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Keywords:

  • head and neck squamous cell carcinoma (HNSCC);
  • cancer-testis antigens;
  • tumor-associated antigens;
  • immunotherapy;
  • gene expression

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cancer-testis (CT) antigens comprise families of tumor-associated antigens that are immunogenic in patients with various cancers. Their restricted expression makes them attractive targets for immunotherapy. The aim of this study was to determine the expression of several CT genes and evaluate their prognostic value in head and neck squamous cell carcinoma (HNSCC). The pattern and level of expression of 12 CT genes (MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, MAGE-C2, NY-ESO-1, LAGE-1, SSX-2, SSX-4, BAGE, GAGE-1/2, GAGE-3/4) and the tumor-associated antigen encoding genes PRAME, HERV-K-MEL, and NA-17A were evaluated by RT-PCR in a panel of 57 primary HNSCC. Over 80% of the tumors expressed at least 1 CT gene. Coexpression of three or more genes was detected in 59% of the patients. MAGE-A4 (60%), MAGE-A3 (51%), PRAME (49%) and HERV-K-MEL (42%) were the most frequently expressed genes. Overall, the pattern of expression of CT genes indicated a coordinate regulation; however there was no correlation between expression of MAGE-A3/A4 and BORIS, a gene whose product has been implicated in CT gene activation. The presence of MAGE-A and NY-ESO-1 proteins was verified by immunohistochemistry. Analysis of the correlation between mRNA expression of CT genes with clinico-pathological characteristics and clinical outcome revealed that patients with tumors positive for MAGE-A4 or multiple CT gene expression had a poorer overall survival. Furthermore, MAGE-A4 mRNA positivity was prognostic of poor outcome independent of clinical parameters. These findings indicate that expression of CT genes is associated with a more malignant phenotype and suggest their usefulness as prognostic markers in HNSCC.

The incidence of squamous cell carcinoma of the head and neck (HNSCC) is greater than 40,000 new cases per year in the United States, and ∼500,000 cases annually world wide. Despite significant advances in early detection and treatment of this cancer, the survival rate for patients with HNSCC has not changed dramatically over the last decades. The majority of patients present with advanced disease and prognosis is usually poor. Loco-regional recurrences are the most frequent cause of treatment failure even after large resections and adjuvant therapy, both of which carry severe long term morbidity for the patient. Current staging criteria, including TNM staging, grading of differentiation, size and site of the neoplasm, are not sufficient for predicting outcome. It is therefore mandatory to identify new prognostic markers to select high-risk patients who may benefit from more aggressive therapy and search for novel therapeutic approaches to reduce the need for mutilating surgery and morbid adjuvant therapy.

The role of cell mediated immunity against cancer has been established for two decades. Numerous antigens coding for immunogenic sequences have been identified in different tumor types (reviewed in1; a peptide database of T-cell-defined tumor antigens can be found at http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm), leading to the development of new strategies for targeted immunotherapy of cancers. Among various classes of tumor associated antigens identified, cancer-testis (CT) antigens are particularly interesting targets for specific immunotherapy. CT genes comprise a large number of genes or gene families, such as MAGE, BAGE, GAGE, SSX, and NY-ESO-1, many of which are mapped to chromosome X (X-CT) (reviewed by Simpson et al.2). They are expressed by human tumors of different histological types but not by normal somatic tissues, with the exception of male germ cells and placenta. Epigenetic mechanisms are at the base of their restricted expression pattern.3, 4 Among the X-linked CT antigens, the MAGE-A family, encoded by 12 highly homologous genes, and NY-ESO-1 family, consisting of NY-ESO-1 and LAGE-1, are the best studied antigens and have been shown to generate both spontaneous and vaccination-induced T-cell mediated responses. In addition to X-CT gene products, tumor associated proteins like PRAME (preferentially expressed antigen on melanoma), HERV-K-MEL, a product related to the env-gene of the endogenous human retrovirus K (HERV-K), and NA-17A, the product of an alternatively spliced N-acetylglucosaminyltransferase V mRNA, also contain epitopes recognized by cytolytic T cells on tumor cells.5–8

A small number of studies have reported a relatively frequent expression of selected CT genes in HNSCC.9–13 However, the small patient number or short follow up time did not allow evaluation of their impact of their expression on survival. In this study, we investigated the correlation of expression of 15 tumor associated antigen-encoding genes, including 12 CT genes, in a cohort of HNSCC patients with known follow up. The genes were chosen based on the capability of their products to generate epitopes recognized by CD8 and/or CD4 T cells. In addition, we sought to determine the impact of individual and combined CT gene expression on clinical outcome. Our findings show frequent coexpression of CT genes in HNSCC of different primary site. Expression of MAGE-A4 and coexpression of several CT genes was associated with poor overall survival. In addition, Cox regression analysis indicated that MAGE-A4 was an independent marker of worse outcome in HNSCC.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients

Fifty-seven tumor samples from 52 patients treated for primary HNSCC were prospectively collected at the Department of Otolaryngology and Head and Neck surgery of Lausanne University Hospital (CHUV), Switzerland. Tumor specimens were collected during initial pretherapeutic endoscopy and immediately snap-frozen. For 11 patients, samples of nearby normal mucosa were collected at the same time. Presence of tumor cells was confirmed in each biopsy sample by a standard haematoxylin-eosin and keratin staining on formalin fixed/paraffin embedded material. Tumor site, histological grade and clinical stage (according to the 2002 IUCC staging system) were prospectively recorded. Following diagnosis, patients were treated by a combination of surgery and chemoradiotherapy, when required, according to standard international treatment guidelines. This study was conducted after approval by the Research Ethics Committee of Lausanne University and conformed to the 1975 Declaration of Helsinki. All patients provided informed consent.

RNA extraction and RT/PCR

Total RNA was isolated from frozen tissue samples using a Nucleospin RNA II kit (Macherey-Nagel) and a Fast-Prep device (Bio 101 Savant; Savant Instruments). RNA (2 μg) was primed with an oligo (dT)18 oligonucleotide and reverse-transcribed with MMLV-RT (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Aliquots of cDNA corresponding to 100 ng input RNA were used for different PCR reactions using a Qiagen Hotstar Taq polymerase Master Kit, except for LAGE-1, NY-ESO-1 and actin PCR, performed with Qiagen Taq polymerase (Qiagen GmbH, Hilden, Germany). cDNA quality was tested by amplification of β-actin in a 21-cycle PCR reaction. The primers, annealing temperature and number of cycles performed are described in Supporting Information Table S1. The number of cycles chosen for BORIS amplification allowed the detection of a 1:500 dilution of a testis sample (0.2%), but not background transcription (not shown).14 For amplification, after an initial denaturation for 15 min (Hotstar Taq polymerase) or 5 min (for other PCR) at 94°C, PCR cycles were performed as follows: denaturation at 94°C for 1 min, 30 sec at the indicated annealing temperature, 1 min at 72°C. A final elongation step was performed at 72°C for 10 minutes. Aliquots of each reaction were size-fractionated on a 1.5% agarose gel and visualized by ethidium bromide staining. Sequence identity of representative PCR products was confirmed by automated sequencing (Microsynth, Switzerland). RNA from SK-Mel-37 cells (a gift from Y. T. Chen, New York), a melanoma cell line expressing high levels of a broad range of CT genes, and NA8-MEL (a gift from F. Jotereau, Nantes, France) was used as positive and negative control, respectively. Quantitative assessment was performed as previously described, using 1:10 dilutions of SK-Mel-37 RNA as reference.15 A threshold level of CT gene expression by tumor cells appears necessary for antigen presentation and recognition by T cells.15, 16 Taking this in consideration, the threshold for sample positivity was set at 1% the expression level of the reference cell line. A tumor “CT score” was calculated by integrating scores of individual CT genes (from 0 to 4+) obtained from semiquantitative analyses. Median X-CT antigen score was 6 (mean 8.6). Tumors with a “CT score” ≥7 were defined as “high CT Score”.

Immunohistochemistry (IHC)

Four-micrometer thick serial sections of formalin-fixed, paraffin-embedded tissue samples were obtained. Expression of proteins of the MAGE-A family was assessed using the anti–pan-MAGE-A antibodies 57B (a gift from G. Spagnoli, Basel, Switzerland) and 6C1.17 Clone 57B, originally raised against MAGE-A3, cross-reacts with several of the homologous MAGE-A proteins, and has been reported to primarily detect MAGE-A4 in melanoma.17–19 NY-ESO-1 and LAGE-1 were detected with the monoclonal antibody D8.38 (a gift from G. Spagnoli).18, 20 Antigen retrieval was performed with microwave treatment in 0.1 M sodium citrate, pH 6.0. Detection was performed with the DAKO EnVision™+ system and DAB as chromogen (DAKO). Nonimmune mouse IgG was used as negative control and sections of testis were used as a positive control. Slides were then analyzed by one of the authors (W.S.) and by a second independent pathologist as control.

Statistical analyses

Statistical analysis was performed with STATA 10 software. The chi-square test and Fisher's exact test were used to evaluate the associations between CT antigen expression and clinico-pathological features, as appropriate. The Kaplan-Meier method was used to estimate overall survival of patients, and differences between groups were compared using the log-rank test. Multivariate analyses were performed using the Cox proportional hazard model to determine the independent contribution of each variable. Covariates with p < 0.08 by univariate analysis were entered in the multivariate analysis. Probability values ≤0.05 were regarded as significant. In case of multiple tumors, a patient was considered CT antigen positive if at least one of the tumors analyzed tested positive.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Over a period of 19 months, 57 primary HNSCC and 11 samples of normal mucosa were collected from 52 untreated patients (39 male and 13 female). Median age was 61 years (range 42–84 years). Table 1 summarizes the clinical and histological characteristics of patients and tumors. Most common localizations of the primary tumors were oral cavity and oropharynx, followed by hypopharynx and larynx. Half of the tumors were moderately differentiated. According to TNM classification, 11 patients had early stage (i.e. I and II) and 41 advanced (i.e. III and IV) cancers.

Table 1. Clinico-pathological characteristics of patients and tumors studied
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Semiquantitative RT/PCR was performed to analyze tumor expression of the cancer testis genes MAGE-A1/3/4/10, MAGE-C2, LAGE-1 and NY-ESO-1, SSX-2 and 4, BAGE, GAGE-1/2 and 3/4. In addition, we studied the expression of the genes coding for the tumor-associated antigens HERV-K-MEL, PRAME, and NA17. Tumor samples were considered as positive when they expressed a given gene at the level of at least 1% that of a reference cell line (see “Material and Methods” section). Representative PCR analyses are shown in Supporting Information Figure S1. Frequency of expression of various genes in tumors is summarized in Figure 1a. MAGE-A4, MAGE-A3, PRAME, and HERV-K-MEL were the most frequently expressed genes and were detected in over 40% of the samples. MAGE-A3 (51%) and MAGE-A4 (60%) were coexpressed in 35% of tumors, and 75% of tumors expressed either gene. MAGE-A1, MAGE-A10, LAGE-1, SSX-4, and GAGE were expressed in 16–30% of tumors, while other genes had lower expression frequency. BAGE and NA-17A were detected only in 1 of the 57 tumors analyzed. Eighty-nine percent of the tumors showed expression of at least one gene in our panel and 81% expressed at least one X-CT gene. Tumors expressed up to 11 of the 12 X-CT genes tested. Frequency of coexpression of CT genes on a patient basis is shown in Figure 1b. Patients had tumors expressing an average of 2.9 X-CT and 3.8 tumor associated antigen-encoding genes. None of the genes tested was expressed in 11 normal mucosa biopsies collected as controls (not shown).

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Figure 1. Expression of CT and other genes in HNSCC. (a) Frequency of expression of the indicated genes, determined by RT-PCR, calculated on a tumor basis (n = 57). (b) frequency of coexpression of the indicated numbers of genes (in any combination) in patients. Light gray, X-CT genes only; dark gray, all genes.

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CT gene expression is independent of BORIS

BORIS (Brother of the Regulator of Imprinted Sites), a testis specific paralog of the DNA binding protein CTCF, has been proposed as a mediator of the induction/derepression of other CT genes in lung cancer cells and dermal fibroblasts.21, 22 We therefore analyzed the expression of BORIS, itself considered a non-X linked CT gene because of its expression pattern, in the HNSCC tumors. BORIS expression was detected in 17% of tumors. To visualize the correlation between expression of various CT genes and BORIS in individual tumors, we grouped the latter into two groups according to BORIS expression (Fig. 2). Within these groups, tumors were further ordered according to the number of CT genes expressed. A correlation was observed between expression of the more frequently expressed MAGE-A3 and A4 and that of various X-linked CT genes (p < 0.05), but not BORIS (p > 0.2). Expression of BORIS however significantly correlated with expression of multiple CT genes (≥4) and MAGE-A10 (p < 0.05). Figure 2 also shows that tumors expressing multiple CT genes generally displayed also quantitatively high expression levels. PRAME, but not HERV-K-MEL, was more frequently expressed in tumors expressing multiple CT antigens (p < 0.05 and p = 0.81, respectively).

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Figure 2. Correlation between expression of BORIS and other CT genes. Results from semiquantitative RT-PCR are shown color-coded as follows: green, negative; yellow to red, low to high levels, respectively, determined as indicated in “Material and Methods” section relative to the reference melanoma cell line SK-Mel-37. Tumor samples are clustered into two groups according to BORIS expression (negative, left; positive, right).

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Expression of MAGE-A and NY-ESO-1 proteins

To verify that MAGE-A genes are also expressed at the protein level, RT-PCR positive tumors were analyzed by IHC using two anti-panMAGE antibodies (clones 57B and 6C1). Eighty-eight percent (38/43) of MAGE-A3 and/or A4 RT-PCR positive tumors showed a positive IHC staining with antibody 57B. Although heterogeneous, ∼¾ of the tumors showed positive staining in over 80% of the cells (not shown). At the cellular level, the staining was both cytoplasmic and nuclear, but a nuclear localization was slightly predominant (not shown). Twenty-one of the 43 tumors tested were also positive with clone 6C1. Using an antibody that recognizes both NY-ESO-1 and LAGE-1, NY-ESO-1 protein family was detected in 7 of the 12 NY-ESO-1 and/or LAGE-1 RT-PCR-positive samples. This staining was predominantly cytoplasmic. In five cases the staining was extensive, with over 75% positive tumor cells. Examples of immunostainings are shown in Figure 3.

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Figure 3. Immunohistochemical detection of MAGE-A and NY-ESO-1 family proteins in HNSCC. (a) representative staining with anti-MAGE-A antibody 57B. The extensive, heterogeneous staining is both cytoplasmic and nuclear. (b), heterogeneous staining, mainly nuclear, with anti-MAGE-A antibody 6C1. Tumors shown in A and B were MAGE-A1/3/4/10 positive by RT-PCR. (c) and (d), staining with anti-NY-ESO-1/LAGE-1 antibody D8.38. Tumor in C shows diffuse staining; D, area with focal staining in a mostly negative tumor. Both tumors were LAGE-1 positive by RT-PCR.

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Correlation between expression of CT genes, PRAME, and HERV-K-MEL and clinico-pathological parameters

There was no statistically significant correlation between CT gene expression (tested as individual genes, gene combinations, or number of coexpressed genes) and gender, clinical stage, tumor localization, differentiation grade, or tumor recurrence (not shown). One exception was the NY-ESO-1 family, which was expressed in moderately and poorly, but not well differentiated tumors (p = 0.0345) and more frequently in hypopharynx/larynx compared to oral cavity/oropharynx (p = 0.014). In addition, MAGE-A4 tended to be preferentially expressed in advanced stage tumors (p = 0.0782). No correlation was found between PRAME or HERV-K-MEL mRNA positivity and any clinico-pathological parameter.

Tumor CT gene expression and survival

The patients in this study had a median follow-up of 27.5 months (range 1–53, mean 26.4), and overall survival at 4 years was 52%. Univariate analysis showed that overall survival significantly correlated with clinical stage, nodal status, and tumor stage, but not tumor localization, differentiation grade, or sex of the patient (Supporting Information Table S2). We investigated the correlation of the mRNA expression of various genes, individually or as combinations, with overall survival. Patients with MAGE-A4 mRNA positive tumors had a significantly poorer outcome compared to those with MAGE-A4 mRNA negative tumors (p = 0.0493, Fig. 4). No significant correlation was observed between survival and positivity for expression of other individual CT genes (Table 2 and Fig. 4b, showing survival curve relative to MAGE-A3 as example). Interestingly, curves of patients with tumors mRNA-positive for NY-ESO-1 family genes leaned towards a poorer outcome (p = 0.161). Expression of HERV-K-MEL and PRAME (42 and 49% positive patients, respectively) had no impact on survival. We next asked whether expression of multiple X-CT genes (in any combination) or quantitative high levels of expression (assessed by calculating a “CT score”, as described in “Material and Methods” section) had an impact on outcome. Coexpression of four or more X-CT genes (n = 17, 33% patients) did indeed correlate with a significantly poorer survival (p = 0.045, Fig. 4c). In addition, a high CT score (n = 24, 46%) was associated with 18% difference in overall survival at 40 months (p = 0.117). Interestingly, patients with tumors negative for all tested X-CT genes (n = 8) had a remarkably good survival, though the log-rank test only indicated a trend (p = 0.100, Fig. 4d). We next performed a multivariate Cox regression analysis to assess whether CT gene expression was prognostic of poor survival independent of clinico-pathological parameters, including clinical and TNM stage. This analysis showed that RT/PCR positivity for MAGE-A4 was an independent prognostic indicators in HNSCC (hazard ratio, 2.949; 95% confidence interval 1.085–8.020; p = 0.034). The association of coexpression of four or more X-CT genes with a higher risk of death did not persist in the multivariate analysis (hazard ratio, 2.057; 95% confidence interval 0.919–4.602; p = 0.079).

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Figure 4. Correlation between X-CT gene expression and overall survival. Kaplan-Meier survival estimates of patients were performed according to RNA expression of (a), MAGE-A4; (b), MAGE-A3; (c), multiple X-CT genes (≥4). (d), survival curves for patients with tumors positive or negative for 1 or more X-CT genes.

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Table 2. Correlation of expression of CT genes with overall survival
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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The identification of tumor specific antigens capable of inducing a specific immune response has raised interest for novel antitumor therapies in various tumor types. CT antigens are amongst the most promising targets for immunotherapy. Our finding that CT gene expression confers a higher risk of poor outcome in HNSCC further strengthens the choice of their products as therapeutic targets in this type of cancer.

We found expression of one or more of the 12 X-CT genes tested in 90% and three or more in over 40% of HNSCC tumor samples. Overall, ∼60% of the patients had tumors expressing at least three of the whole panel of genes investigated in this study. The X-linked CT genes MAGE-A4, MAGE-A3, together with PRAME and HERV-K-MEL, were the most frequently expressed. Of the genes studied, only four were detected at a frequency below 10%. NY-ESO-1, encoding one of the most immunogenic CT antigens, was among the latter. However, together with its homolog LAGE-1, which codes for identical HLA-A2 restricted epitopes, it was expressed in approximately a quarter of tumors. The frequencies of expression of individual genes in HNSCC were similar to those reported in previous studies.10–12 Expression of MAGE-A and NY-ESO-1 protein families was confirmed by IHC staining in the majority of RT-PCR positive tumors. Heterogeneous and scattered staining pattern for CT proteins has been frequently observed in tumors and could have implications for targeted immunotherapy. In this study, most tumors showed an extensive staining for both MAGE-A and NY-ESO-1 family proteins, indicating that CTA positive cells are not rare. Kienstra et al. found that less than half of the MAGE-A1 and A3 RT-PCR positive tumors were also positive by immunohistochemistry, however, the antibodies used were not specified.12 Concordant with our results, a recent immunohistochemical study has reported the expression of MAGE-A (detected by clone 57B) and NY-ESO-1 family proteins in 70% and 30% of pharyngeal tumors, respectively.23 These results are consistent with the relatively high CTA expression levels detected by RT-PCR, similar to those observed in melanoma. Altogether our study, to our knowledge the larger, in terms of number of genes coding for immunogenic products and patients, confirms that a large proportion of patients with HNSCC of different site could receive specific immunotherapy targeting multiple antigens.

Three quarters of the tumors expressed MAGE-A3 and/or A4, and expression of other X-linked CT genes was significantly correlated with these genes. A similarly coordinated expression of CT genes has been reported for nonsmall cell lung cancer.24 Interestingly, coexpression of multiple CT genes also associated with high mRNA levels (Fig. 2), suggesting that high transcriptional activity is associated with the extent of DNA demethylation. The exact mechanism underlying this observation is not clear at present. Expression of CT genes, especially those encoded in the X-chromosome, is strictly confined to germline and placenta. Methylation of CpG islands in CT gene promoters is the primary silencing mechanism in healthy somatic tissues. Activation of CT gene expression in tumors is thought to result from demethylation of these sequences.3, 4 BORIS, the product of a CT gene located on chromosome 20, has been recently suggested by Vatolin et al. as an essential mediator of CT gene derepression, particularly MAGE-A1.22 In addition, BORIS has been implicated in NY-ESO-1 expression in lung cancer cells.21 In HNSSC, the frequency of BORIS expression was only 17% and contrasted with the frequent expression of X-linked CT genes. As a comparison, a parallel BORIS analysis applied to metastatic melanoma samples yielded a frequency of ∼50% (Rimoldi D., unpublished observation). Expression of CT genes, particularly MAGE-A3 and 4, but also MAGE-A1 and NY-ESO-1, was observed in the absence of BORIS. Conversely, BORIS positive tumors did not necessarily express high levels of other CT genes. Thus, BORIS expression does not seem to be sufficient or necessary for the expression of other CT genes in HNSCC, although we cannot rule out that a transient expression of BORIS may precede their activation. Similar to our results, a lack of association between BORIS expression and MAGE-A1 activation has been reported in cutaneous melanoma,14 and thus the “gate keeper” for the expression of CT genes in these cancers still remains to be identified.

Overall, we did not find a correlation between expression of CT genes, either individually or in combination, and clinical parameters (including TNM staging) or characteristics of the primary tumor. This is in agreement with results of most previous studies on HNSCC.9, 12, 23, 25 A positive correlation between expression of two or more of a panel of nine genes, including MAGE-A and NY-ESO-1 families, and higher tumor stage was reported by Figueiredo et al. in a study on 33 HNSCC patients.11 There was no correlation in our cohort between CT gene expression and tumor or clinical stage, except for a marginal association of MAGE-A4 with advanced TNM stage. It should be noted however that in our, as well as previous HNSCC studies, late stage patients were a majority, thus firm conclusions on correlations with stage await results from larger studies. Eura et al (n = 83) found that the expression levels of individual MAGE-A genes varied with tumor localization and degree of differentiation, though no common pattern could be drawn.10 The only significant correlation of CT gene expression and tumor grade in our cohort was the lack of NY-ESO-1 and LAGE-1 in well differentiated tumors. This is at odds with a recent report that shows similar expression of these proteins in grade 1 and 2 pharyngeal tumors, but lower frequency in grade 3 ones.23 This discrepancy may be due to the different localizations of tumors studied (grade 1 tumors in our study were mostly from the oral cavity). A lower frequency of NY-ESO-1 expression in low histological grade tumors has also been observed in urinary tract cancer,26 while the opposite has been reported for esophageal cancer.27 Altogether, the correlation between differentiation grade and expression of NY-ESO-1 family genes remains unclear.

A major finding of this study is the correlation between CT gene expression (assessed by RT/PCR) and clinical outcome. Both MAGE-A4 expression and coexpression of multiple X-linked CT genes (at least 4 of the 12 analyzed) significantly correlated with poor survival. More importantly, the former emerged as a potential new prognostic indicator. These findings further strengthen the choice of CT antigens as immunotherapy targets in this type of cancer. A previous report on a smaller group of patients found no correlation between mRNA expression of CT genes, including the MAGE-A family, and tumor recurrence or metastasis, though clinical parameters and follow-up were not specified.11 Our study is the first one to evaluate the effect of individual CT genes on survival of HNSCC patients. Association of expression of MAGE-A proteins with a poorer disease-free survival in patients with pharyngeal squamous cell carcinoma, has been shown in a recent immunohistochemical study, although the difference was not statistically significant.23

While coexpression of multiple X-linked CT genes correlated with poor overall survival, it appears that the different genes may not equally contribute to outcome. This was evident for the MAGE-A family. MAGE-A4 and A3 were expressed at similar frequencies, yet only expression of the former had an impact on the patients' survival (Figs. 4a and 4b). This is similar to results by Shigematsu et al.28 showing that expression of MAGE-A4, but not MAGE-A3 or NY-ESO-1, as determined by RT/PCR, was predictive of poor survival in nonsmall cell lung cancer patients.29, 30 Gure et al. identified MAGE-A3 and NY-ESO-1, but not MAGE-A4, as independent markers of poor prognosis for adenocarcinoma of the lung.24 These discrepancies may be related to the different patient populations. Expression of MAGE-A4 protein, as detected by antibody 57B, has been reported to be an independent marker of poor survival for serous ovarian cancer patients31 and to associate with progression of noninvasive bladder cancer to muscle invasive tumors.32 Caution however must be applied in interpreting positive 57B staining as MAGE-A4 positivity, as the antibody can also recognize other MAGE-A proteins.17, 18 Expression of other CT genes has also been generally associated with poor prognosis,24, 33, 34 with only few studies revealing a positive effect.27, 35 None of the CT genes tested in this study was associated with better survival. Noteworthy, the small group of patients whose tumors tested negative for RNA of all 12 X-linked CT genes tested appeared to have a particularly good outcome, though this did not reach statistical significance. The severe prognosis of patients with tumors expressing MAGE-A4 or multiple CT genes suggests that these patients may require more intense follow-up and aggressive therapy.

At present, how the expression of MAGE-A4 and other CT genes translates into poor clinical outcome can only be a subject of speculation. Products of CT genes may confer a highly malignant phenotype to the tumor or resistance to chemo/radiotherapy. Alternatively, expression of these genes may be coinduced with that of others in a subset of tumors with a more aggressive behavior. Although the function of CT proteins remains poorly understood, different MAGE-A proteins have been reported to associate with p53 containing complexes and inhibit DNA damage-induced apoptosis, lending support to the former hypothesis.36–38 However, other reports indicated that MAGE-A4 may actually promote apoptosis.39, 40 Further studies are clearly needed to establish the direct contribution, if any, of MAGE-A4 and other CT gene products to a more malignant phenotype in HNSCC.

In addition to X-linked CT genes, PRAME and HERV-K-MEL, both coding for in vivo generated CTL epitopes,5, 7, 8, 41, 42 are interesting candidates for specific immunotherapy of HNSCC. Because of its restricted expression and its epigenetic regulation, PRAME, a gene located on chromosome 22 (reviewed in43, 44), is sometimes considered as a non-X-linked CT gene (e.g. in CTdatabase, at http://www.cta.lncc.br/). In this regard, it is interesting that PRAME expression correlated with that of multiple X-linked CT genes in our tumor series. The frequency of expression of PRAME in this study (49%) confirms frequencies reported in smaller studies (39 and 42%).8, 11 While PRAME expression has been reported as a predictor of both poorer and better patient outcome (e.g. in breast cancer and promyelocytic leukemia, respectively),34, 45, 46 it had no influence on survival in our cohort of HNSCC patients. We are the first to report the extent of HERV-K-MEL expression in a large series of HNSCC tumors. HERV-K-MEL is a spliced env sequence from a HERV-K pseudogene expressed in over 80% of benign and malignant melanocytic lesion.5 Normal tissue expression has been reported to be confined to testis and, at a low level, normal skin. Spliced env and rec mRNAs from HERV-K genes have been detected in other cancers.47 While promoter demethylation has been implicated in some tumors in the activation of related HERV-K sequences,48 the mechanism of activation of HERV-K-MEL is not known. In HNSCC tumors, expression of HERV-K-MEL was independent of CT gene expression, suggesting that different mechanisms are involved in the activation of CT genes and endogenous viral sequences. Similar conclusions have been drawn in melanoma. As HERV-K products can elicit immune responses, they may have biological implications in HNSCC.

In conclusion, this study showed a coordinated activation of different CT genes in HNSCC and established an association between expression of MAGE-A4 and multiple X-CT antigens with poor survival. The value of MAGE-A4 as an independent prognostic marker should be confirmed in a larger prospective study.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. G. Spagnoli for the gift of antibodies and Dr. M. Faouzi for help with statistical analyses. D.R. was supported by a grant from the Conrad N. Hilton Foundation to the Ludwig Institute for Cancer Research, Ltd. P.R. and L.B. are supported by grants from the Swiss National Science Foundation and the Swiss Cancer League, respectively.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional supporting information may be found in the online version of this article.

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IJC_25607_sm_SuppFig1.pdf150KSupporting Figure 1
IJC_25607_sm_SuppTable-1.doc45KSupporting Table 1
IJC_25607_sm_SuppTable-2.doc46KSupporting Table 2

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