Companion Diagnostics and Cancer Biomarkers Molecular

Cancer
Therapeutics
A Comprehensive Expression Analysis of Cancer
Testis Antigens in Head and Neck Squamous Cell
Carcinoma Revels MAGEA3/6 as a Marker for
Recurrence
Fernando Tadeu Zamune
r1, Bruno Takao Real Karia1, Cleyton Zanardo de Oliveira2,
Carlos Roberto dos Santos3, Andre
Lopes Carvalho3, and Andre
Luiz Vettore1,4

Abstract
Despite significant advances in the treatment of head and 85% of the tumors expressed at least one of these five CTAs. The
neck squamous cell carcinoma (HNSCC), the survival rate has mRNA positivity of CXORF48, MAGEB6, and CRISP2 presented

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not changed in the last decades. Therefore, the development of significant associations with recognized clinical features for
novel therapeutic strategies is pursued. Cancer–testis antigens poor outcome. Furthermore, MAGEA3/6 positivity was associ-
(CTA) are strong immunogenic proteins with a tumor-restricted ated with significantly better disease-free survival (DFS, P ¼
expression pattern, and are considered ideal targets for tumor- 0.014), and the expression of this antigen was shown to be an
specific immunotherapeutic approaches. In this study, using independent prognostic factor for tumor recurrence. In conclu-
an in silico approach, we selected, among 139 previously sion, one of five selected CTAs is expressed in at least 85% of the
described CTA, candidates to be evaluated in 89 HNSCC and HNSCCs, suggesting a possible usage as target for immuno-
20 normal mucosa samples. SPANX-CD (71.9%), MAGEB2 therapeutic approaches, and the mRNA-positivity for MAGEA3/
(44.9%), MAGEA1 (44.9%), MAGEB6 (32.6%), and CXORF48 6 is shown to be an independent marker for DFS. Mol Cancer Ther;
(27.0%) were found frequently expressed in HNSCC, and over 14(3); 828–34.
2015 AACR.

Introduction gens (CTA) represent promising targets due to their high immu-
nogenicity and specific expression pattern (3–5). To date, more
Head and neck squamous cell carcinoma (HNSCC) affects
than 200 genes encoding CTAs have been identified and cataloged
600,000 new patients worldwide each year (1). Despite significant
in a dedicated database (CTDatabase— http://www.cta.lncc.br/
advances in therapy, the survival rate for these patients has not
index.php; ref. 6). As spontaneous antitumor immune responses
improved markedly over the past 30 years (2). Late diagnosis and
can be elicited by CTAs, they are promising candidates for cancer
frequent locoregional recurrences are the most frequent causes of
immunotherapy and, in recent years, gained importance in the
treatment failure. Therefore, the development of new therapeutic
development of cancer vaccine–based clinical trials (7). The
approaches and their integration into current forms of treatment,
expression of these antigens has been detected in various types
which include surgery, radiotherapy, and chemotherapy, are
of malignant tumors of different histological types, but rarely in
pursued to improve this prognosis.
normal somatic tissues, with the exception of immunoprivileged
The recent discovery of tumor antigens has opened new doors
gametogenic tissues. CTAs are described as frequently expressed in
for specific tumor-targeted treatments using passive or active
melanomas, multiple myeloma, glioblastomas, and carcinomas
immunotherapeutic strategies. In particular, cancer–testis anti-
of the bladder, lung, ovarian, and liver (8–14). A moderate
expression could be detected in breast and prostate tumors
(5, 15), and they are rarely expressed in renal cell carcinoma,
1
Laboratory for Cancer Molecular Biology, Department of Biological colon cancer, lymphomas, and leukemias (9, 15–17). Few studies
~ o Paulo, Diadema, Brazil. 2Biosta-
Sciences, Federal University of Sa
tistics Department, Pio XII Foundation, Barretos Cancer Hospital, with small sample sizes have detected CTA expression in HNSCCs
Barretos, Brazil. 3Department of Head and Neck Surgery, Pio XII (18–26).
Foundation, Barretos Cancer Hospital, Barretos, Brazil. 4Program in In light of these considerations, the current study provides a
Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School,
Singapore. comprehensive evaluation of CTA gene expression in a large
number of HNSCC cases to assess their potential as targets for
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
immunotherapy and to evaluate their prognostic significance.

Corresponding Author: Andre
L. Vettore, Laborato
rio de Biologia Molecular do

Câncrer, Rua Pedro de Toledo, 669 - 11o andar - L11B, Vila Clementino, S~ao Paulo, Materials and Methods
SP, Brazil 04039-032. Phone: 55-11 5576 4848, ext. 2000; E-mail: Patients
andre.vettore@gmail.com
This retrospective study involved tissue specimens from 89
doi: 10.1158/1535-7163.MCT-14-0796 patients with HNSCC who underwent tumor resection between

2015 American Association for Cancer Research. 2007 and 2010 at the Barretos Cancer Hospital (Sao Paulo,

828 Mol Cancer Ther; 14(3) March 2015

 CTA Expression in HNSCC

Brazil). These samples were available at the Tissue Bank of the Statistical analyses
Hospital. Only patients diagnosed with primary HNSCC, not Statistical analysis was performed using the statistical software
previously treated, presenting with tumors at oral cavity, pharynx, SPSS 19.0 for Windows. To assess the association between the
or larynx and treated with curative intent were included in the anatomical and pathologic variables of patients and the molec-
study. All samples were checked microscopically for the presence ular findings in the tumor samples, the c2 or Fisher exact tests were
of neoplastic tissue and the absence of contaminating normal performed, as appropriate. Survival curves were calculated by the
mucosa. In addition, 20 oral mucosa tissue samples from healthy Kaplan–Meier method, and differences between groups were
donors undergoing odontological and preprosthetic surgeries compared using the log-rank test. Overall survival (OS) was
were used as normal controls. All tissue samples were snap-frozen measured as the time interval between the date of the initial
in liquid nitrogen within 30 minutes after resection and stored at treatment for the primary tumor and the date of the last follow-up
80 C until the RNA extraction. or death, whereas the disease-free survival (DFS) was defined as
The medical records of patients were reviewed for standard the time interval between the date of initial treatment and the date
demographic data, clinical and pathologic information, and of the diagnosis of the first recurrence. For evaluation of the
outcome of the disease. Smoking was defined as use of tobacco, independent contribution of significant clinical and molecular
chewable or smoked, for at least 1 year continuously. Alcohol use variables on DFS or OS, all factors with significance in the Kaplan–
was defined as intake of more than 2 alcoholic drinks per day, for Meier analysis (P  0.2) were tested in the multivariate analyses
at least 1 year continuously. Tissue sampling and study design using the Cox proportional hazard model. Results were calculated
were approved by the Institutional Review Board of the Barretos with 95% confidence intervals (CI). For all analyses, we consid-
ered statistical significance when P value <0.05.

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Cancer Hospital.

RNA extraction and RT-PCR

Results
Total RNA was isolated using Trizol reagent (Life Technolo-
gies), following the manufacturer's recommendations. After Patient characteristics and clinical predictors
extraction, RNA samples were quantified in a spectrophotometer The clinical and histological features of the 89 patients with
(NanoDrop ND1000; Thermo Fisher Scientific), and the quality HNSCC enrolled in this study are presented in Table 1. They were
of the RNA was checked by electrophoresis on 1% agarose gels. mainly males (86.5%), with age ranging from 32 to 82 years
Two micrograms of each RNA sample was subjected to cDNA (median, 58.9 years). Tobacco or alcohol consumption (current
synthesis using the SuperScript III First-Strand Synthesis System or former) was reported by 90.7% and 73.5%, respectively.
(Invitrogen). The cDNA obtained was diluted 10-fold before use. Primary tumor sites included oral cavity (68.5%), oropharynx/
The mRNA expression of the selected CTA in tumor and control
samples was determined by RT-PCR using primer sequences and
amplification conditions described on the CTDatabase (http:// Table 1. Demographic and clinical characteristics of patients with HNSCC
www.cta.lncc.br/index.php), except for PRAME (ref. 27; Supple- included in the study (n ¼ 89)
mentary Table S1). Testicular tissue was used as a positive control, Characteristics n (%)
and the ACTB gene was used as loading control. MAGEA3 and Age
MAGEA6 share more than 98% similarity in their sequences and, <55 years 31 (34.8)
55 years 58 (65.2)
for this reason, the primers used in this study were not able to
Gender
discriminate the expression of these two genes. The same is valid Male 77 (86.5)
for SPANX-C and SPANX-D genes. Electrophoresis gels were Female 12 (13.5)
visualized by SYBR Safe (Invitrogen) staining using the EP Alpha- Tobacco consumption
Imager (Alpha Innotech). Each RT-PCR experiment was per- Yes 78 (90.7)
formed in duplicate using the same cDNA sample. All cases with No 8 (9.3)
Alcohol consumption
a detectable band on both replicates were considered positive. The
Yes 61 (73.5)
intensities of the PCR products were heterogeneous, and some No 22 (26.5)
specimens yielded only faint bands. These were scored positive Tumor site
only if the result could be reproduced by a repeated RT-PCR. Cases Oral cavity 61 (68.5)
with very low transcript levels that were not reproducibly positive Oro/hypopharynx 14 (15.7)
were regarded as negative. Larynx 14 (15.7)
T stage
T1/T2 16 (18.2)
Selection of CTA genes T3/T4 72 (81.8)
We defined a strategy to select, among the CTAs cataloged in the N stage
CTDatabase, the most promising ones to have their mRNA N0 39 (50.0)
N1/N2/N3 39 (50.0)
positivity evaluated in HNSCC samples. Firstly, we mined the
Extracapsular spread
Oncomine (http://www.oncomine.org) and SAGE Anatomic Yes 21 (28.4)
Viewer (http://cgap.nci.nih.gov/SAGE/AnatomicViewer) data- No 53 (71.6)
bases to search for CTA genes expressed in HNSCCs. After that, Perineural invasion
we evaluated high-throughput expression data (EST, MPSS, Yes 21 (24.7)
CAGE, and RT-PCR experiments) previously provided by Hof- No 64 (75.3)
Lymphovascular invasion
mann and colleagues (28). In the end, we also accessed published
Yes 14 (17.1)
data of CTA mRNA expression in cancer cell lines (CTDatabase), No 68 (82.9)
as well as, information retrieved from the literature.

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r et al.
Zamune

Table 2. Comparison of CTA expression in 89 HNSCC cases and 20 normal control samples
CTAs Control, n (%) HNSCC, n (%) Sensitivity % (95% CI) Specificity % (95% CI)
PRAME 1 (5.0) 71 (79.8) 79.8 (69.93–87.55) 95 (75.13–99.87)
SPANX-CD 1 (5.0) 64 (71.9) 71.9 (61.38–80.93) 95 (75.13–99.87)
MAGEA4 0 (0.0) 54 (60.7) 60.7 (49.75–70.87) 100 (83.16–100)
MAGEA3/6 0 (0.0) 50 (56.2) 56.2 (45.25–66.68) 100 (83.16–100)
MAGEA12 0 (0.0) 44 (49.4) 49.4 (38.66–60.25) 100 (83.16–100)
MAGEB2 0 (0.0) 40 (44.9) 44.9 (34.38–55.86) 100 (83.16–100)
MAGEA1 1 (5.0) 40 (44.9) 44.9 (34.38–55.86) 95 (75.13–99.87)
MAGEA9 0 (0.0) 36 (40.5) 40.5 (30.17–51.38) 100 (83.16–100)
MAGEC2 1 (5.0) 35 (39.3) 39.3 (29.13–50.25) 95 (75.13–99.87)
MAGEB6 0 (0.0) 29 (32.6) 32.6 (23.02–43.34) 100 (83.16–100)
CXORF48 1 (5.0) 24 (27.0) 27.0 (18.10–37.42) 95 (75.13–99.87)
MAGEC1 1 (5.0) 14 (15.7) 15.7 (8.87–24.98) 95 (75.13–99.87)
CRISP2 2 (10.0) 10 (11.2) 11.2 (5.52–19.69) 90 (68.30–98.76)

hypopharynx (15.7%), and larynx (15.7%). Most tumors were Next, we evaluated the mRNA positivity of the remaining 19
presented at advanced stages (T3/T4, 81.8%). Among 78 patients CTA genes in 17 HNSCC samples. LIPI (0%), PAGE5 (5.9%), SSX2
with data available, metastatic cells could be detected in lymph (5.9%), XAGE2 (5.9%), OIP5 (11.8%), and XAGE3 (11.8%) were

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nodes of 39 patients (50%) and 21 (28.4%) of them presented rarely expressed in HNSCC and were set aside. By the end, 13 CTA
extracapsular spread. Perineural invasion was present in 21 cases genes that could better distinguish HNSCC from control samples
(24.7%) among 85 patients with available data. Lymph vascular were selected to be tested in the prevalence set (n ¼ 89) and
invasion was observed in 14 (17.1%) cases. control subjects (n ¼ 20).
MAGEC1 and CRISP2 were found rarely expressed in the
HNSCC specimens (15.7% and 11.2%, respectively). However,
Selection of CTAs
we were able to identify 11 CTA genes whose expression was
To select CTA-candidate genes with high probability to be
highly associated with HNSCC cases (PRAME 79.8%, SPANX-CD
expressed in HNSCC, an in silico screening was performed using
71.9%, MAGEA4 60.7%, MAGEA3/6 56.2%, MAGEA12 49.2%,
all the 139 CTA genes cataloged on the CTDatabase to interrogate
MAGEB2 44.9%, MAGEA1 44.9%, MAGEA9 40.5% MAGEC2
the NCBI-CGAP EST and SAGE databases, Oncomine microarray
39.3%, MAGEB6 32.6%, and CXORF48 27.0%; Table 2).
data collection, published data of CTA mRNA expression in cancer
According to the CTDatabase, the expression of MAGEB2,
cell lines (CTDatabase), and high-throughput expression data
MAGEA1, MAGEB6, SPANX-CD, and CXORF48 can be detected
provided by Hofmann and colleagues (28). By the end, we were
in several tumors, whereas it is absent in normal tissues. For
able to select 36 CTA genes for the assessment of gene expression
this reason, they are described as "testis-restricted" CT anti-
in a context of high expression in HNSCC samples and absence in
gens. Notably, in our study, 85.4% of the examined HNSCC
normal mucosa (Supplementary Table S2). Of note, previous
cases showed expression of at least one of these five CTAs.
studies have already reported the expression of 11 of those
Among them, 20.2% of the cases expressed only one of these
selected CTAs (CTAGE1, LIPI, MAGEA1, MAGEA12, MAGEA2,
CTA genes, whereas coexpression of two, three, four, or five
MAGEA3/6, MAGEA4, MAGEC1, MAGEC2, PRAME, and SSX2) in
antigens was detected in 23.5%, 18.1%, 18.1%, and 5.5% of
HNSCCs, whereas the presence of transcripts from the remaining
the HNSCC cases, respectively. Evaluating different panels
25 CTAs (ACTL8, CABYR, CASC5, CEP290, CRISP2, CSAG2,
formed by combinations of these five "testis-restricted" CT
CXORF48, HSPB9, MAGEA9, MAGEB2, MAGEB6, NXF2, OBP2B,
antigens expressed in HNSCCs, we observed that sensitivity of
OIP5, OTOA, PAGE5, PBK, SPANX-CD, TAF7L, TDRD1, TFDP3,
the panels can range from 57.3% to 85.4%, whereas the
TSSK6, VENTXP1, XAGE2, and XAGE3) has never been described
specificity observed can be as high as 100% in some combina-
in head and neck cancers.
tions of genes (Table 3).

Analysis of CTA gene expression in HNSCC Prognostic value of CTA expression in HNSCC
Based on the above results, 36 CTA genes were selected to be Significant associations between demographical and clinical
evaluated in HNSCC and normal mucosa samples using an RT- characteristics (age, gender, tobacco consumption, alcohol
PCR analysis. Because of the scarcity of RNA quantity of many consumption, tumor site, T stage, N stage, perineural invasion,
samples and the high number of genes selected, it would be lymphovascular invasion, and extracapsular spread) of the 89
virtually impossible to evaluate all possible candidate-genes in all patients with HSNCC enrolled in this study and the mRNA
samples. So, we firstly decided to conduct a discovery study, and expression of 11 selected CTA genes were evaluated. This
then a more limited set of "best" CTA genes would be used in the analysis showed MAGEA12 expression associated with non-
prevalence set of samples. The first step was to verify the expres- smokers (P ¼ 0.028) and SPANX-CD mRNA positivity corre-
sion pattern of the 36 selected CTA genes in mucosa samples lated with age <55 years (P ¼ 0.005). The lack of MAGEB6
collected from healthy individuals (controls, n ¼ 10). This anal- expression was associated with the absence of extracapsular
ysis showed that 17 CTAs (ACTL8, CABYR, CASC5, CEP290, spread (P ¼ 0.005), whereas CXORF48 expression was associ-
CSAG2, CTAGE1, HSPB9, MAGEA2, NXF2, OBP2B, OTOA, PBK, ated with perineural (P ¼ 0.023) and lymphovascular invasions
TAF7L, TDRD1, TFDP3, TSSK6, and VENTPX1) were expressed in (P ¼ 0.003). The detection of CRISP2 transcripts was also
more than 20% of the normal controls, indicating low specificity. associated with lymphovascular invasion (P ¼ 0.006; Supple-
Therefore, these CTAs were excluded from the study. mentary Table S3). No other significant correlation could be

830 Mol Cancer Ther; 14(3) March 2015 Molecular Cancer Therapeutics
 CTA Expression in HNSCC

Table 3. Best combination of CTA genes for antigen detection in patients with HNSCC (20 normal controls and 89 HNSCCs)
Genes Specificity % (95% CI) Sensitivity % (95% CI)
MAGEB2-SPANX-MAGEB6-MAGEA1-CXORF48 85.0 (62.11–96.79) 85.4 (76.32–91.99)
MAGEB2-SPANX-MAGEB6-MAGEA1 90.0 (68.30–98.76) 83.1 (73.73–90.25)
MAGEB2-SPANX-MAGEB6-CXORF48 90.0 (68.30–98.76) 83.1 (73.73–90.25)
MAGEB2-SPANX-MAGEA1 90.0 (68.30–98.76) 82.0 (72.45–89.36)
MAGEB2-SPANX-CXORF48 90.0 (68.30–98.76) 82.0 (72.45–89.36)
MAGEB2-SPANX-MAGEB6 95.0 (75.13–99.87) 79.8 (69.93–87.55)
MAGEB2-SPANX 95.0 (75.13–99.87) 78.6 (68.69–86.63)
MAGEB2-MAGEB6 100.0 (83.16–100) 57.3 (46.30–67.74)

observed between the expression of the selected CTA genes and and short survival (33). Besides, studies have associated lower
the patient's characteristics. rates of survival with immune system depression in patients with
In addition, we investigated the correlation of the OS and HNSCC (34). These facts point up an involvement of the immune
DFS with clinical and molecular variables. As expected, the OS response in the control of HNSCC and highlight the potential
was better for those patients with early T stage (86.6% T1/T2 vs. value of cancer immunotherapy for these patients (34, 35).
41.4% T3/T4, P ¼ 0.002), without perineural invasion (65.4% In the present study, we surveyed the landscape of CTA expres-
absent vs. 31.7% present, P ¼ 0.038) and with a tendency to sion in HNSCC by conducting a deep screening of the CTA listed
without metastasis (64.1% N0 vs. 43.7 N1/N2/N3, P ¼ 0.065;

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in the CTDatabase. A small number of studies have reported a
Table 4). relatively frequent, but sometimes conflicting, expression of
The analyses of OS were not able to identify any significant selected CTA genes in HNSCC (18–25). According to them,
association with the mRNA expression of the 11 selected CTA MAGEA3 expression could be detected in 43% to 72% of HNSCC
genes. However, the 5-year DFS analyses showed that 23.8% of cases evaluated in five studies, whereas MAGEA4 gene product was
the patients with HNSCC expressing MAGEA3/6 presented detected in 27% to 60% of tumors examined by four independent
recurrences, whereas relapses were detected in 65.9% of the research groups. MAGEA1 is expressed in a considerable propor-
patients with no expression of this CTA, and this difference was tion of HNSCCs (30%–40%), as well as PRAME (42%–49%), NY-
significant (P ¼ 0.014; Fig. 1). ESO-1 (6%–33%), MAGEC1 (28%–48%), MAGEC2 (10%–
To detect independent predictors of survival, an analysis of 33%), MAGEA12 (49%), SSX1 (45%), and SSX4 (30%). Other
prognostic variables based on the Cox proportional hazards CTAs reported as occasionally expressed in HNSCC are MAGEA2,
model was performed involving significant clinical features (age, MAGEA10, BAGE, SCP1, GAGE, and SSX2. Consistent with this,
gender, alcohol consumption, tumor site, T stage, N stage, peri- we also found MAGEA1 (44.9%), MAGEA4 (60.7%), MAGEA3
neural invasion, lymphovascular invasion, and extracapsular (56.2%), MAGEC1 (15%), MAGEC2 (39.3%), and MAGEA12
spread) and molecular variables (MAGEA3/6 and MAGEA12 (49.4%) frequently expressed in HNSCC samples. Further, we
mRNA positives) associated with survival probability. In 5-year were able to detect PRAME transcripts in 79.8% of the HNSCC
DFS, this multivariate analysis revealed that gender (P ¼ 0.032; samples, a frequency higher than observed previously. Worth
HR ¼ 3.21; 95% CI, 1.11–9.30) and MAGEA3/6 expression (P ¼ mentioning, this is the first study to identify a frequent expression
0.008; HR ¼ 0.30; 95% CI, 0.128–0.732) remained as indepen- of CXORF48, MAGEA9, MAGEB2, MAGEB6, CRISP2, and SPANX-
dent predictors of recurrence (Table 5). CD in HNSCC.
Several clinical trials have evaluated different CTAs as targets for
anticancer vaccines in patients with lung cancer, prostate, ovarian,
Discussion and melanoma (12, 36–42). In HNSCC, a clinical trial conducted
Over the last years, diagnosis and management of patients by the University of Maryland School of Medicine, Baltimore,
with HNSCC have improved through combined efforts in United States, is evaluating a vaccine targeting MAGEA3 and
surgery, radiotherapy, and chemotherapy, but long-term sur- HPV-16 antigens (NCT00257738, http://clinicaltrials.gov/). Our
vival rate is still around 50% (29). Therefore, novel forms of results show a large proportion of patients with HNSCC expres-
treatment are urgently needed, and immunotherapy represents sing at least one of five CTAs, and this expression seems to be
an approach that has yet to be fully explored in HNSCC. restricted to tumor tissues. Taken collectively, these data suggest
Immunotherapy is a promising cancer treatment due to the that patients with HNSCC expressing CTAs might be eligible for
potential for persistence of the antitumor effect caused by future immunotherapy approaches targeting multiple antigens;
immunologic memory (30). In this way, immunotherapeutic however, the demonstration of immunogenicity in the human
intervention could help in the control of the HNSCC progres- host is crucial for a CTA to be considered as a potential cancer
sion and relapse. There is a growing interest in applying vaccine target. Hence, further studies are necessary to evaluate
immunotherapy approaches to different tumors, encouraged whether some of the CTAs highlighted in this study are able to
by the recent FDA approval for Sipuleucel-T in prostate cancer elicit coordinated humoral and cell-mediated responses in
and Ipilimumab for metastatic melanoma (31, 32). patients with HNSCC. It will also be important to verify the
Although there are few studies in this field, evidences show that presence of IgG antibodies against these antigens in sera of these
the weakening of the immune response may play an important patients.
role in the installation, progression, and recurrence of HNSCC. The identification of human tumor antigens could be impor-
For instance, solid organ transplant recipients who are diagnosed tant not only for the analysis of antitumor immune responses and
with HNSCC while receiving high doses of immunosuppressive the development of immunotherapy, but also for the identifica-
progress extremely poorly, with advanced diagnosis of the tumor tion of molecular markers useful for diagnosis or prognosis

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Table 4. OS and DFS rates according to the clinical and molecular Table 4. OS and DFS rates according to the clinical and molecular
variables variables (Cont'd)
5-year 5-year 5-year 5-year
Variables OS (%) P DFS (%) P Variables OS (%) P DFS (%) P
Age, years MAGEC1
<55 67.7 0.084 55.7 0.616 Yes 71.4 0.564 79.5 0.387
55 45.9 65.4 No 51.6 59.8
Gender CRISP2
Male 56.4 0.075 62.8 0.115 Yes 70.0 0.603 80.0 0.552
Female 37.0 54.5 No 53.6 59.9
Tobacco consumption
Yes 58.6 0.569 63.2 0.255
No 62.5 57.1
Alcohol consumption
determination (43). Probably, due to the small sample sizes
Yes 57.9 0.912 71.9 0.130 analyzed, all except one of the previous studies failed in establish-
No 65.0 44.6 ing a clear correlation between CTA expression and HNSCC
Tumor site prognosis. Cuffel and colleagues (21) showed that patients with
Oral cavity 49.7 0.461 51.2 0.114 tumors expressing MAGEA4 or multiple CTA genes had a poorer
Oropharynx/hypopharynx 64.3 76.9
OS, and MAGEA4 mRNA positivity was associated with poor
Larynx 70.5 91.7
outcome independent of clinical parameters. Our results reveal,
T stage
for the first time, a significant association between MAGEB6

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T1/T2 86.6 0.002 55.3 0.542
T3/T4 41.4 65.6 expression and the tumor extracapsular spread. The biologic
N stage significance of the extracapsular spread in the outcome of the
N0 64.1 0.065 60.9 0.355 patients is referred as a manifestation of a more aggressive
N1/N2/N3 43.7 58.0 neoplasm providing higher rates of recurrence (44–47). In
Perineural invasion
addition, CRISP2 and CXORF48 expression was correlated with
Yes 31.7 0.038 58.3 0.150
No 65.4 64.7
the presence of lymphovascular invasion, a recognized clinical
Lymphovascular invasion factor of poor prognosis. Based on these findings, we can speculate
Yes 50.0 0.172 70.7 0.632 an association between the expression of these CT antigens
No 59.3 63.7 and a more malignant phenotype, suggesting their usefulness
Extracapsular spread as prognostic markers in HNSCC.
Yes 42.9 0.229 56.6 0.139 Interestingly, our study showed the positive correlation
No 53.6 64.9
between MAGEA3/6 expression and better disease outcome.
PRAME
Yes 55.5 0.862 54.7 0.216
Although most of previous studies have reported an association
No 50.0 83.0 of CTA expression with poor outcome in multiple myeloma,
SPANX-CD ovarian, lung, head and neck, and gastric cancers (9, 11,
Yes 52.3 0.496 62.5 0.916 21, 48, 49), Sharma and colleagues (10) described a positive
No 58.9 61.5 association between expression of CT10 and improved survival
MAGEA4 for patients with urothelial carcinoma. Along the same line,
Yes 50.8 0.427 60.8 0.721
Freitas and colleagues presented the mRNA positivity of CT
No 62.3 62.0
MAGEA3/6
Yes 57.0 0.579 76.2 0.014
No 53.1 43.1 1.0
MAGEA12
Yes 54.3 0.826 69.5 0.130
No 55.6 55.0 0.8
MAGEB2
Yes 49.8 0.348 76.3 0.263
No 58.6 53.0 0.6
DFS (%)

MAGEA1
Yes 49.9 0.151 65.0 0.698
No 55.0 60.2 0.4
MAGEA9
Yes 47.9 0.623 60.9 0.948
No 59.0 62.4 0.2
MAGEA3/6 (+)
MAGEC2
Yes 54.0 0.367 67.9 0.453 MAGEA3/6 (−)
No 51.2 58.0
0.0
MAGEB6
Yes 50.7 0.496 74.0 0.601 0.00 20.00 40.00 60.00
No 57.2 56.4 Follow-up (months)
Cxorf48
Yes 41.7 0.683 57.4 0.415 Figure 1.
No 58.1 63.8 Correlation between MAGEA3/6 expression and DFS of patients with HNSCC.
Kaplan–Meier survival estimates of patients were performed according to
(Continued on the following column)
mRNA positivity of MAGEA3/6 (P ¼ 0.014).

832 Mol Cancer Ther; 14(3) March 2015 Molecular Cancer Therapeutics
 CTA Expression in HNSCC

Table 5. Results of multivariate analysis of selected prognostic factors for DFS used a frozen tissue cohort in which a selection bias existed as a
(gender, tumor site, N stage, and MAGEA3/6 expression) result of the selection of specimens with large volume tumors
Categories HR (95% CI) P appropriate for frozen tissue collection.
Gender In conclusion, this study, one of the largest studies reported to
Male 1 (Ref.)
Female 3.21 (1.11–9.30) 0.032
date evaluating CTA expression in HNSCC, identified 11 CTA
Tumor site genes frequently expressed in HNSCC. Five of them are consid-
Oral cavity 1 (Ref.) ered as "testis-restricted" CTAs and, in the future, should be
Oropharynx/hypopharynx 0.59 (0.17–1.99) 0.396 evaluated as immunotherapy targets for this neoplasm. Further-
Larynx 0.146 (0.02–1.12) 0.065 more, on the basis of our results, the expression of MAGEA3/6
MAGEA3/6
is an independent predictor of DFS in patients with HNSCC,
Yes 1 (Ref.)
No 0.30 (0.12–0.73) 0.008
and the mRNA positivity of MAGEB6, CRISP2, and CXORF48 is
associated with bad prognosis for these patients.

Disclosure of Potential Conflicts of Interest

antigens as an independent predictor of better OS for patients No potential conflicts of interest were disclosed.
with glioblastoma (14). Additional prospective studies with
independent cohorts of patients with HNSCC are needed to Authors' Contributions
confirm the positive correlation between MAGEA3/6 expression Conception and design: A.L. Carvalho, A.L. Vettore

Downloaded from http://aacrjournals.org/mct/article-pdf/14/3/828/2240189/828.pdf by guest on 07 March 2023

and better disease outcome, but, based on our results, we can Development of methodology: F.T. Zamun
er, C.Z. de Oliveira
speculate that the expression of these CTA in head and neck Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): F.T. Zamun
er, B.T.R. Karia, C.R. dos Santos
tumors may have elicited a spontaneous immune response and
Analysis and interpretation of data (e.g., statistical analysis, biostati-
this could impact favorably the prognosis. stics, computational analysis): F.T. Zamun
er, B.T.R. Karia, C.Z. de Oliveira,
The biologic function of MAGEA3 remains poorly understood, A.L. Carvalho
but this antigen is found frequently expressed in different cancer Writing, review, and/or revision of the manuscript: F.T. Zamun
er, C.R. dos
types. Yang and colleagues proposed that the expression of Santos, A.L. Carvalho, A.L. Vettore
MAGEA3 could protect cells from programmed cell death and Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): B.T.R. Karia
contribute to tumor development by stimulating the cell survival
Study supervision: C.R. dos Santos, A.L. Carvalho, A.L. Vettore
and proliferation (50).
Some points in this report warrant emphasis. First, this study
identifies 11 CTA genes frequently expressed in HNSCC, and five Grant Support
This work was supported by Fundaç~ao de Amparo a Pesquisa do Estado de
of them are reported as "testis-restricted" CTAs. Second, different S~ao Paulo. F.T. Zamun
er was recipient of scholarship from Fundaç~ao de Amparo
panels formed by combinations of these "testis-restricted" CTAs a Pesquisa do Estado de S~ao Paulo—FAPESP, and B.T.R. Karia was recipient of
can show up to 85.4% of positivity for at least one member of the scholarship from Coordenaç~ao de Aperfeiçoamento de Pessoal de Nível Super-
panel, making them potential targets for immunotherapy ior—CAPES. A.L. Carvalho has a National Counsel of Technological and
approaches. Third, MAGEB6, CRISP2, and CXORF48 expressions Scientific Development (CNPq) scholarship.
The costs of publication of this article were defrayed in part by the
are significantly correlated with well-known poor outcome clin-
payment of page charges. This article must therefore be hereby marked
ical features. Fourth, the MAGEA3/6 expression is an independent advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
marker for tumor recurrence. On the other hand, there are also this fact.
some limitations to the present study, such as the limited patient
number evaluated and the fact of these patients were not consec- Received October 27, 2014; accepted November 25, 2014; published
utively and prospectively collected for the study. Furthermore, we OnlineFirst January 6, 2015.

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