Cancer-testis gene expression profiling in ...

research paper

Research paper

Cancer Biology & Therapy 12:3, 191-197; August 1, 2011; © 2011 Landes Bioscience

Cancer-testis gene expression profiling in esophageal squamous cell carcinoma Identification of specific tumor marker and potential targets for immunotherapy

Mohammad Mahdi Forghanifard,1,2,* Mehran Gholamin,2 Moein Farshchian,2 Omeed Moaven,2,3 Bahram Memar,4 Mohammad Naser Forghani,5 Ezzat Dadkhah,2 Hossein Naseh,4 Meysam Moghbeli,2 Reza Raeisossadati2 and Mohammad Reza Abbaszadegan2,* Department of Biology, Science and Research Branch; Islamic Azad University; Tehran, Iran; 2Human Genetic Division; Immunology Research Center; Avicenna Research Institute; Mashhad University of Medical Sciences; Mashhad, Iran; 3Department of Surgery; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA; 4 Department of Pathology; 5Department of Surgery; Omid Hospital; MUMS; Mashhad, Iran

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Keywords: esophageal squamous cell carcinoma, cancer testis antigens, real-time PCR, tumor marker, cancer vaccine Abbreviations: ESCC, esophageal squamous cell carcinoma; CTAs, cancer testis antigens

Cancer-testis antigens (CTAs) are often specifically expressed in cancer cells and under normal conditions are only considered to be expressed in the germ line cells and the placenta. CTAs are potential targets for cancer immunotherapy and therefore necessitates their expression profiling. The expression profile of LAGE1, MAGE-A4 and NY-ESO1, their possible correlations and interaction, and the clinicopathological associations of each marker were studied. RNA was extracted from fresh esophagectomy tissues of 41 esophageal squamous cell carcinoma (ESCC) patients prior to any other therapeutic intervention. The relative mRNA expression of LAGE1, MAGE-A4 and NY-ESO1 was assessed with the real-time reverse transcription-polymerase chain reaction (RT-PCR) 5‘ nuclease assay. The overexpression of LAGE1, MAGE-A4 and NY-ESO1 was found in 39, 90.2 and 41.4% of ESCC samples respectively. Of the patients, 97.5% showed an overexpression of at least one CTA. The relative expression of MAGE-A4 was directly associated with lymph node metastasis and the stage of the tumor (p < 0.05). A significant direct correlation was also detected between the MAGE-A4/ LAGE1 and MAGE-A4/NY-ESO1 levels of gene expression. MAGE-A4 is identified as a specific biomarker of ESCC with a possible oncogenic role contributing to tumor progression. Interactions between MAGE-A4, LAGE1 and NY-ESO1 and their significant clinical consequences introduce these CTAs as appropriate targets for a polyvalent cancer vaccine.

Introduction Esophageal squamous cell carcinoma (ESCC) is the second leading cause of cancer-related deaths in northeastern Iran, an area located on the esophageal cancer belt.1 In the absence of efficient screening and early diagnostic tools, patients often remain undiagnosed until the advanced stages when common therapeutic modalities are virtually ineffective and the overall survival rate is low.2 Consequently novel therapeutic modalities are needed to combat the odds of survival. Among these, one of the most attractive is immunotherapy, which relies on targeting tumor specific antigens.3 Thus, the identification of these markers in the tumor is crucial. As a subgroup of tumor specific antigens, cancer-testis antigens (CTAs) have a distinct pattern of expression, which, under normal conditions, is mostly confined to the placenta and immature cells of the gonads.4 While the aberrant expression of CTAs has been frequently observed in a variety of cancer cells, a causal link has yet

to be clarified. Through the observation of these genes’ expression in mesenchymal cells,5 a link between the cellular processes of selfrenewal and tumorigenicity has been reported and may point to the role of these genes in the development of cancer.6 MAGE-A4, LAGE1 and NY-ESO1 are among the major CTAs targeted in several studies.7-10 Contradictory data have been reported regarding the role of MAGE-A4 in the cell biology of different tumors. It has been shown that MAGE-A4 contributes to transcriptional control in different ways.11,12 By binding to the transcription factor PIAS2, the c-terminal fragment of MAGE-A4 suppresses transcription at target promoters.13 This fragment is the active portion in various interactions, including histone deacetylase 1 (HDAC1) and SNW1,14 at promoters leading to a localized transcriptional repression. Moreover, the c-terminal fragment of MAGE-A4 prevents p21-mediated cell cycle arrest through its inhibition of the binding of the transcription factor Miz-1 to the p21 promoter and the blocking of p21 upregulation.13 In contrast

*Correspondence to: Mohammad R. Abbaszadegan and Mohammad M. Forghanifard; Email: [email protected] and [email protected] Submitted: 12/13/10; Revised: 04/16/11; Accepted: 05/03/11 DOI: 10.4161/cbt.12.3.15949 www.landesbioscience.com

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Figure 1. Scatter plot representative of descriptive analysis of relative gene expression distribution of LAGE1, MAGE-A4 and NY-ESO1 in the patients with ESCC. The Y axis indicates the relative gene expression, and the X axis represents the patients. Relative mRNA expression of more than two-fold in tumor tissue is considered as overexpression, less than minus two-fold as underexpression, and the range in-between is deﬁned as normal. Patient 9 (T2N1, distal tumor with poorly differentiated pathologic grade) did not show overexpression in any of the studied CTA. All other patients have overexpression of at least one of the CTAs.

to its oncogenic roles, the carboxyl-terminal portion of MAGE-A4 induces apoptosis through p53-dependent and p53-independent pathways.15 MAGE-A4 interacts with the liver’s oncoprotein gankyrin and suppresses its oncogenic activity.16 MAGE-A4 can bind PSMD10, an oncoprotein associated with the 19S regulatory cap of proteasomes.17 Contrary to MAGE-A4’s suggested oncogenic roles, these findings propose that MAGE-A4, in some malignancies, functions as a tumor suppressor protein rather than an oncoprotein. The short peptide sequences of CTA epitopes elicit immunogenicity in cancer. In light of this, these tumor-specific antigens are highly attractive targets for immunotherapy.18 Determining the expression patterns of CTA and their clinicopathological significance will not only offer a better understanding of their impact on the process of tumorigenesis,19 but also provide essential preliminary steps to identify specific markers for cancer immunotherapy. In this study, a comparative real-time PCR 5' nuclease assay was applied to quantitatively compare the expression profile of MAGE-A4, LAGE1 and NY-ESO1 in tumoral cells to the corresponding normal tissue margin. The possible correlations and interactions between these and the clinicopathological associations of each marker were studied. Results The expression of cancer-testis antigens was analyzed in 41 newly diagnosed, fresh frozen tumors and their normal margins by reverse transcription and real time-polymerase chain

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reaction (PCR) amplification. The mean age ± SD of the enrolled patients was 61.44 ± 12.83 (ages ranging between 37–84). The Male:Female ratio was 1:1 (22:19). The clinicopathological characteristics of the patients are presented in Table 2. Upregulation of LAGE1, MAGE-A4 and NY-ESO1 in ESCC. The mRNA expression levels of LAGE1, MAGE-A4 and NY-ESO1 in the tumoral tissue was compared to the corresponding resected tumor-free esophageal epithelium by real-time RT-PCR. Descriptive analysis of relative gene expression distribution of LAGE1, MAGE-A4 and NY-ESO1 in the patients with ESCC is represented as a scatter plot in Figure 1. Overexpression of LAGE1, MAGE-A4 and NY-ESO1 mRNA was detected in 39, 90.2 and 41.4% of tumor specimens respectively. Except for one (patient 9 in Fig. 1), all other patients showed overexpression of at least one of the CT antigens. Association of MAGE-A4 and clinicopathological features. In order to show the impact of CTA expression on pathologic indices of poor prognosis, we assessed the correlation between metastasis to lymph nodes, tumor depth of invasion and tumor stage. We found that the expression of MAGE-A4 significantly correlated with the metastasis of the tumor cells to lymph nodes (p < 0.05). Moreover, association between the number of involved lymph nodes (0.739 ± 1.25) and mean relative expression of MAGE-A4 was studied and a significant correlation was observed (p < 0.05). There was a significant correlation between the expression of MAGE-A4 mRNA and the stage of the tumor (p < 0.01). The expression of LAGE1 and NY-ESO1 genes was not associated with any clinicopathological features. Correlations between the expression levels of MAGE-A4, LAGE1 and NY-ESO1 and clinicopathological parameters are shown in Table 3 and 4. Correlation between LAGE1, MAGE-A4 and NY-ESO1 gene expressions. Correlation analysis between LAGE1, MAGE-A4 and NY-ESO1 mRNA expression was performed by Pearson’s correlation test (Table 5). Significant direct correlation was detected between the expressions of LAGE1 and MAGE-A4 genes (p < 0.01, correlation coefficient: 0.411). Compared to others, patients with concomitant overexpression of MAGE-A4 and LAGE1 showed significantly higher levels of MAGE-A4 expression (12.3 ± 26.51 vs. 7.34 ± 6.15, p < 0.001) and LAGE1 expression (5.98 ± 5.04 vs. 0.29 ± 1.95, p = 0.021). Figure 2 illustrates this correlation in a regression plot. The mRNA expression of MAGE-A4 was significantly associated with that of NY-ESO1 (p < 0.05, correlation coefficient: 0.368). Patients with concomitant overexpression of MAGE-A4 and NY-ESO1 had significantly higher levels of MAGE-A4 expression (11.99 ± 5.97 vs. 7.15 ± 6.46, p = 0.021) and NY-ESO1 expression (9.13 ± 6.30 vs. 0.25 ± 1.38, p < 0.001). Correlation is represented as a regression plot in Figure 3. No significant association between the expression levels of LAGE1 and NY-ESO1 was identified. To better understand the gene-gene associations, the impact of MAGE-A4 overexpression was assessed after having excluded the cases without MAGE-A4 overexpression. The expression levels of LAGE1 in the MAGE-A4 overexpressed group was compared with that of patients with normally expressed MAGE-A4.

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Figure 2. Regression plot illustrating correlation between level of MAGE-A4 and LAGE1 expression. (p = 0.411, p value = 0.008).

NY-ESO1, were observed in cases with concomitant expression of both genes. In order to show the impact of pathologic indices of poor prognosis, i.e., metastasis to lymph nodes, tumor depth of invasion and tumor stage, the association between expressions of CTAs, in tumors with different depth of tumor (T), tumors with or without node metastasis and different stages of tumors were compared. Coexpression of CTAs was more frequently observed in tumors having a greater depth of invasion, i.e., invasion to the adventitia (T3/T4, p < 0.05) and in tumor with node metastasis (N1, p < 0.05). Consequently, both of these associations were only observed in more advanced stages (stage III/IV: p < 0.05). To determine the impact of tumor grade of differentiation on concomitant expression of genes, coexpression of genes were assessed in different pathologic grades. A significant correlation between the coexpression of LAGE1 and MAGE-A4 in all patients was observed in only well-differentiated tumor tissues (p = 0.014, correlation coefficient: 0.776). Additionally, MAGE-A4 and NY-ESO1 coexpression was significantly correlated in moderately differentiated tumors (p < 0.001, correlation coefficient: 0.669). The expression levels of LAGE1 and NY-ESO1 were strongly correlated in poorly differentiated tumors (correlation coefficient: 0.707) at a level close to statistical significance (p = 0.076). Discussion

Figure 3. Regression plot illustrating correlation between level of MAGE-A4 and NY-ESO1 expression. (ρ = 0.368, p value = 0.018).

LAGE1 was significantly expressed at higher levels in the patients with overexpressed MAGE-A4 (5.98 ± 5.04 vs. -0.63 ± 1.50, p < 0.001). Similarly, to understand the impact of LAGE1 expression on MAGE-A4, it should be noted that MAGE-A4 was significantly expressed at higher levels in the patients with overexpressed LAGE1 (12.32 ± 6.51 vs. -0.13 ± 0.18, p < 0.001). The same set of analysis was performed for the MAGE-A4/ NY-ESO1 association and similar results were obtained. By excluding cases with no MAGE-A4 overexpression, it was observed that the expression level of NY-ESO1 was significantly higher if both genes were coexpressed (9.13 ± 6.30 vs. -0.51 ± 1.07, p < 0.001), whereas the difference in the expression of MAGE-A4 was not significant. Similarly, when samples without NY-ESO1 overexpression were excluded, higher levels of MAGE-A4, but not

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Cancer testes antigens have been part of different preclinical and clinical immunotherapy trials, and are promising targets for cancer vaccines. However, the lack of sufficient data on the expression profiles of CTAs in esophageal cancer points to a need for a detailed study prior to their application as targets for a cancer vaccine. Few reports have predominantly focused on the protein expression of CTAs20-23 and, to some extent, mRNA expression with conventional methods.24-27 In this study, we applied the more sensitive and accurate technique of real-time PCR and demonstrated a high frequency of MAGE-A4, NY-ESO1 and LAGE1 overexpression in ESCC. Amongst these, MAGE-A4 overexpression was observed in more than 90% of the ESCC patients, thus introducing this tumor-specific antigen as a sensitive and specific biomarker in ESCC. Tumor expression of MAGE-A4 has been detected in various human malignancies.10,28-33 Quillien et al. reported the highest expression of MAGE-A4 in 71% of their examined ESCC samples. This discrepancy can be explained by the advantages of Real-Time over traditional RT-PCR methods and higher sensitivity in the detection of the mRNA overexpression.21,34 The quantitative analysis of mRNA on fresh tissues in this study, which is the first report in ESCC, is more sensitive for expression analysis. In addition, most of the previous studies were performed on archived tumors and, furthermore, did not exclude patients who had obtained adjuvant therapy. These two factors may considerably influence mRNA expression. The expression level of MAGE-A4 was correlated with parameters indicating the progression of the tumor. It was not only correlated to the metastasis of the tumor to the lymph nodes, but the number of involved lymph nodes was also associated with the level of MAGE-A4 expression. This is consistent with previous


Table 1. Primer and probe sequences used for real-time RT-PCR Primer sequence (5’→3’) LAGE1 MAGE-A4 NY-ESO1 GAPDH

Probe sequence

Forward: gcc tgc ttc agt tgc aca tc Reverse: cgg acc agc tcc gct tc Forward: cca cta cca tca gct tca ctt gc Reverse: ctt ctc gga aca agg act ctg c Forward: tgc ttg agt tct acc tgc ca Reverse: tat gtt gcc gga cac agt gaa Forward: gaa ggt gaa ggt cgg agt c Reverse: gaa gat ggt gat ggg att tc

Table 2. Clinicopathological features of patients Total (41)

Males (22)

Females (19)

6

6

-

FAM-cga tgc ctt tct cgt c-TAMRA

61

FAM-agg caa ccc aa tga ggg ttc cag c-TAMRA

108

FAM-agg atg ccc cac cgc ttc cc-TAMRA

137

FAM-caa gct tcc cgt tct cag cc-TAMRA

225

Table 3. Correlation between expression level of MAGE-A4 and metastasis to lymph nodes and tumor stage MAGE-A4

Tumor invasion T1, 2 T3, 4

35

16

19

MRE ± SD

Lymph node metastasis N0

23

12

11

N1

17

10

7

N2

1

-

1

26

15

11

III/IV

15

7

8

WD

9

4

5

MD

25

16

9

PD

7

2

5

Upper

1

-

1

Middle

25

13

12

Lower

15

9

6

Grade

Tumor location

Tumor size 1–4 cm

23

12

11

4–8 cm

18

10

8

WD, Well-differentiated; MD, moderately-differentiated; PD, poorly differentiated.

studies on SCC malignancies, which reported an association between MAGE-A4 expression and lymph node metastasis in lung and vulvar cancers.20,30 Putz et al. showed the expression of MAGE-A on all of the micrometastatic cell lines in an established working model for human micrometastases.35 Along with the previous studies, our results provide further proof that upregulation of MAGE-A4 may contribute to enhancement of invasive and metastatic ability of tumor cells. Direct correlation between the level of MAGE-A4 expression and number of involved lymph nodes, brings up the assumption that not the expression itself, but also the level of expression and probably its impact on downstream products will lead to a higher level of metastasis to nodes. It is expected that the quantity of the downstream products of MAGE-A4 have influence on the ability of cells to spread and metastasize to the lymph nodes and overexpression, per se, may

194

N0

7.28 ± 5.91

N1

11.28 ± 6.99

0.040

Stage

Stage I/II

p value

Node metastasis

I/II

6.96 ± 5.76

III/IV

12.65 ± 6.68

0.006

MRE, Mean relative expression; SD, Standard deviation.

not be the ample factor to exert this role. Moreover, we showed that higher levels of MAGE-A4 expression correlated with advanced tumor stage, which is consistent with previous reports.36-39 The exact mechanism by which expression of MAGE-A4 and other CTAs results in poor clinical outcome is not well described.40 The precise role that CT antigens might play in cancer cell biology has yet remained to be clarified. It is not exactly understood whether reactivation of their genes is simply an anomaly of deregulated expression or is associated with the proliferative and metastatic potential of the tumor.5 In line with several recent reports in various malignancies,41-43 our data support the later hypothesis since it appears that the overexpression of MAGE-A4 contributes to the progression of ESCC. Conflicting functions are suggested for MAGE-A4 in different malignancies. This includes a role as a transcriptional suppressor and a tumor suppressor in one entity, and functioning as an oncoprotein in another entity.13-16,28-30 Associated with development of tumor in ESCC, MAGE-A4 may be involved in transcriptional suppression of tumor suppressor genes or activation of oncogenes. Crosstalk between MAGE family and different cell signaling pathways has been proposed.44 The MAGE-A4 protein has been implemented in the repression of p53 activity.45 Marcar et al. have recently suggested a mechanism of p53 repression by MAGE-A. Through interaction with peptides located within the DNA binding surface of the core domain of p53, MAGE-A can disrupt the association of p53 with its recognition site in chromatin.46 Moreover; they showed that silencing of MAGE-A expression upregulates several p53-responsive genes including p21 and MDM2. MAGE-A4 is also associated with Rb and competes with p16 to bind to cyclin-dependent kinase 4 (CDK4), thus increases both the phosphorylation and degradation of Rb.47 We


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Table 4. Correlation between number of involved lymph nodes and relative expression of MAGEhave previously suggested a possible crossA4, LAGE1 and NY-ESO1 talk between p53-MDM2 and p16/Rb 48 Number of involved lymph nodes* pathways. MAGE-A4 could be potenPearson correlation p value tially introduced as an intermediate molecule of this crosstalk.45,47,48 Along with MAGE-A4 0.376 0.015 the previous reports, our observations NY-ESO1 0.016 0.924 bring up a hypothesis that MAGE-A4 LAGE1 0.152 0.343 may block the arrests of cell cycle and *Mean ± SD of involved lymph nodes is 0.739 ± 1.25. promote proliferation and inhibit apoptosis through the inhibition of p16/Rb and Table 5. Concomitant mRNA expressions in 41 patients and a ssociations between gene p53. The possible intermediate role should expressions be further studied in detail to support this LAGE1 NY-ESO1 hypothesis. MAGE-A4 14 (34.14%) 16 (39.02%) Higher level of MAGE-A4 expression was observed to be associated with higher p value 0.008 0.018 levels of NY-ESO1 and LAGE1 expresPearson correlation 0.411 0.368 sion. Correlation between the expressions NY-ESO1 8 (19.51%) of CTAs has been reported in other maligp value 0.99 nancies as well.49 Glazer et al. documented Pearson correlation 0.001 a significant, coordinated coexpression of six CTAs, including MAGE-A4 and NY-ESO1, in the SCC subtype of non-small cell lung cancer. It expression at the mRNA level will lead to membrane presentahas been suggested that coordinated promoter demethylation is tion of the antigens. associated with the concomitant aberrant expression of CTAs.51 In summary, MAGE-A4, LAGE1 and NY-ESO1 are freHowever, demethylation alone may not explain the activation quently overexpressed in ESCC. MAGE-A4 is a tumor-specific of different CTAs in various cancers and alternative transcrip- biomarker that is significantly overexpressed in the majority of tional and even post-transcriptional mechanisms that control ESCC patients. MAGE-A4 is associated with the development gene expression may contribute.18,21 On top of the correlations of tumors through advanced stages, playing a possible oncogenic between the gene expressions, it has been observed that overex- role. The expression of MAGE-A4 is correlated with the levels pression of each gene exerted a significant impact on the levels of of LAGE1 and NY-ESO1 expression. The clinicopathological expression of other CTAs that was concomitantly overexpressed. consequences of these associations suggest a possible functional These correlations were associated with unfavorable parameters interaction between these CTAs. A combination of these CTAs responsible for tumor progression and pathologic indices of poor could be a beneficial target for designing a polyvalent vaccine for prognosis, including the depth of invasion, metastasis to lymph ESCC immunotherapy. nodes and the tumor stage. The observed coordinated coexpresMaterial and Methods sion and association with advanced tumors are consistent with reports in other malignancies.40,52 Each correlation was associated with a distinct pathologic behavior of the tumor. These findings Study population and sample collection. A total of 41 historaise the possibility of the role of some functional interactions, to logically confirmed ESCC patients were enrolled in the study be responsible for the correlation between the overexpression of following an informed consent and prior to receiving any therathe CTAs, in addition to the general regulatory impact of pro- peutic interventions. Fresh esophageal tissues were obtained from moter demethylation. MAGE-A4 is suggested as an interesting the patients, who had undergone surgery at Omid Hospital of target for ESCC immunotherapy, especially in advanced tumors Mashhad University of Medical Sciences (MUMS), the referral since it expresses at a considerably high frequency in ESCC oncology hospital of northeastern Iran. Fresh tumoral and nortumors and these expression levels are associated with unfavor- mal specimens were microdissected and microscopically examined to ensure that all the included samples contained more than able clinicopathological parameters of the tumor. The correlation with LAGE1 and NY-ESO1 and their influ- 70% tumor cells, with rare or no infiltrating cells. These were ence on tumor progression, strongly suggests that the use of these kept in an RNA later solution (Qiagen, Hilden, Germany) at CTAs makes a more efficient option for a polyvalent vaccine -20°C until extraction. resulting in a more extended and optimum clinical outcomes. The ethics committee of MUMS approved the study and all The polyvalent vaccine simultaneously introduces the antigens patients gave their informed consent. to the immune system and is expected to elicit a specific immune RNA extraction, cDNA synthesis and quantitative RT-PCR. response against these antigens, which selectively targets the RNA was extracted from the normal and tumoral tissues of cells containing these antigens i.e., tumoral cells. To validate the the resected esophagus with the RNeasy Mini kit (Qiagene, applicability of these antigens as suitable targets, future experi- Hilden, Germany). Reverse transcription of total RNA was perments must be performed to confirm that the increased CTA formed using oligo dT in first-strand synthesis kit (Fermentas,



Lithuania) according to the manufacturer’s protocols. cDNA was amplified on the Stratagene Mx-3000P real-time thermocycler (Stratagene, La Jolla, CA) with primers and probes presented in Table 1. Real-time PCR was carried out with the Maxima probe (Fermentas), containing ROX as a reference dye. The following thermal cycling program was applied: 10 min at 95°C and 40 cycles of 15 s at 95°C, 30 s at 60°C and 45 s at 72°C. Data were normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression via the comparative threshold cycle method. The PCR efficiency for GAPDH, LAGE1, MAGE-A4 and NY-ESO1 was measured using standard curves generated by the serial dilution of cDNA and it ranged from 95–100. All experiments were performed in triplicates. More than a 2-fold fluorescence intensity of mRNA expression in the tumor tissue in comparison with corresponding normal tissues was considered as overexpression for each gene. Less than a minus 2-fold intensity indicated underexpression. The range in between was interpreted as normal. Statistical analysis. Statistical analysis was performed using the SPSS 16.0 statistical package (SPSS, Chicago, IL). The correlation between the expression levels of the genes, and References 1. Gholamin M, Moaven O, Memar B, Farshchian M, Naseh H, Malekzadeh R, et al. Overexpression and interactions of interleukin-10, transforming growth factorbeta and vascular endothelial growth factor in esophageal squamous cell carcinoma. World J Surg 2009; 33:1439-45. 2. Mousavi SM, Gouya MM, Ramazani R, Davanlou M, Hajsadeghi N, Seddighi Z. Cancer incidence and mortality in Iran. Ann Oncol 2009; 20:556-63. 3. Vergati M, Intrivici C, Huen NY, Schlom J, Tsang KY. Strategies for cancer vaccine development. J Biomed Biotechnol 2010; In press. 4. Kalejs M, Erenpreisa J. Cancer/testis antigens and gametogenesis: a review and “brain-storming” session. Cancer Cell Int 2005; 5:4. 5. Cronwright G, Le Blanc K, Gotherstrom C, Darcy P, Ehnman M, Brodin B. Cancer/testis antigen expression in human mesenchymal stem cells: downregulation of SSX impairs cell migration and matrix metalloproteinase 2 expression. Cancer Res 2005; 65:2207-15. 6. Costa FF, Le Blanc K, Brodin B. Concise review: cancer/testis antigens, stem cells and cancer. Stem Cells 2007; 25:707-11. 7. Bandic D, Juretic A, Sarcevic B, Separovic V, Kujundzic-Tiljak M, Hudolin T, et al. Expression and possible prognostic role of MAGE-A4, NY-ESO-1 and HER-2 antigens in women with relapsing invasive ductal breast cancer: retrospective immunohistochemical study. Croat Med J 2006; 47:32-41. 8. Dhodapkar MV, Osman K, Teruya-Feldstein J, Filippa D, Hedvat CV, Iversen K, et al. Expression of cancer/testis (CT) antigens MAGE-A1, MAGE-A3, MAGE-A4, CT-7 and NY-ESO-1 in malignant gammopathies is heterogeneous and correlates with site, stage and risk status of disease. Cancer Immun 2003; 3:9. 9. Hudolin T, Kastelan Z, Derezic D, Basic-Jukic N, Cesare Spagnoli G, Juretic A, et al. Expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1 cancer-testis antigens in fetal testis. Acta Dermatovenerol Croat 2009; 17:103-7. 10. Yoshida N, Abe H, Ohkuri T, Wakita D, Sato M, Noguchi D, et al. Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens and T cell infiltration in non-small cell lung carcinoma and their prognostic significance. Int J Oncol 2006; 28:1089-98.

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correlation between the gene expression level and continuous variables (age, tumor size, number of involved lymph nodes) were assessed by Pearson’s correlation or the nonparametric Spearman rank correlation, depending on the results. The association between gene expression status and categorical variables was also analyzed by the χ2 test or Fisher’s exact test. Moreover, comparison of expression level of each gene between different categorical data was performed with t-test (lymph node metastasis, tumor depth of invasion, tumor location and tumor stage) or ANOVA (tumor grade of differentiation). A p value of < 0.05 was considered to be statistically significant. Acknowledgments

The authors gratefully acknowledge Azadeh A‘rabi and Martha Ghahraman in the Division of Human Genetics for their technical assistance, and also the colleagues from the departments of surgery and pathology at Omid Hospital for their help in gathering and preparing the tissue specimens. This study was supported by a grant from Mashhad University of Medical Sciences (#88098). The results described in this paper were part of a Ph.D. dissertation.

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33. Sarcevic B, Spagnoli GC, Terracciano L, Schultz-Thater E, Heberer M, Gamulin M, et al. Expression of cancer/ testis tumor associated antigens in cervical squamous cell carcinoma. Oncology 2003; 64:443-9. 34. Jungbluth AA, Stockert E, Chen YT, Kolb D, Iversen K, Coplan K, et al. Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE1 antigen in formalin-fixed paraffin embedded lung tumours. Br J Cancer 2000; 83:493-7. 35. Putz E, Witter K, Offner S, Stosiek P, Zippelius A, Johnson J, et al. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases. Cancer Res 1999; 59:241-8. 36. Yamanaka K, Miyake H, Hara I, Gohji K, Arakawa S, Kamidono S. Expression of MAGE genes in renal cell carcinoma. Int J Mol Med 1998; 2:57-60. 37. Toh Y, Yamana H, Shichijo S, Fujita H, Tou U, Sakaguchi M, et al. Expression of MAGE-1 gene by esophageal carcinomas. Jpn J Cancer Res 1995; 86:714-7. 38. Patard JJ, Brasseur F, Gil-Diez S, Radvanyi F, Marchand M, Francois P, et al. Expression of MAGE genes in transitional-cell carcinomas of the urinary bladder. Int J Cancer 1995; 64:60-4. 39. Brasseur F, Rimoldi D, Lienard D, Lethe B, Carrel S, Arienti F, et al. Expression of MAGE genes in primary and metastatic cutaneous melanoma. Int J Cancer 1995; 63:375-80. 40. Cuffel C, Rivals JP, Zaugg Y, Salvi S, Seelentag W, Speiser DE, et al. Pattern and clinical significance of cancer-testis gene expression in head and neck squamous cell carcinoma. Int J Cancer; 128:2625-34.


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