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LABORATORY INVESTIGATION
Vol. 82, No. 6, p. 775, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
The Cancer-Testis Gene, NY-ESO-1, Is Expressed in
Normal Fetal and Adult Testes and in Spermatocytic
Seminomas and Testicular Carcinoma In Situ
Anne-Pascale Satie, Ewa Rajpert-De Meyts, Giulio C. Spagnoli, Sébastien Henno,
Laurence Olivo, Grete Krag Jacobsen, Nathalie Rioux-Leclercq, Bernard Jégou, and
Michel Samson
GERM-INSERM U. 435 (A-PS, BJ, MS), Université de Rennes I, Campus de Beaulieu, Rennes, France; Department
of Growth and Reproduction (ER-DM), Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark;
Departments of Surgery and Research (GCS), University of Basel, Switzerland; Service d’Anatomopathologie (SH,
LO, NR-L), CHU Ponchaillou, Rennes, France; and Department of Pathology (GKJ), Gentofte University Hospital,
Gentofte, Denmark
SUMMARY: Cancer/testis genes are potential targets for therapeutic genetic and immunologic approaches, and are highly
expressed in a large variety of human cancers. However, they are not expressed in normal tissues, with the exception of the testis.
The NY-ESO-1 gene is the most recently identified member of the cancer/testis family and its product is one of the most
immunogenic tumor antigens. We used immunohistochemistry to investigate the expression of NY-ESO-1 in healthy human
prenatal and adult testes and in 59 human testicular tumors of different subtypes. We found that NY-ESO-1 was expressed from
18 weeks until birth in human fetal testes. In the adult testis, NY-ESO-1 was strongly expressed in spermatogonia and in primary
spermatocytes, but not in post-meiotic cells or in testicular somatic cells. NY-ESO-1 was not expressed in the Sertoli cells, Leydig
cells, classical seminomas, or nonseminomatous germ cells in the 59 testicular tumors. In contrast, NY-ESO-1 was expressed
both in carcinomas in situ, which are the earliest stage of testicular tumors (7 of 15 cases), and in spermatocytic seminomas,
which are believed to be derived from spermatogonia or primary spermatocytes (8 of 16 cases). We conclude that NY-ESO-1 is
a marker that can be used to follow the early progression of testicular tumorigenesis when the tumors present a similar pattern
of expression to the cells from which they originated, although the later tumors cease to express NY-ESO-1. (Lab Invest 2002,
82:775–780).
esticular cancer is the most common form of
cancer in men aged between 15 and 40 years. Its
occurrence has been reported to have increased in a
number of industrialized western countries (Adami et al,
cancers and in the testis, but not in other organs. We
recently demonstrated that MAGE-A4, a member of the
CT family, is expressed in human testicular germ cells
throughout development. MAGE-A4 is expressed in
classical seminomas and in carcinomas in situ (CIS), but
not in nonseminomas; thus it is possible to distinguish
between the different types of testicular tumor (Aubry et
al, 2001). Here, we focused on NY-ESO-1, which was
recently characterized and shown to be expressed in
normal testes (Jungbluth et al, 2001). Its product is one
of the most immunogenic tumor antigens defined to date
in the CT family (Jager et al, 2000). We also analyzed the
expression of NY-ESO-1 in fetal and adult testes and in
a large variety of different testicular tumors, including
classical seminomas, CIS, nonseminomatous germ cell
tumors (NSGCT), spermatocytic seminomas, and non-
germ-cell tumors derived from Leydig and Sertoli cells.
T
1
994; Moller, 1993; Osterlind, 1986). The etiology of
testicular cancer is still very poorly understood. How-
ever, it is generally acknowledged that the carcinogenic
process may be initiated before birth (Depue et al, 1983;
Henderson et al, 1979). This highlights the importance of
identifying specific testicular markers for each type of
testicular tumor and for the early stages of neoplastic
transformation. We aimed to identify such early markers
by focusing on the members of the cancer/testis (CT)
family as they are expressed both in a large variety of
DOI: 10.1097/01.LAB.0000017169.26718.5F
Received February 15, 2002.
This work has been supported by INSERM, the Ministère de l’Education
Nationale de la Recherche et de la Technologie, the Fondation pour la
Recherche Médicale, the Association pour la Recherche sur le Cancer, the
Ligue nationale contre le cancer and the Région Bretagne.
Address reprint requests to: Dr. Michel Samson, Université de Rennes I,
GERM-INSERM U. 435, Campus de Beaulieu, 35042 Rennes cedex,
Bretagne, France. E-mail: michel.samson@rennes.inserm.fr
Results
Western Blot Analysis
Six monoclonal antibodies (mAbs), namely B9-8, D8-
38, B4-2, D1-13, D8-10, and B10-3, raised against the
recombinant NY-ESO-1 fusion protein (Schultz-Thater
Laboratory Investigation • June 2002 • Volume 82 • Number 6 775

Satie et al
et al, 2000), were tested on human testicular extracts
the D8-38 antibody. As expected, no staining was
observed in somatic tumors (1 Sertoli cell and 2 Leydig
cell tumors; data not shown). Surprisingly, no reaction
was detected in the 12 classical seminomas (Fig. 4A)
or in the 12 NSGCT (Fig. 4B, Table 1). In contrast,
specific immunostaining was observed in 7 of 15 CIS
specimens screened (Fig. 4E: NY-ESO-1-positive CIS
and Fig. 4G: NY-ESO-1-negative CIS). To confirm that
CIS was present in all seminiferous tubules, including
those that were negative for NY-ESO-1, we used an
antibody against placental-like alkaline phosphatase
(
T) and the recombinant NY-ESO-1 protein (r). B9-8,
D8-38, B4-2, D8-10, and B10-3 recognized both the
recombinant and native proteins. However, in our
conditions, D1-13 failed to recognize the target pro-
teins. When the recombinant protein was used, a
protein with an apparent molecular weight of 40 kDa
was revealed, whereas a band of 22 kDa was detected
in the testis extracts (Fig. 1).
Immunohistochemical Analysis of the Expression of
NY-ESO-1 in the Adult Testis
(
(
PLAP), which is routinely used to identify CIS cells
Giwercman et al, 1991). We showed that all of the CIS
D8-38 was selected for the immunohistochemical
experiments because it presented a strong reactivity
with the testicular extracts (Fig. 1). No staining was
observed with the control mAb in the normal testis
samples were PLAP-positive (Fig. 4, F and H). Further-
more, 8 of 16 spermatocytic seminomas tested NY-
ESO-1-positive (Fig. 4C: NY-ESO-1-positive sper-
matocytic seminoma and Fig. 4D: NY-ESO-1-negative
spermatocytic seminoma). The cytoplasm of cancer
cells was also stained in a similar way to the germ cells
from normal testes.
(
Fig. 2A), but strong and specific staining was ob-
served within the seminiferous tubules (Fig. 2B). The
intensely labeled cells were identified as spermatogo-
nia (Fig. 2C) and primary spermatocytes (Fig. 2D). This
staining was localized in the cytoplasm. Spermatids
(
post-meiotic cells) and Sertoli cells (somatic cells)
Discussion
were consistently negative. Furthermore, no staining
was observed in the seminiferous tubules from pa-
tients with Sertoli-cell-only syndrome (Fig. 2E).
The so-called CT genes are attractive targets for
antigen-specific cancer therapy. A strategy based on
spontaneous antibody responses to tumor associated
antigens (SEREX) was recently used to identify a new
CT antigen, named NY-ESO-1. This antigen is one of
the most immunogenic tumor antigens defined to
date, because it induces spontaneous humoral and
CD8ϩ T cell responses against NY-ESO-1 in 40% to
50% of patients with advanced NY-ESO-1–express-
ing tumors (Jager et al, 2000). More recently, Kurash-
ige et al (2001) showed that there is a positive corre-
lation between the expression of NY-ESO-1 and the
immunogenicity associated with the tumor grade in
transitional cell carcinoma (Kurashige et al, 2001) and
in melanoma (Goydos et al, 2001). These data suggest
that NY-ESO-1 is a marker for the most advanced
cancerous stages. Here, we used a number of mAbs
directed against NY-ESO-1 (Schultz-Thater et al,
2000) to investigate the expression of NY-ESO-1 in the
human testis at different developmental stages, and in
various types of testicular tumors. As previously re-
ported, the NY-ESO-1 mAbs specifically recognized
the recombinant NY-ESO-1 protein (Schultz-Thater et
al, 2000) but Western blotting showed that they also
recognized the natural protein in testicular extracts.
Our results for normal germ cells confirmed those of
Jungbluth et al (2001), who showed that the expres-
sion of NY-ESO-1 was localized to spermatogonia in
the cells. Moreover, our investigations revealed that
NY-ESO-1 was strongly expressed in pachytene sper-
matocytes, but not in post-meiotic germ cells. As with
MAGE-A4 (Aubry et al, 2001), the NY-ESO-1 gene was
no longer expressed by the time of the second meiotic
division. The NY-ESO-1 labeling was strictly limited to
germ cells, as demonstrated by the absence of signal
in the somatic cells within the normal testis with
complete spermatogenesis, and in Sertoli-cell-only
tubules. We studied the expression and localization of
NY-ESO-1 at different developmental stages. NY-
Immunohistochemical Localization of NY-ESO-1 in the
Fetal Testis at Different Stages of Development
Paraffin-embedded testis sections from 12-, 13-, 14-,
16-, 18-, 19-, 22-, 24-, 26-, 28-, 32-, 34-, and 40-
week-old human fetuses were labeled with the D8-38
antibody to investigate the localization of NY-ESO-1 at
different developmental stages. No NY-ESO-1 stain-
ing was observed in the embryonic gonad before 16
weeks (Fig. 3A), but a few NY-ESO-1-positive cells
were seen in the gonads of the 18-week-old fetus (Fig.
3B). The number of stained cells increased after 18
weeks and was maximal in the 40-week-old fetal
gonad (Fig. 3C). At all stages studied, the NY-ESO-1-
positive cells were cytoplasmic, strictly located in the
tubules, and presented the morphologic characteris-
tics of gonocytes (Fig. 3C insert), the precursors of
spermatogonia.
NY-ESO-1 Expression in Testicular Tumors
We used immunohistochemistry to investigate the
expression of NY-ESO-1 in 59 testicular tumors using
Figure 1.
Western blot analysis of recombinant NY-ESO-1 (r) and a protein extract from
human testicles (T) using different monoclonal antibodies (1:40) raised against
NY-ESO-1. The study was performed using 10 ng of recombinant NY-ESO-1
protein (r) or 40 ␮g of protein extracted from whole human testes. The
reactions were revealed using horseradish peroxidase conjugated to an
antibody. M: molecular mass markers (in kDa).
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76 Laboratory Investigation • June 2002 • Volume 82 • Number 6

NY-ESO-1 in Germ Cell Testicular Tumors
Figure 2.
Immunolocalization of NY-ESO-1 in the adult human testis. All tissues were fixed in paraformaldehyde. NY-ESO-1 was located using the monoclonal antibody D3-38
and an avidin-biotin peroxidase complex. The testis sections were either obtained from patients with no testicular diseases (A and B) or from patients suffering from
Sertoli-cell-only (SCO) syndrome (E). These sections were incubated with the anti-NY-ESO-1 antibody (B and E) or control IgG1 (A); scale bar ϭ 20 ␮m. A specific
NY-ESO-1 signal was located within the seminiferous tubules. The interstitial tissue (*) was consistently NY-ESO-1–negative (B). The positive cells were identified
as being spermatogonia (Sg) (C) and pachytene spermatocytes (PS) (D); scale bar ϭ 100 ␮m. Sertoli cells (S) were not labeled. None of the cell types in the SCO
testis were NY-ESO-1–positive (E).
ESO-1 was expressed from 18 weeks on in the
spermatogonia precursors, gonocytes. A similar ex-
pression pattern was reported for MAGE-A4 (Aubry et
al, 2001). Interestingly, the SSX protein (synovial sar-
coma on X chromosome) is expressed in spermato-
gonia, primary spermatocytes, and gonocytes from at
least the 17th week of development on (Stoop et al,
PCR-based approach used by Yuasa et al (2001)
found that NY-ESO-1 was expressed in some semi-
nomas. This discrepancy could result from the con-
tamination of the cancerous testicular tissues used by
Yuasa et al (2001) with the normal testicular tissue that
always surrounds the tumor and which contains sper-
matogonia and primary spermatocytes, which express
NY-ESO-1.
2001). This suggests that this stage is crucial in the
production of several proteins involved in the devel-
opment of the gonad.
In our conditions, NY-ESO-1 was only expressed in
47% of CIS, whereas PLAP was in all CIS. This
observation reveals the molecular heterogeneity of
CIS. It is possible that the seminomas and NSGCT
that did not express NY-ESO-1 had lost NY-ESO-1
during tumor progression. Consequently, the CIS cells
that were positive for NY-ESO-1 may have retained
the gonocyte-like phenotype, whereas those that were
negative for NY-ESO1 could be pre-invasive precur-
sors that are already partly differentiated toward sem-
inomas or NSGCT. Spermatocytic seminomas, which
are thought to be derived from spermatogonia/sper-
matocytes (Stoop et al, 2001), expressed NY-ESO-1,
as did the spermatogonia and primary spermatocytes
The demonstration that NY-ESO-1 is expressed in
gonocytes suggests that this CT can be used as a
marker to follow testicular carcinogenesis. Indeed,
NY-ESO-1 was specifically expressed in 7 of 15 CIS
and in 8 of 16 spermatocytic seminomas studied. CIS
are considered to be gonocytes that were mispro-
grammed and are the earliest cellular stages of sem-
inomas. Spermatocytic seminomas seem to have de-
rived from spermatogonia and/or spermatocytes. In
contrast, NY-ESO-1 was not expressed in somatic cell
tumors (Sertoli and Leydig), in nonseminomatous
germ cell tumors, or in seminomas. However, the
Laboratory Investigation • June 2002 • Volume 82 • Number 6 777

Satie et al
ESO-1 positive. Similarly, although nearly all of the
spermatocytic seminoma samples studied were
MAGE-A4-positive (Rajpert-De Meyts et al, 2002), only
50% were NY-ESO-1-positive. Thus, a better under-
standing of the expression patterns of the different CT
members should allow us to precisely classify the
different types of the CIS-derived testicular tumors
and their developmental stages.
In conclusion, we have shown that NY-ESO-1 is
specifically expressed in some CIS and in spermato-
cytic seminomas but not in seminomas, NSGCT, or
somatic cell tumors (derived from Sertoli and Leydig
cells). NY-ESO-1 can be used as a marker to improve
our understanding of the cellular origin of malignant
cells and the progression of the tumors.
Materials and Methods
Tissue Specimens and Clinicopathologic Data
Fifty-nine specimens of testicular tumors and tissues
were obtained from patients with testicular cancer
who underwent surgery at one of several hospitals in
France, Denmark, and Sweden. The series included
12 seminoma specimens, 13 NSGCT specimens with
various histologic components, 15 CIS specimens, 16
spermatocytic seminoma specimens, 1 Sertoli cell
tumor, and 2 Leydig cell tumors (Table 1). Three
normal testis specimens were used as positive con-
trols. Sections of fixed gonads from 12- to 40-week-
old fetuses were obtained from the pathology depart-
ment of Ponchaillou Hospital, France. These tissue
specimens were isolated after induced or spontane-
ous abortions when routine autopsies found no signs
of disease or developmental disorders. The study was
approved by local ethical committees.
Western Blot Analysis of NY-ESO-1
Proteins were subjected to SDS-PAGE on 12.5%
polyacrylamide gels. We transferred 40 ␮g of crude
protein extracts from human testes or 10 ng of recom-
SQ
binant NY-ESO-1 protein onto Immunobilon-P
(PVDF) membranes (Millipore, Saint Quentin en Yve-
lines, France) as described by Towbin et al (1992). The
membranes were blocked overnight at 4° C in 0.02 M
Tris buffered saline (TBS) supplemented with 3% BSA.
After washing with TBS, the membranes were incu-
bated for 2 hours at room temperature with mAbs
directed against NY-ESO-1 (working dilution, 1:40) in
TBS supplemented with 0.1% Tween and 1% BSA.
After washing in TBS-Tween 0.1%, the membranes
were incubated with a 1/5,000 dilution of horseradish
peroxidase conjugated anti-mouse antibody (Amer-
sham Pharmacia Biotech, Les Ulis, France). After
several washes in TBS-Tween 0.1%, antibody binding
was detected by use of ECL (Amersham Pharmacia
Biotech). Kodak Biomax films were exposed to the
blots for 15 minutes.
Figure 3.
Immunolocalization of NY-ESO-1 in human fetal male gonad. Sections of
human 16-week-old (A), 18-week-old (B), and 40-week-old fetal gonads (C)
were incubated with the D3-38 antibody. The dotted lines indicate the
sex-cords in A, B, and C. Arrows show positively labeled cytoplasmic
gonocytes (B and C). Scale bars ϭ 20 ␮m. The scale bar in the insert ϭ 50
␮
m.
in normal tissue. A heterogeneous pattern of NY-
ESO-1 expression in spermatocytic seminoma might
indicate the molecular heterogeneity of this tumor,
perhaps reflecting different stages of the tumor pro-
gression. Interestingly, although NY-ESO-1 and
MAGE-A4 belong to the same CT family and share
similar characteristics, they also present specific fea-
tures. Whereas most (87%) CIS samples were MAGE-
A4-positive (Aubry et al, 2001), only 47% were NY-
Immunohistochemical Localization of NY-ESO-1
Tissue sections were deparaffinized and rehydrated.
They were then incubated for 5 minutes in 3% H O to
2
2
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78 Laboratory Investigation • June 2002 • Volume 82 • Number 6

NY-ESO-1 in Germ Cell Testicular Tumors
Figure 4.
Immunolocalization of NY-ESO-1 in a range of testicular tumors. Sections of human testicular tumors were fixed in paraformaldehyde and incubated with either an
anti-NY-ESO-1 (D3-38) antibody (A to E, and G) or an anti-placental-like alkaline phosphatase antibody (F and H). Seminomas (A), nonseminomatous germ cell tumors
(
(
B), spermatocytic seminomas (D), and tubules presenting CIS (G) were negative for NY-ESO-1. Spermatocytic seminoma (C) and some cancer-in-situ
CIS)–containing tubules (E) were positive for NY-ESO-1. Tubules presenting cancer-in-situ were positive for placental-like alkaline phosphatase (F and G). Scale bars
ϭ 100 ␮m.
Table 1. Expression of NY-ESO-1 in Testicular Tumors
with a 1:500 dilution of peroxidase-conjugated
streptavidin (DAKO) for 30 minutes. After a final rinse
with TBS, the sections were incubated with amino-
ethyl-carbazole substrate (DAKO) for 30 minutes to
reveal the specific red staining. The nuclei were coun-
terstained with Hemalun Masson solution. The sec-
tions were photographed using an Olympus AX60TF
microscope with monochromatic objectives (Olym-
pus, Paris, France), coupled to a digital macro camera
Type of testicular tumor
n
Positive staining
Seminoma
NSGCT
CIS
Spermatocytoma
Sertoli cell tumor
Leydig cell tumor
17
12
15
16
1
0
0
7
8
0
0
2
(Kigamo, Metis, France).
block endogenous peroxidase activity, rinsed in TBS,
and incubated twice for 10 minutes in TBS supple-
mented with 1% BSA to block the nonspecific sites.
The tissue sections were incubated overnight at 4° C
with either a 1:50 dilution (in 1% BSA) of the anti-NY-
ESO-1 antibody (mouse monoclonal antibody D8-38)
or with 1 ␮g/ml mouse IgG1 (DAKO, Glostrup, Den-
mark) as a negative control. All subsequent steps were
performed at room temperature. After several washes
with TBS, the sections were incubated for 2 hours with
a biotinylated rabbit anti-mouse IgG at a dilution of
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