Virchows Arch (2014) 465:567–577 DOI 10.1007/s00428-014-1635-1
ORIGINAL ARTICLE
Expression pattern of clinically relevant markers in paediatric germ cell- and sex-cord stromal tumours is similar to adult testicular tumours Christiane Hammershaimb Mosbech & Terje Svingen & John Erik Nielsen & Birgitte Groenkaer Toft & Catherine Rechnitzer & Bodil Laub Petersen & Ewa Rajpert-De Meyts & Christina Engel Hoei-Hansen
Received: 10 April 2014 / Revised: 28 June 2014 / Accepted: 14 July 2014 / Published online: 30 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Paediatric germ cell tumours (GCTs) are rare and account for less than 3 % of childhood cancers. Like adult GCTs, they probably originate from primordial germ cells, but the pattern of histopathological types is different, and they occur predominantly in extragonadal sites along the body midline. Because they are rare, histology of paediatric GCTs is poorly documented, and it remains unclear to what extent they differ from adult GCTs. We have analysed 35 paediatric germ cell tumours and 5 gonadal sex-cord stromal tumours from prepubertal patients aged 0–15 years, to gain further knowledge, elaborate on clinical-pathological associations and better understand their developmental divergence. The tumours were screened for expression of stemness-related factors (OCT4, AP-2γ, SOX2), classical yolk sac tumours (YSTs; AFP, SALL4), GCTs (HCG, PLAP, PDPN/D2-40), C. H. Mosbech : T. Svingen : J. E. Nielsen : E. Rajpert-De Meyts : C. E. Hoei-Hansen Department of Growth and Reproduction, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark B. G. Toft Department of Pathology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark C. Rechnitzer Department of Paediatrics, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark B. L. Petersen Department of Pathology, University Hospital Roskilde, Roskilde, Denmark C. E. Hoei-Hansen (*) Department of Paediatrics, University Hospital Hillerød, Dyrehavevej 29, 3400 Hillerød, Denmark e-mail:
[email protected]
as well as markers for sex-cord stromal tumour (PDPN, GATA4). All YSTs expressed AFP and SALL4, with GATA4 present in 13/14. The majority of teratomas expressed SOX2 and PDPN, whereas SALL4 was found in 8/13 immature teratomas. Adult seminoma markers AP-2γ, OCT4, SALL4 and PDPN were all expressed in dysgerminoma. We further report a previously unrecognised pathogenetic relationship between AFP and SALL4 in YST in that different populations of YST cells express either SALL4 or AFP, which suggests variable differentiation status. We also show that AP2γ is expressed in the granulosa layer of ovarian follicles and weakly expressed in immature but not in mature granulosa cell tumours. Our findings indicate that the expression pattern of these antigens is similar between paediatric and adult GCTs, even though they develop along different developmental trajectories. Keywords AP-2γ/TFAP2C . GATA4 . Germ cell tumour . Paediatric . SALL4 . SOX2 . PDPN
Introduction Germ cell tumours (GCTs) account for about 3 % of cancers in children 0–16 years of age. They have received much less attention than adult GCTs. Although it has been postulated that both paediatric and adult GCTs arise from an immature germ cell, they differ in that histological subtypes vary in terms of age, site of presentation and genetic profile [1]. Consequently, knowledge gained from studies on adult GCTs does not necessarily apply to paediatric GCT. Paediatric GCTs develop directly from primordial germ cells (PGCs) without going through precursor stages [2, 3]. This is in contrast to adult testicular GCTs, which develop from pre-
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invasive carcinoma in situ (CIS) with exception of spermatocytic seminomas [1, 4, 5]. Likewise, tumours in dysgenetic gonads develop from CIS or from intermediate gonadoblastoma [6, 7]. To address a significant gap in available data, we studied clinical and histopathological characteristics of a large group of rare paediatric GCTs with a panel of markers, previously used to facilitate diagnosis of pre-invasive and gonadal and extragonadal GCTs in adults. These markers have only been sporadically evaluated in paediatric GCTs. We chose tumours located along the body midline, including malignant GCTs (dysgerminoma/seminoma and yolk sac tumours, YST) and benign GCTs (immature and mature teratomas) and included sex-cord stromal tumours (SCST; granulosa, Sertoli and Leydig cell tumours) for comparison. As histological markers, we chose stem cell-related transcription factors OCT4, AP-2γ (TFAP2C), and SOX2, YST markers such as alpha-fetoprotein (AFP) and SALL4, and GATA4, a sex-cord stromal marker. To study cell type, we used the primordial and neoplastic germ cell markers placental-like alkaline phosphatase (PLAP), podoplanin (PDPN) and the Sertoli/granulosa cell markers PDPN and anti-Müllerian hormone (AMH).
development with aminoethyl-carbazole substrate for 4– 20 min. Images were captured using NanoZoomer 2.0 HT scanner (Hamamatsu Photonics, Germany) and evaluated by CHM, JEN, TS and CEHH using a semi-quantitative system describing staining intensity (Table 3). Immunofluorescence experiments were carried out as previously described [9] in two steps using as fluorescent secondary donkey antibodies Alexa 488 labeled anti-mouse, anti-rabbit and anti-goat, Alexa 568 labeled anti-goat and anti-mouse and Alexa-647 labeled anti -rabbit (Life Techologies, Denmark) at 1:600 dilution. Sections were counterstained 15 s with 4,6-diamidino-2phenylindole (DAPI,Soln. 12; ChemoMetec A/S, Denmark), mounted using Prolong Gold Antifade reagent (P36930; Life Technologies) and imaged with an Olympus BX61microscope and captured using Cell Sense Dimensions V1.6 software (Olympus Ltd., UK). Image processing was with Adobe Photoshop.
Materials and methods
Clinical data were available from 27/37 patients (summarised in Table 1). Tumour TNM stage was 15 T1N0M0, 7 T2N0M0, 5 T3N0M0 (all pelvic/ovarian tumours), one T1N1M1 (sacrococcygeal mature teratoma), one T2N1M1 (sacrococcygeal YST) and one T3N1M1 (ovarian YST). Of the YSTs 12/13 presented with elevated serum (AFP range 446 to >60.500 μg/L). One patient with mature teratoma and one with immature teratoma also had elevated AFP levels. All patients underwent surgery; eight received chemotherapy (carboplatinum, etoposide and bleomycin), and one patient with a Sertoli–Leydig cell tumour received cisplatinum+ etoposide. Mean follow-up was 103 months (assessed August 2013). Complete remission was reached for 22 patients; four relapsed followed by complete remission, and one patient died. The two relapsing patients had a mature teratoma without detectable yolk sac elements, whereas the recurring tumours were YSTs.
Tissue samples Patients were between 0 and 15 years of age and diagnosed at Rigshospitalet in 1995–2010. Tumour specimens were collected following surgery, fixed in 4 % formalin, dehydrated and embedded in paraffin. Tissues were re-evaluated for this study by experienced pathologists (BLP and BGT). Patient records were reviewed by CHM and CEHH. We included 40 samples from 37 patients, consisting of 13 YSTs, 11 immature teratomas, 8 mature teratomas, 1 dysgerminoma, 2 mixed tumours (YST+IT, IT+MT), 3 juvenile granulosa cell tumours, 1 Sertoli–Leydig and 1 Sertoli cell tumour (Table 1). All pre-pubertal testicular tissues showed a histological pattern without spermatids. As control tissues, we used normal testis, adult testis with CIS, embryonal carcinoma and placenta. The study was approved by the Regional Committee for Medical Research Ethics in Denmark (no. H-4-2012-161). Histology and immunohistochemistry Tissue sections (4 μm) were haematoxylin and eosin (H&E) stained. Immunoperoxidase staining was performed as described previously [8] using the antibodies listed in Table 2. We used a three-layered protocol with biotinylated secondary antibodies goat anti-rabbit, goat anti-mouse (Histostain kits, 956143B and 956543B; Invitrogen, CA, USA) and donkey anti-goat (The Binding Site, AB360, UK), followed by colour
Results Distribution of the tumours, clinical course and treatment response
Yolk sac tumours We studied 13 YST specimens and one YST-element from a mixed tumour (Fig. 1). AFP was detected in all YSTs (Fig. 1, A; Table 3), mainly with positive staining of >50 % of the total area. SALL4 was detected in all YSTs (Fig. 1, B) with strong staining of the majority of neoplastic cells in 11 tumours and moderate staining of approximately one third of tumour cells in two YSTs. GATA4 showed nuclear staining in 13/14 specimens (Fig. 1, D and E) in both AFP-positive and AFPnegative cells (Fig. 1, I). PLAP strongly stained small areas
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Table 1 Clinical data Histology
Location
Sex Age in years
TNM
Serum markers at diagnosis (AFPb), μg/L
Follow Main up time, outcome monthsc
1
YST
Testis
M
6/12
T1N0M0
6,781
155
CR
2
YST
Testis
M
10/12
T1N0M0
8,334
154
CR
3
YST
Testis
M
10/12
T1N0M0
7,219
109
CR
4
YST
Testis
M
11/12
T1N0M0
446
70
CR
5
YST
Testis
M
1
T1N0M0
447
68
CR
6
YST
Testis
M
2
T1N0M0
4,104
116
CR
7
YST
Testis
M
3
T1N0M0
850
159
CR
8
YST
Ovary
F
12
T3N0M0
>60,500
87
CR
9
CR
YST
Ovary
F
14
T3N1M1
>60,500
95
10 YST
Ovary
F
15
T3N0M0
Normal
61
CR
11 YST
Vagina
F
1
T1N0M0
2,649
114
CR
12 IT
Testis
M
0 (1 day)
T1N0M0
Normal
170
CR
13 IT
Ovary
F
9
T2N0M0
Normal
90
CR
14 IT
Sacrococcygeal
F
0 (2 days)
n.a.
n.a.
145
CR
15 IT
Sacrococcygeal
M
0 (3 days)
n.a.
n.a.
136
CR
16 IT
Sacrococcygeal
F
0 (1 day)
n.a.
n.a.
45
CR
17 IT
Sacrococcygeal
F
8/12
T1N0M0
Normal
49
CR
18 IT
Retroperitoneal
F
3/12
n.a.
n.a.
39
CR
19 IT
Neck
M
0 (1 day)
n.a.
n.a.
126
CR
20 IT
Abdominal metastasis
F
12
n.a.
Died
n.a.
–
21 IT+MT metastasis Mediastinum+metastasis left lung F
8/12 metastasis 1 T2N0M0
n.a.
60
Relapse+CR
22 IT+relapsea
>60,500+140
123
Relapse+CR
Sacrococcygeal
F
2
T1N0M0
23 MT
Ovary
F
2
T3N0M0
Normal
72
CR
24 MT
Sacrococcygeal
F
0 (3 day)
n.a.
n.a.
76
CR
25 MT
Sacrococcygeal
F
0 (3 day)
n.a.
n.a.
77
CR
26 MT
Sacrococcygeal
F
0 (4 months)
n.a.
n.a.
66
CR
27 MT
Sacrococcygeal
F
1
T1N1M1
>60,500
50
CR
28 MT+YST relapse
Sacrococcygeal
M
2/12, relapse 2
T2N0M0+T2N0M0 Normal, relapse >60,500 189
Relapse+CR
29 MT+YST relapse
Sacrococcygeal
F
T2N0M0+T2N1M1 n.a., relapse 26,815
171
Relapse+CR
30 DG
Abdominal
F
0 (3 days), relapse 3 13
T3N0M0
Normal
121
CR
31 Mixed: YST+IT
Retroperitoneal
F
0 (2 days)
n.a.
n.a.
163
CR
32 Mixed: IT+MT
Ovary
F
8
T1N0M0
Normal
201
CR
33 Gra
Testis
M
1/12
T1N0M0
Normal
182
CR
34 Gra
Ovary
F
4
T2N0M0
Normal
51
CR
35 Gra
Ovary
F
12
n.a.
n.a.
77
CR
36 Ser-Ley
Ovary
F
6
T2N0M0
Normal
57
CR
37 Ser
Ovary
F
10
T1N0M0
Normal
52
CR
One boy had a primary tumour and a recurrent tumour with different histology, as was the case for two girls, which explains the mismatch between the number of patients and tumour samples. In one girl, tissue was available from the metastasis only and not from the primary tumour M male, F female, YST yolk sac tumour, IT immature teratoma, MT mature teratoma, DG dysgerminoma, Gra granulosa cell tumour, Ser Sertoli cell tumour, Ser-Ley Sertoli–Leydig cell tumour, n.a. not available, CR complete remission a
Specimens from the recurrent tumour not available
b
Only alpha-fetoprotein serum levels are noted as β-human choriogonadotropin (HCG) levels were normal for all patients
c
Follow up time was defined as time for diagnosis to last control session
in two YSTs (Fig. 1, C) and weakly a few cells in four additional tumours. PDPN staining turned out negative in all YSTs; only vascular structures stained (Fig. 1, G, arrow).
MAGE-A4 only showed cytoplasmic staining of prespermatogonia in remnant seminiferous tubules in YST of testicular origin, but tumour cells were negative (Fig. 1, H,
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Table 2 List of antibodies used in this study Antibody
Origin
Dilution
Retrieval buffer
Cat. no.
Source
AMH AFP HCG PLAP SALL4 OCT4 OCT4a AP-2γ GATA4 SOX2 PDPN MAGE-A4
Mouse Rabbit Rabbit Mouse Mouse Mouse Goat Mouse Goat Goat Mouse Mouse
1:150 1:10,000 1:10,000 1:100 1:400 1:100 1:200 1:40 1:250 1:250 1:100 1:800
Urea/TEG TEG TEG TEG TEG TEG TEG Urea/TEG CIT/TEG TEG CIT CIT
A008 A0231 M7191 sc-101147 sc-5279 sc-8629 sc-12762 sc-1237 AF2018 M3619 Clone 57B
Gift from R. Cate (France) DAKO, Hamburg, Germany DAKO, Hamburg, Germany DAKO, Hamburg, Germany Santa Cruz Biotechnology, CA, USA Santa Cruz Biotechnology, CA, USA Santa Cruz Biotechnology, CA, USA Santa Cruz Biotechnology, CA, USA Santa Cruz Biotechnology, CA, USA R&D systems, MN, USA DAKO, Hamburg, Germany Gift from G. Spagnoli (Switzerland)
a
Indicates the use of goat anti-OCT4 in respective figure panels to separate the use of two antibodies against OCT4. Where two retrieval buffers are listed, the former was used for peroxidase staining and the latter for IF experiments
inset). The mixed tumour (YST + immature teratoma) expressed AFP, SALL4 and GATA4 in yolk sac elements. The initial single-label immunoperoxidase experiments on serial sections of YST showed AFP and SALL4 expression in mutually exclusive regions. Double-label immunofluorescence experiments on single-sections confirmed that cells strongly expressing AFP express very little or no SALL4, while cells strongly expressing SALL4 rarely express AFP (Fig. 1, J–L). Immature and mature teratomas We examined 11 immature teratomas, eight mature teratomas, one mixed immature+mature teratoma and an immature teratoma element from a mixed YST+immature teratoma. The mature teratomas were all negative for most of the histological markers, but eight specimens showed PDPN staining typically in elements containing mesenchyme or squamous and columnar epithelium. Weak to moderate SOX2 expression was also observed in most mature teratomas. Heterogeneous histology of teratomas was also reflected in protein expression (Fig. 2, A). SOX2 was moderately expressed in all immature teratoma specimens mostly in primitive neuroepithelial and endodermal structures (Fig. 2, B). Small foci with AFP expression were seen in four immature teratomas (Fig. 2, C), and very few or even single OCT4-positive cells were noted in epithelial-like structures in four immature teratomas (Fig. 2, D). PDPN was expressed in undifferentiated structures in all but two immature teratomas, but also in developing cartilage (Fig. 2, E). AP-2γ, which was not detected in any other GCT type except dysgerminoma, was expressed in a subset of immature teratomas, typically in epithelial elements such as skin adnexal structures (Fig. 2, G and H) and also in granulosa cells of ovarian tumours (Fig. 2, I).
Dysgerminoma We found expression of OCT4, SALL4, AP-2γ and PDPN throughout the dysgerminoma from a 13-year-old girl (Fig. 3, A, B, E, F and G), whereas PLAP showed strong expression in ~70 % of the sample (Fig. 3, H). Strong HCG expression was observed in single syncytiotrophoblastic giant cells (Fig. 3, C and D). Weak GATA4 expession was observed in some regions (not shown). SALL4 expression was further validated by immunofluorescence (Fig. 3, I), as was cellular colocalisation of OCT4 and PDPN (Fig. 3, J–L). Sex-cord stromal tumours All SCSTs, three granulosa cell tumours, one Sertoli-Leydig and one Sertoli cell tumour, showed strong GATA4 expression (Fig. 4, A in a testicular granulosa cell tumour from a 1month-old boy and Fig. 4, B, in an ovarian Sertoli cell tumour from a 10-year-old girl). In the granulosa cell tumour, AMH was expressed in Sertoli cells of immature seminiferous tubules adjacent to the tumour (Fig. 4, C and G) and in regions localised around granulosa elements within the tumour (Fig. 4, D and H). AMH expression was not observed in the Sertoli cell tumour (Fig. 4, I and J). Strong PDPN expression was evident in vascular structures between the testicular cords, with weaker cytoplasmic staining of the Sertoli cells (Fig. 4, E), whereas Sertoli-derived neoplastic cells of the less differentiated tumour showed stronger PDPN staining, albeit not as strong as interspersed vascular structures (Fig. 4, F). Both tumours displayed robust GATA4 expression in the Sertoli/ granulosa cell regions (Fig. 4, G–J). Protein expression in the entire panel of specimens is summarised in Table 3. Table 4 lists marker expression profiles in paediatric tumours from previously published studies.
N
M F
AFP
13 3
9
1
IT
MT
Dysgerminoma
1
0
0
3
1
1
1
1
2
1
0/1 negative
0/1 negative
0/3 negative
0/1 negative
14/14 ++/+++ 10–90 % 10 4/13 +/+++ 1–20 % 8 0/9 negative
6
0/9 negative
6/14 +/+++
