancreas ductal epithelial cell line and volunteer lymphocytes from healthy volunteers) were PCR (−). Accordingly, MAGEA3 gene expression was recorded as a binary result (+/−) for all PDAC specimens.

Table I. Pancreatic Cancer Demographics
Clinicopathologic factorsN
Total patients57
 Men30
 Women27
Age (yr)
 <503
 50–7027
 >7027
UICC/TNM stages
 pI18
 pII39
T-stage
 pT19
 pT241
 pT37
Lymph node metastasis
 pN021
 pN136
Pathologic grade
 Well9
 Moderate25
 Poor23
Tumor size (cm)
 <28
 2–544
 >55
Perineural invasion
 None18
 Present39
Lymphovascular invasion
 None41
 Present16

MAGEA3 gene expression was detected in the PDAC specimens of 25 (44%) patients. All 57 patients were then stratified into 2 groups based on MAGEA3 expression. Clinicopathologic factors of MAGEA3 negative and positive patients were compared (Table II). All patients in the cohort had negative surgical margins by H&E. The groups were similar for all other factors except for PDAC tumor grade, with a higher number of differentiated tumors in the MAGEA3 negative group. Kaplan–Meier curves were constructed to determine whether MAGEA3 gene expression correlated with survival in these groups. A significant difference in overall survival was present between patients with MAGEA3 positive vs negative gene expression (median 16 vs 33 months, respectively; log-rank, p = 0.032) (Fig. 2). During a median follow-up time of 15 months, 35 (61%) patients had succumbed to disease.

Figure 2.

Kaplan–Meier curves showing the difference in survival between patients with MAGEA3 negative and positive pancreatic cancers.

Table II. Comparison of Clinicopathologic Factors Between MAGEA3 Negative and Positive Patients
Clinicopathologic factorsMAGEA3p-value1
Negative (n = 32)Positive (n = 25)
  • 1

    Comparison for age was performed by Student's t-test, all other comparisons were assessed by Fisher's Exact test.

Age (yr)  0.58
 Mean ± SD67 ± 1269 ± 9 
 Range42–9054–84 
Gender  1.0
 Female1512 
 Male1713 
UICC/TNM stage  0.15
 pI135 
 pII1920 
Primary tumor  0.61
 pT154 
 pT22219 
 pT352 
Lymph node metastasis  0.27
 pN0147 
 pN11818 
Tumor size (cm)  0.43
 0–274 
 >2–52318 
 >523 
Pathologic grade  0.028
 Well72 
 Moderate169 
 Poor914 
Perineural invasion  0.78
 Absent117 
 Present2118 
Lymphovascular invasion  0.14
 Absent2615 
 Present610 

To determine the prognostic significance of MAGEA3 gene expression, clinicopathologic data, including age, gender, stage, tumor extent, lymph node metastasis, tumor grade and size, perineural invasion and lymphovascular invasion were compared by univariate analysis for the 2 groups (Table III). By univariate analysis, MAGEA3 and tumor grade were significant prognostic factors for poor survival (HR 2.1, 95% CI: 1.0–4.4, p = 0.041; and HR 3.7, 95% CI: 1.8–7.6, p = 0.0004, respectively). On multivariate analysis, only poorly-differentiated histologic tumor grade remained a significant prognostic factor for poor survival (HR 6.6, 95% CI: 1.8–23.6, p = 0.0007). Lymph node metastasis, a prognostic factor for poor survival in large cohort studies was not significant here.36, 37

Table III. Univariate and Multivariate Analysis
Clinicopathologic Factors#Death/nUnivariate analysisMultivariate analysis
Hazard ratio (95% CI)p-valueHazard ratio (95% CI)p-value
Age  NS NS
 <502/31.0   
 50–7017/271.1 (0.3–4.7)   
 >7016/271.3 (0.3–5.5)   
Gender  NS NS
 Female16/271.0   
 Male19/301.1 (0.6–2.1)   
Stage  NS NS
 p110/181.0   
 p225/391.5 (0.7–3.1)   
Tumor extent  NS NS
 pT14/91.0   
 pT227/411.5 (0.5–4.4)   
 pT34/71.1 (0.3–4.4)   
Lymph node disease  NS NS
 pN011/211.0   
 pN124/361.6 (0.8–3.2)   
Tumor size (cm)  NS NS
 <25/81.0   
 2–525/440.8 (0.3–2.2)   
 >55/52.4 (0.8–8.5)   
Grade
 Well4/91.0   
 Moderate11/252.2 (0.6–8.0)   
 Poor20/233.7 (1.8–7.6)0.00046.6 (1.8–23.6)0.0007
Perineural invasion  NS NS
 Absent10/181.0   
 Present25/391.6 (0.8–3.5)   
Lymphovascular invasion  NS NS
 Absent25/411.0   
 Present10/161.3 (0.6–2.8)   
MAGEA3 expression    NS
 No20/321.0   
 Yes15/252.1 (1.0–4.4)0.041  

Immunohistochemistry

IHC was performed on representative PEAT sections of MAGEA3 gene expression positive and negative PDAC specimens (n = 14). IHC of specimens having absent MAGEA3 gene expression by qRT demonstrated no detectable immunostaining. In our study, matched histologically benign pancreas and colon served as negative control specimens. Distinct patterns of membranous and cytoplasmic immunostaining for MAGEA3 protein were observed in undifferentiated cancer cells in the PDAC specimens (Fig. 3a), whereas little or no staining was present in representative desmoplastic tissues (Fig. 3b) or well-differentiated tissues (Fig. 3c).

Figure 3.

(a) Representative MAGEA3 protein expression in undifferentiated pancreatic cancer cells by immunohistochemistry followed by staining with hematoxylin at ×400 magnification. There was absence of immunostaining in (b) desmoplastic tissues next to malignant cells and absence of immunostaining in (c) well-differentiated tissues at ×200 magnification.

Discussion

As antigens with expression patterns unique to many tumors, cancer testis antigens from the MAGEA family have garnered attention as potential targets for vaccine-based immunotherapy of cancer.9, 10 The lack of gene expression of the encoding genes in healthy tissues theoretically ensures strict tumor-specific targeted immune responses after patient vaccination. Recently, clinical trials have been initiated with MAGEA-derived immunogens.38, 39, 40, 41 Therefore, the determination of patients eligible for immunization with a defined MAGEA antigen mandates analysis of tumors for expression of the MAGEA gene along with the appropriate HLA specificity. Whether the appropriate criteria have been met can be readily tested by HLA typing and by RT-PCR on RNA extracted from tumor samples.

Expression of the MAGE family of cancer testis antigens has been detected in several epithelial cancers of varying embryologic origin.17, 18, 19, 20 The role of these MAGE antigens during embryogenesis, cellular growth and differentiation is still not entirely clear since their initial discovery, and their function in epithelial cancers is largely unknown.4 In these cancers, clinical correlations have been identified as a result of differential expression patterns. Some reports have noted that expression of MAGE antigens was associated with worse pathological tumor stage or poor clinical outcomes.42, 43 In contrast, Hansel et al.44 discovered that absence of MAGEA1 expression indicated a worsened prognosis. In our current report, we detected MAGEA3 gene expression in a large percentage of our patient cohort (44%). Patients whose tumors expressed MAGEA3 had an ∼50% decrease in overall survival compared to tumors with absent expression.

The considerable difference in survival relative to MAGEA3 gene expression was significant by univariate analysis. When clinicopathologic factors were adjusted, MAGEA3 expression was no longer significant (Table III). Because of the large percentage of deaths (n = 20 of 23; 87%) in patients with poorly differentiated tumors, tumor grade overshadowed MAGEA3's role as a prognostic factor for survival in this patient cohort. Exclusion of tumor grade from the multivariate analysis identifies MAGEA3 as a significant prognostic factor for poor survival (HR 2.1, 95% CI 1.0–4.4; p = 0.04). Furthermore, there is a significant association of MAGEA3 gene expression with tumor grade. Only 24% (n = 11 of 45) of well- and moderately differentiated tumors demonstrated MAGEA3 gene expression, whereas 61% (n = 14 of 23) of poorly differentiated tumors were MAGEA3 positive (χ2, p = 0.035). The implication is that MAGEA3 protein contributes to poor survival through its expression in primarily poorly differentiated pancreatic tumors.

Recent studies illustrate that epigenetic mechanisms may regulate the differential patterns of MAGE gene expression.25 This mechanism may account for the varying levels of MAGEA3 gene expression by qRT in our pancreatic cancer cell lines (Fig. 1). A demonstration of the epigenetic silencing of MAGEA3 was reported by Sigalotti et al.,45 who utilized 5-aza-2′-deoxycytidine to reverse methylation silencing of the MAGE genes. This finding is informative since the MAGEA3 gene on the X-chromosome has methylated CpG islands in normal somatic tissues.46, 47 However, additional unknown epigenetic events may regulate MAGEA3 expression and, therefore, require further studies. It is possible that variable silencing of MAGEA3 gene expression by epigenetic mechanisms may decrease levels of gene expression to such degrees, that it cannot be detected with semi-quantitative methods. This explanation may account for the discrepancies in results between the report by Kubuschok et al.26 and our study. Their group, using gel-based RT-PCR, noted MAGEA3 gene expression in only 2 of 10 pancreatic cancer cell lines and in none of their pancreatic cancer biopsies.

Current therapeutic options, which include chemotherapy, monoclonal antibodies and radiation therapy remain limited in efficacy, although new agents and regimens show promise.48 MAGEA3, however, has been shown to be immunogenic in humans, eliciting both cellular and antibody responses.49, 50, 51, 52 Various peptide vaccines are under investigation and have shown to elicit specific immune responses, and more importantly clinical responses.41, 52, 53, 54 In this report, we evaluated patients with limited disease, who were candidates for curative resection. Therefore, our patient cohort may not be representative of the 80–90% of patients with pancreatic cancer, who typically present with unresectable disease.55 It is unknown, whether MAGEA3 gene expression is differentially expressed in the primary and metastatic lesions. We are currently accruing patients to evaluate whether MAGEA3 gene expression has prognostic value in patients with advanced disease.

Pancreatic cancer is an appropriate target for MAGE immune-based treatment strategies, specifically for those patients with tumors that express MAGEA3. In a recent trial, 25 tumor-bearing HLA-A1 melanoma patients were immunized with a MAGEA3 peptide presented by HLA-A1, where objective regression of metastases was observed in 7 patients.38 Three of these regressions were complete. No adverse side effects are expected in germline cells because HLA molecules are not present at the surface of these cells. In conclusion, the clinical characteristics of MAGEA3, its absence in normal tissues and association with worse outcomes make it an attractive target for immune-based treatment strategies in patients with pancreatic cancer.

We, the authors of this study, certify that we have not entered into any agreement that interferes with our access to the data of this research study, our ability to analyze the data independently, our preparation of the manuscript and our publication of this manuscript.

We, the authors of this study, have no financial interests to declare.

Acknowledgements

We would like to thank Dr. K. Washington at the Cooperative Human Tissue Network, Vanderbilt University School of Medicine (Nashville, TN, USA) for their assistance in obtaining pancreas specimens and we would like to thank the Tissue Procurement Core Laboratory of the Department of Pathology at UCLA for their expertise and support in this project.

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