Oncogene (2004) 23, 8552–8556& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00
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Aggrus: a diagnostic marker that distinguishes seminoma from embryonalcarcinoma in testicular germ cell tumors
Yukinari Kato1,2, Isoji Sasagawa3, Mika Kaneko2, Motoki Osawa2, Naoya Fujita1
and Takashi Tsuruo*,1,4
1Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan;
Department of Experimental and Forensic Pathology, Yamagata University School of Medicine, 2-2-2, Iida-nishi, Yamagata
990-9585, Japan; 3Department of Urology, Yamagata University School of Medicine, 2-2-2, Iida-nishi, Yamagata 990-9585,
Japan; 4Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1, Kami-Ikebukuro, Toshima-ku,
Tokyo 170-8455, Japan
2

Aggrus (also known as T1a/podoplanin) is a membranesialoglycoprotein whose function in tumors is unknown.
We recently determined that Aggrus possessed the abilityof inducing platelet aggregation and that its expression
was frequently upregulated in colorectal tumors. Thus,
Aggrus expression might be associated with tumorinduced platelet aggregation and tumor metastasis. Here
we show, by means of cancer proﬁling array and real-time
PCR, that aggrus mRNA expression is frequently
upregulated in testicular germ cell tumors when comparedwith the surrounding normal tissue. Immunohistochemical
staining revealed that Aggrus protein expression was
detected in 10 of 11 seminomas (90.9%), but its
expression was not observed in embryonal carcinomas(0/4; 0%). Speciﬁc markers for seminomas have not beenreported, and Aggrus is a potential diagnostic marker for
seminomas and may be associated with malignancies of
the testis.
Oncogene (2004) 23, 8552–8556. doi:10.1038/sj.onc.1207869
Published online 13 September 2004
Keywords: Aggrus; seminoma; embryonal carcinoma;
germ cell tumor

Testicular germ cell tumors (GCTs) comprise less than
2% of all human malignancies and are the most frequentcancer in young males between the ages of 20 and 40
years (Moller, 1993; Adami et al., 1994). Over the past
several decades, the incidence of GCTs has been
increasing in the Western world (Bergstrom et al.,1996). GCTs comprise large numbers of histologicallydifferent forms and can be practically divided into two
broad categories: seminoma and nonseminomatous germ
cell tumors (NSGCTs) (Moller, 1993; Adami et al.,1994). NSGCT is an umbrella designation that includes*Correspondence: T Tsuruo, Laboratory of Cell Growth andRegulation, Institute of Molecular and Cellular Biosciences, The
University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan; E-mail: ttsuruo@iam.u-tokyo.ac.jp
Received 21 December 2003; revised 4 May 2004; accepted 4 May 2004;
published online 13 September 2004

tumors of one histological type, such as embryonalcarcinoma, as well as those with more than one
histological type. However, there are combined or mixed
GCTs that are composed of elements from bothseminomas and NSGCTs. Chromosomal and molecularstudies have shown that seminoma is capable ofdifferentiation into embryonic (embryonal carcinoma,teratoma) and extraembryonic (yolk sac tumor, choriocarcinoma) tissues (de Jong et al., 1990; Fogel et al.,1990). GCTs of all types are frequently associated withcarcinoma in situ (CIS). Therefore, both seminoma and
NSGCTs are thought to arise from cytologically identical
CIS lesions, indicating a common cell for all GCTs.
Seminoma and NSGCTs present with distinctive
clinical features, but they also differ with respect totherapy and prognosis (Moller, 1993; Adami et al.,1994). Seminoma, which is extremely radiosensitive andtends to remain localized for long periods, has the best
prognosis. More than 95% of patients with stage I or II
disease can be cured. Among NSGCTs, histologic
subtype does not inﬂuence the prognosis signiﬁcantly,
and hence they are treated as one group. Although
NSGCTs do not share the same prognosis as seminoma,
90% of patients with NSGCTs can achieve complete
remission with aggressive chemotherapy, and most canbe cured (Moller, 1993; Adami et al., 1994). With theavailability of effective treatment, it is important toclarify histological subtype in testicular tumors. However, clinical differentiation between various types oftesticular GCTs is imperfect because there are no
speciﬁc clinical features of the testicular masses produced by tumors of different histological types. Hence,
the immunohistochemical staining after total orchiectomy is very important for diagnosis, and the distinction
between seminoma and NSGCT is essential for treatment of testicular tumors.
Mouse Aggrus is a 44-kDa sialoglycoprotein (gp44),
which is expressed on the tumor cell surface with aplatelet aggregation-inducing ability (Watanabe et al.,1988). Our generated 8F11 monoclonal antibodyrecognizing mouse Aggrus could neutralize tumorinduced platelet aggregation (Watanabe et al., 1988).
Recently, we revealed that mouse Aggrus is identical toAggrus expression in seminoma
Y Kato et al8553

T1a/podoplanin, whose function in tumors is unknown(Kato et al., 2003). The molecules have putative
extracellular and transmembrane domains and a shortcytoplasmic tail with putative PKC and cAMP phosphorylation sites (Ma et al., 1998). Aggrus is highly
O-glycosylated and contains sialic acid (Ma et al., 1998),
and its expression was found in lymphatic endotheliumor alveolar type I cells (Wetterwald et al., 1996; Williams
et al., 1996). Sequence analysis indicates that Aggrus
does not share common domains with other protein
families, of known function, that predict its function.

Therefore, the physiological role of Aggrus has yet to be
determined. Recently, Ramirez et al. (2003) generated
T1a null mice containing a targeted mutation in the T1agene locus. Homozygous null mutant mice died immediately after birth, due to respiratory failure (Ramirez
et al., 2003). The T1a/ mice also had defects inlymphatic vasculature formation and were characterizedby congenital lymphedema (Schacht et al., 2003).
We previously cloned human Aggrus (Kato et al.,
2003; GenBank Accession No. AB127958). Althoughmouse and human Aggrus proteins have only 39%

Figure 1 Increased aggrus mRNA expression in testicular tumors. (a–c) Human aggrus mRNA expression in tumor (T) and normal(N) tissues in the same patient was assessed by hybridizing 32P-labeled human aggrus cDNA to the Cancer Proﬁling Array or Cancer
Proﬁling Array II (BD Biosciences) as described previously (Kato et al., 2003). The radioactivity of each dot was visualized (a) andquantiﬁed (b and c). (b) The relative aggrus mRNA expression level in each dot was normalized by measuring the radioactivity of each
dot after hybridization with 32P-labeled ubiquitin cDNA. (c) The T/N ratio was calculated by dividing normalized radioactivity oftumor samples with that of normal tissues in the individual patients. Bars, average of the human aggrus mRNA expression (b) and T/Nratio in each tissue (c). Statistical signiﬁcance of aggrus mRNA expression in normal and tumor tissues was performed using paired
t-test. Asterisk indicates the signiﬁcant difference: *Po0.05. The expression level of aggrus mRNA of tumor versus normal tissues in
the prostate, bladder, and kidney was not signiﬁcantly different (NS). (d) The aggrus transcripts in testicular tumor tissues (patients 11,
12, and 17) and normal testicular tissues (Normal-1 and -2) were quantiﬁed by performing real-time PCR. Total RNAs were preparedfrom frozen sections of patients 11, 12, and 17 employing an RNeasy mini prep kit (Qiagen Inc.). The ﬁrst-strand cDNA wassynthesized by Moloney murine leukemia virus reverse transcriptase (USB Corp.) via priming nine random oligomers according to the
manufacturer’s instructions. PCR was performed using the human aggrus sense (50 -GGAAGGTGTCAGCTCTGCTC-30 ), human
aggrus antisense (50 -CGCCTTCCAAACCTGTAGTC-30 ), human b-actin sense (50 -ACTCTTCCAGCCTTCCTTCCTG-30 ), andhuman b-actin antisense (50 -ATCTCCTTCTGCATCCTGTCGG-30 ) oligonucleotides. Real-time PCR was carried out using the
QuantiTect SYBR Green PCR (Qiagen). PCR conditions were 951C for 15 min (one cycle), followed by 40 cycles of 941C for 15 s, 551C
for 25 s, and 721C for 20 s. Subsequently, a melting curve program was applied, with continuous ﬂuorescence measurement. Standard
curves for aggrus and b-actin templates were generated by serial dilution of the PCR products (1  108 copies/ml to 1  103 copies/ml).
The expression level of aggrus was normalized by estimating the quantity of the b-actin transcript. The vertical bars represent s.d.
values of duplicate determinations
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Aggrus expression in seminoma
Y Kato et al8554

amino-acid identity, human Aggrus also had the ability
to induce platelet aggregation (Kato et al., 2003).
Moreover, the rat Aggrus homolog had platelet
aggregation-inducing capability (Kato et al., 2003).
Thus, under pathological conditions, Aggrus could be
involved in the platelet aggregation induced by tumor
cells. In most patients with intestinal tumors, Aggrus
mRNA and protein expression was enhanced (Katoet al., 2003). However, the role of Aggrus in GCTs has
never been reported. In the present study, we investigated the expression of Aggrus in the testicular tumors.

Expression of aggrus mRNA in testicular GCTs
We estimated human aggrus expression in humantumors using the Cancer Proﬁling Array II. In thisarray, normalized cDNA pairs from 160 primary tumor
tissues and corresponding normal tissues from individual patients were immobilized on a nylon membrane.
The increase in aggrus mRNA expression was observedin almost all testicular GCTs, compared with thecorresponding normal testicular tissue (Figure 1a andTable 1). After hybridization with 32P-labeled ubiquitin
cDNA, human aggrus expression level in each dot was
normalized (Figure 1b). The average expression levels ofhuman aggrus mRNA (relative aggrus/ubiquitin radioactivity) in testicular GCTs and normal tissue wereabout 7.6 and 1.8, respectively (Figure 1b). The aggrus
mRNA expression of tumor versus normal tissues intestis was signiﬁcantly increased (Po0.05). In contrast
to testicular GCTs, aggrus mRNA expression was

signiﬁcantly lower in prostate, bladder, and kidney
tumors (Figures 1a and b). Calculating T/N (tumor/
normal) ratios of aggrus mRNA expression in theindividual patients conﬁrmed the considerably elevatedexpression in testicular GCTs (Figure 1c). The averageT/N ratio was about 4.3 (n ¼ 10). We also estimatedhuman aggrus expression in intestinal tumors (colon andrectum) using the Cancer Proﬁling Array and found theelevated expression of aggrus in intestinal tumors whencompared with corresponding normal tissues (Figure 1c).
This result was consistent with our previous report using
Cancer Proﬁling Array II (Kato et al., 2003). The
average T/N ratios of human aggrus in colon (n ¼ 34)
and rectum (n ¼ 18) tumor patients were about 4.0 and
3.72, respectively. In contrast, T/N ratio in prostate(n ¼ 4), bladder (n ¼ 5), kidney (n ¼ 10), vulva (n ¼ 5),
trachea (n ¼ 3), liver (n ¼ 3), uterus (n ¼ 10), cervix(n ¼ 10), thyroid gland (n ¼ 10), skin (n ¼ 10), pancreas(n ¼ 7), and stomach (n ¼ 10) varied from patient topatient (Figure 1c), and the average expression ratios inthese organs were about 1.2, 1.3, 0.8, 2.2, 1.1, 1.9, 1.1,
1.8, 1.3, 1.3, 1.8, and 1.7, respectively. Thus, aggrus
might be a marker for not only intestinal tumors but
also testicular GCTs.
To conﬁrm the result of array analysis, we performed
real-time PCR with cDNA prepared from the frozen
tissues of three testicular tumor patients who underwent
surgery for testicular GCT in the Yamagata University
Hospital (patients 11, 12 and 17, Table 1) and normal
testicular tissues (Figure 1d). Two normal testis specimens (Normal-1 and -2) were obtained from patients
who underwent bilateral orchiectomy (castration).
Quantitative PCR analysis revealed that human aggrus

Table 1 Patient information of the samples used in this study
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

Year
Unknown
22
45
69
32
54
54
42
38
28
31
27
34
34
28
35
18
35
33
16
31
61
34
34
47

Race

Stage

Type

T/N

TT679

Caucasian
Asian
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
Asian
unknown

T1N0M0T2N0M0T1N0M0T1N0M0T1N0M0T1N0M0T1N0M0Unknown
Unknown
T3N0M0T2N2M0T1N1M1
Unknown
T2N0M0T2N2M0T1N0M1
T2N3M1
T1N0M0T1N0M1
T2N3M0T1N0M0T1N0M0T1N0M0T2N0M0T1N0M0Seminoma
SeminomaEC
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma
EC
EC
EC
EC
Seminoma
Seminoma
Seminoma
Seminoma
Seminoma

1.76
5.19
1.53
1.88
3.07
7.72
1.20
5.56
11.20
4.00
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D

N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive

EC: embryonal carcinoma, N/D: not determined
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Aggrus expression in seminoma
Y Kato et al8555

mRNA expression was clearly enhanced in testicular
GCTs derived from patients 11 and 12, but not frompatient 17. In the normal testicular tissue, almost no
aggrus mRNA expression could be detected. The
average amounts of aggrus mRNA in samples frompatients 11, 12, and 17 were about 80, 50, and 0.43 times
as much as those in normal testicular samples (Normal-1
and -2).

Expression of Aggrus protein in seminoma tissue
As shown in Figure 1d, aggrus mRNA expression was
detected in seminoma patients (patients 11 and 12,
Table 1), but not in an embryonal carcinoma patient(patient 17, Table 1). Thus, we checked the immunohistochemical reactivity of an anti-Aggrus polyclonal
antibody (TT679) to specimens from testicular GCTs.
We stained 15 testicular tumors (11 seminomas and four
embryonal carcinomas) (Table 1). As shown in Table 1,
this study included 14 patients who underwent surgery
for testicular GCT (from 1990 to 2002) in the Yamagata
University Hospital (patients 11–24) and one additional
human testicular tumor specimen purchased from
Novagen (patient 25). We found that 10 of the 11(90.9%) seminomas showed strong immunoreactivity to
TT679 (Figure 2a and Supplementary Figure 1). Only
patient 15 seminoma sample showed negative TT679
reactivity (Table 1 and Supplementary Figure 1).
Although seminomas are divided into three histologicalsubtypes (classical, anaplastic, and spermatocytic), all
the 11 seminomas studied in this study were diagnosed
as classical subtypes. Thus, its negative TT679 reactivity
was not dependent on histological subtypes. The reason
why patient 15 sample showed negative TT679 reactivity
should be solved in future. In contrast, no immunoreactivity was detected in the embryonal carcinomas(Figure 2a and Supplementary Figure 1). Furthermore,
Aggrus was not found in other NSGCTs, such as yolksac tumors (n ¼ 1) and teratomas (n ¼ 4) (data notshown). Western blot analysis conﬁrmed that Aggrus
protein was expressed in seminomas but not inembryonal carcinomas or normal testicular tissue(Figure 2b).
Aggrus is identical to lymphatic marker podoplanin(Wetterwald et al., 1996). We thus stained lymphatic
vessels in normal colorectal tissues by TT679 antibody
in combination with an antibody to lymphatic marker
Prox1 protein. Prox1-positive lymphatic endothelial
cells were strongly stained by TT679 antibody (Supplementary Figure 2). This result suggests that lymphatic
vessels expressed Aggrus/podoplanin on their surface.
Although seminoma tissues used in this study could be
stained by TT679 antibody, they could not be stained byan anti-Prox1 antibody (data not shown). Moreover,seminoma tissues showed no immunoreactivity toantibodies against other lymphatic marker proteins
LYVE-1 and VEGFR-3 (data not shown). These results
indicate that TT679 stained seminomas but not lymphatic vessels in seminoma tissues. NSGCTs have

Figure 2 Increased Aggrus protein expression in testicular tumors.
(a) Immunohistochemical staining of Aggrus in testicular tumors.
A polyclonal antibody to human Aggrus (TT679) was added to thedeparafﬁnized and rehydrated specimens (1 mg/ml) for 4 h at 231C.
Specimens were further developed using the DAKO CSA system(DakoCytomation Co.). Seminoma tissues from patients 11 and 12,
and embryonal carcinoma tissues from patients 17 and 18 (Table 1).
Bars, 10 mm. (b) Western blot analysis of Aggrus protein in
testicular tumors was performed as described previously (Katoet al., 2003). Tumor tissues from patient 11 (lane 1) and patient 17(lane 2), and normal testicular tissues of two prostate tumor
patients (Normal-1, lane 3; Normal-2, lane 4) were solubilized andimmunoblotted with an anti-human Aggrus polyclonal TT679
antibody (upper panel) or an anti-b-Actin antibody (Sigma, lower
panel)

clinically useful, speciﬁc markers such as AFP or
HCG. Seminomas used in this study could not be
stained by anti-HCG and anti-AFP antibodies (Figures
3b and c, respectively). These results indicate that
Aggrus could be a diagnostic marker for seminoma.
According to Izquierdo et al. (1995), c-kit protooncogene is highly expressed in 28 of the 28 seminomas.
However, nine of 29 NSGCTs (32%) also have
immunoreactivity to an anti-c-kit antibody. The tumor
antigen MAGE-A4, which can be recognized by tumorspeciﬁc cytolytic T lymphocytes, has been reported to be
expressed in seminomas but not in NSGCTs (Richie,2003). However, Cheville and Roche (1999) and
Jungbluth et al. (2000) reported that MAGE-A4positive seminomas were only 42 and 70%, respectively.
Our results suggest that the Aggrus protein was
speciﬁcally expressed in seminomas (10/11, 90.9%) but
not in embryonal carcinomas (0/4, 0%). Thus, Aggrus
might have an important role in the malignancies oftestis and be useful for diagnosis of seminoma. Further
studies, including careful clinical follow-ups of seminoma patients, are needed to evaluate the signiﬁcance ofAggrus in disease prognosis.
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Y Kato et al8556
Acknowledgements
This study was supported in part by a special grant for
Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We are
grateful to Dr Horiuchi, Department of Experimental andForensic Pathology, Yamagata University School of Medicine,Japan, for kind assistance.
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Figure 3 Immunohistochemical staining of Aggrus, AFP, and
HCG in seminomas. Seminoma specimens of patient 11 were
stained with an anti-human Aggrus antibody (TT679, a), an antiAFP antibody (DakoCytomation, b), and an anti-HCG antibody(DakoCytomation, c) at a concentration of 1 mg/ml for 4 h at 231C.
Then the specimens were further developed using the DAKOEnVision System (DakoCytomation). Bars, 100 mm

Oncogene