- Email: [email protected]
Ovarian Brenner tumour: A morphologic and immunohistochemical analysis suggesting an origin from fallopian tube epithelium
European Journal of Cancer (2013) 49, 3839–3849
Available at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Ovarian Brenner tumour: A morphologic and immunohistochemical analysis suggesting an origin from fallopian tube epithelium Elisabetta Kuhn a,⇑, Ayse Ayhan d, Ie-Ming Shih a,b,c, Jeffrey D. Seidman e, Robert J. Kurman a,b,c a
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA Department of Gynecology and Obstetrics, The Johns Hopkins Medical Institutions, Baltimore, MD, USA c Department of Oncology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA d Department of Pathology, Seirei Mikatahara Hospital, Hamamatsu, Japan e Department of Pathology and Laboratory Medicine, Washington Hospital Center, Washington, DC, USA b
Available online 5 September 2013
KEYWORDS Brenner tumour Pathogenesis Histogenesis Ovarian tumour Metaplasia Transitional Tuboperitoneal junction
Abstract Background: Brenner tumours (BTs), like other epithelial ovarian tumours, are thought to develop from the ovarian surface epithelium. Aim and Methods: We hypothesised that BTs arise from transitional metaplasia near the tuboperitoneal junction which, when embedded in the ovary as Walthard cell nests, may progress to BTs. The aim of this study was to validate this hypothesis by a morphologic and immunohistochemical (IHC) analysis. Results: The IHC analysis revealed that fallopian tube secretory cells, transitional metaplasia, Walthard cell nests and the epithelial component of BTs shared a similar IHC profile, consistently expressing AKR1C3 (an enzyme involved in androgen biosynthesis) and androgen receptor, but not calretinin. The tumour stromal cells that immediately surrounded the epithelial nests showed strong expression of calretinin, inhibin and steroidogenic factor 1 (markers of steroidogenic cells) in the majority of BTs. Using a highly sensitive immunofluorescent staining method, we detected small groups of cilia in transitional metaplasia and Walthard cell nests, multifocal stretches of cilia and/or ciliated vacuoles in benign BTs and well-developed cilia in atypical proliferative BTs. Conclusions: Our findings suggest a tubal origin of BTs through transitional metaplasia and Walthard cell nests, based on their anatomic proximity, similar IHC profile and the presence of cilia. In addition, we hypothesise a role of androgenic stimulation in the pathogenesis of BT, based on the IHC staining pattern of calretinin, inhibin and steroidogenic factor 1 expressed in the luteinised stromal cells surrounding the epithelial nests of the tumours, and
⇑ Corresponding author: Address: Department of Pathology, Johns Hopkins University, 1550 Orleans Street, CRBII, Room 376, Baltimore, MD 21231, USA. Tel.: +1 410 502 7774; fax: +1 410 502 7943. E-mail address: [email protected] (E. Kuhn).
0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.08.011
3840
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
AKR1C3 and androgen receptor expressed in both the epithelial and stromal components. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Described by Brenner in 1907 as oophoroma folliculare ovarii [1], Brenner tumours (BTs) account for 5% of benign ovarian tumours. They are composed of nests of epithelial cells resembling urothelium, which are surrounded by dense fibromatous stroma and are believed to arise from the ovarian surface epithelium (OSE) that undergoes transitional-type metaplasia. In recent years the origin of ovarian epithelial tumours, as being primarily derived from the OSE, has been challenged. It is now believed that most serous tumours are derived from fallopian tube epithelium (FTE), and that endometrioid and clear cell tumours, which are derived from endometriosis, ultimately originate from endometrial tissue by retrograde menstruation [2,3]. This new model of ovarian carcinogenesis is consistent with the Mu¨llerian-type appearance of serous, endometrioid and clear cell neoplasms. However, the epithelium of BTs does not display a Mu¨llerian phenotype and therefore their origin remains enigmatic. We previously described the junction between the FTE and the mesothelium of the tubal serosa, the socalled ‘tuboperitoneal junction’, and reported that transitional cell metaplasia (TM) is frequently found in this location [4–6]. We hypothesised that BTs arise from TM which, when it invaginates into the paratubal or ovarian tissue, is termed a ‘Walthard cell nest’ (WC). When embedded in the ovary these nests develop into a BT. In keeping with this hypothesis, Seidman et al. found an association of WCs with BT and identified that WCs are 79% more common in women with BT compared with controls (50% and 28%, respectively) [6]. However, this association was partially mitigated by the fact that the identification of WCs was correlated with increased tissue sampling [6]. The aim of the present study was to evaluate the morphologic and immunohistochemical features of tuboperitoneal junction, TM, WCs, BTs and atypical proliferative BTs, in an attempt to more clearly understand their relationship.
available slides were reviewed and clinicopathological data collected. This study was approved by the Institutional Review Board of the Johns Hopkins Medical Institutions. 2.2. Immunohistochemistry Immunohistochemistry (IHC) was performed using the primary antibodies and conditions shown in Table 1. Formalin-fixed, paraffin-embedded sections were immunostained using protocols described previously [7–9] and detailed in the Supporting information, Supplementary methods. 2.3. Description of the selected antibodies PAX2, PAX8 and WT1 are markers of Mu¨llerianlineage expressed by secretory cells of the fallopian tube [10–12]. CEA is a sialomucin present in mucin-producing cells, that has been identified in BTs but never studied in TM or WCs. Expression of the sex-steroid receptors androgen receptor (AR), oestrogen receptor (ER) and progesterone receptor (PR) was examined because they are the main effectors of sex-steroid hormone action. Calretinin, inhibin and steroidogenic factor 1 (SF1) were selected as markers of sex-cord differentiation and steroidogenesis. Calretinin is also a reliable marker for mesothelium. In order to gain insight into which cells are capable of sex-steroid hormone production, we immunostained for CYP19 (aromatase) and AKR1C3 (HSD17 b5), key enzymes in the production of oestrogens and androgens respectively [13,14]. 2.4. Immunofluorescence Cilia were visualised with double-colour immunofluorescent staining with a-tubulin (green) for microtubules and c-tubulin (red) for basal bodies using a protocol described in the Supporting information, Supplementary methods. 3. Results
2. Materials and methods 3.1. Morphologic and IHC findings 2.1. Case selection A total of 17 ovaries, 38 fallopian tubes, 25 peritoneal mesothelium, 17 TMs, 56 WCs, 50 BTs and seven atypical proliferative BTs were retrieved from the pathology files of the Johns Hopkins Hospital (Baltimore, MD), Washington Hospital Center (Washington, DC) and Seirei Mikatahara Hospital (Hamamatsu, Japan). All
The detailed correlation of the morphologic and IHC findings is shown in Table 2 and representative images in Figs. 1–3. 3.1.1. Ovary 3.1.1.1. Ovarian stroma. It is composed of densely packed spindle cells with scant cytoplasm except
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3841
Table 1 Primary antibodies, sources and conditions used in this study. Antibody
Symbol
Clone
Vendor
Address
Cat#
Dilution
Stain type
Instrument
Aldo–keto reductase family 1, member C3 Androgen receptor
AKR1C3
NP6.G6.A6
St. Louis, MO
LS-B2295
1:10000
Manual
NA
AR
ER179
Sigma– Aldrich Epitomics
3184-1
1:400
Manual
NA
Calretinin
CRT
Polyclonal
Cell Marque
232A-78
Predilute
Automated
Carcinoembryonic antigen
CEA
TF-3H8-1
Ventana
Burlingame, CA Hot Springs, AK Tucson, AZ
760-2507
Predilute
Automated
Carcinoembryonic antigen
CEA
Polyclonal
Dako
A0115
1:500
Automated
Cytochrome P450 Aromatase Oestrogen receptor
CYP19 ER
H4 6F11
Manual Automated
INH PAX2 PAX8 PR
Polyclonal Polyclonal 3G11 16
1:25 1:100 1:100 Predilute
Automated Automated Automated Automated
Steroidogenic factor 1 Wilms tumour 1
SF1 WT1
N1665 6F-H2
MCA2077S RTU-ER6F11 0100-0549 71-6000 10336-1-ap RTUPGR-312 434200 348M-98
1:800 Predilute
Alpha-inhibin Paired box 2 Paired box 8 Progesterone receptor
AbD Serotec Leica Microsystems AbD Serotec Invitrogen Invitrogen Leica Microsystems Invitrogen Cell Marque
1:100 Predilute
Automated Automated
BenchMark XT BenchMark XT BenchMark XT NA BenchMark XT Bond Max Bond Max Bond Max BenchMark XT Bond Max BenchMark XT
immediately surrounding follicles where the theca cells are larger and contain more abundant cytoplasm. In non-specific ovarian stroma WT1 was positive in 5/17 (29%), calretinin in 11/17 (65%), inhibin in 10/16 (63%) and SF1 in 15/15 (100%) cases. ER was expressed in 13/18 (72%), PR in 16/19 (84%), whereas AR in 16/16 (100%) cases. AKR1C3 was present in 6/9 (67%) cases but CYP19 was not detected. CYP19 was consistently strongly positive in granulosa-lutein cells of the corpus luteum, granulosa cells, theca interna cells and AKR1C3 in theca cells and luteinised stromal cells. 3.1.1.2. Ovarian surface epithelium. It was composed of flattened cells with elongated bland nuclei which, in foci, were cuboidal. Occasionally short stretches of ciliated epithelium were found between the predominant population. WT1 was positive in 16/16 (100%), calretinin in 15/16 (94%), ER in 10/16 (63%), PR in 11/17 (65%) and AR in 0/15 cases. Inhibin was negative in all cases but SF1 was positive in 10/15 (67%) cases. AKR1C3 and CYP19 were negative in all cases. 3.1.2. Peritoneal mesothelium Peritoneal mesothelium was composed of flattened epithelium; ciliated cells were not identified. The only positive markers were WT1 in 23/24 (96%) and calretinin in 23/23 (100%) cases. 3.1.3. Fallopian tube epithelium 3.1.3.1. Secretory cells. These cells were columnar with bland nuclei arranged perpendicular to the basement membrane and were interspersed with ciliated cells.
Carpinteria, CA Raleigh, NC Bannockburn, IL Raleigh, NC Carlsbad, CA Carlsbad, CA Bannockburn, IL Carlsbad, CA Hot Springs, AK
All cells were positive for PAX2, PAX8 and WT1. Except for a single case (3%) that was positive for calretinin, all the other cases were negative for calretinin, inhibin and SF1. ER was expressed in 37/37 (100%), PR in 31/38 (82%) and AR in 31/31 (100%) cases. AKR1C3 was present in 16/16 (100%) cases, but CYP19 was not detected. 3.1.3.2. Ciliated cells. These cells, like secretory cells, are columnar and arranged perpendicularly to the basement membrane. They contain large oval to round nuclei and multiple apical cilia. PAX2 was not expressed whereas PAX8 was expressed in 2/26 (8%) and WT1 in 30/35 (86%) cases. Calretinin, inhibin and SF1 were not detected. ER was detected in 37/37 (100%), PR in 31/38 (82%) and AR in 1/31 (3%) cases. AKR1C3 was present in 16/16 (100%) cases but CYP19 was not detected. 3.1.4. Transitional metaplasia The cells are identical to those that comprise WCs (see below). These small foci of cells are located on the tubal serosa and are generally smaller than WCs. They are frequently found near the tuboperitoneal junction. PAX2 was present in 1/9 (11%), PAX8 in 2/5 (40%) and WT1 in all nine (100%) cases. Calretinin, inhibin and SF1 were not detected. ER and PR were expressed in 1/10 (10%), whereas AR in 9/9 (100%) cases. Moderate AKR1C3 was present in 4/4 (100%) cases but CYP19 was not detected. 3.1.5. Walthard cell nests As noted above the cells in WCs are identical to those that comprise TM with bland nuclei, often
3842
Table 2 Immunohistochemical results. Antibody
Gene Symbol
Markers of mucinous differentiation Carcinoembryonic CEA antigen monoclonal Carcinoembryonic CEA antigen polyclonal
OSE Pos/tot (%)
Stroma Pos/tot (%)
3/12 (25) 3/16 (19) 16/16 (100)
0/14 (0) 0/16 (0) 5/17 (29)
0/6 (0) 0/6 (0)
Fallopian tube epithelium
Transitional metaplasia Pos/tot (%)
Walthard cell nest Pos/tot (%)
Benign Brenner tumour
Atypical proliferative Brenner tumour
Epithelium Pos/tot (%)
Stroma Pos/tot (%)
Epithelium Pos/tot (%)
Stroma Pos/tot (%)
Secretory cells Pos/tot (%)
Ciliary cells Pos/tot (%)
0/23 (0) 3/15 (20) 23/24 (96)
34/34 (100) 26/26 (100) 35/35 (100)
0/34 (0) 2/26 (8) 30/35 (86)
1/9 (11) 2/5 (40) 9/9 (100)
1/37 (3) 2/27 (7) 32/48 (67)
0/38 (0) 0/40 (0) 4/41 (10)
0/38 (0) 0/40 (0) 13/41 (32)
0/3 (0) 0/3 (0) 0/3 (0)
0/3 (0) 0/3 (0) 3/3 (100)
0/6 (0)
0/4 (0)
0/7 (0)
0/7 (0)
NA
NA
1/16 (6)
0/16 (0)
NA
NA
0/6 (0)
0/4 (0)
0/7 (0)
0/7 (0)
1/1
3/4 (75)
17/17 (100)
0/17 (0)
NA
NA
11/17 (65) 10/16 (63) 15/15 (100)
23/23 (100) 0/24 (0) 0/20 (0)
1/30 (3) 0/30 (0) 0/25 (0)
0/30 (0) 0/30 (0) 0/25 (0)
0/7 (0) 0/7 (0) 0/6 (0)
0/38 (0) 0/41 (0) 0/33 (0)
0/42 (0) 0/42 (0) 0/41 (0)
35*/42 (83) 34*/42 (81) 39*/41 (95)
0/4 (0) 0/4 (0) 0/1 (0)
4*/4 (100) 4*/4 (100) 1*/1 (100)
0/7 (0)
6/9 (67)
0/6 (0)
16/16 (100)
16/16 (100)
4/4 (100)
21/21 (100)
26/26 (100)
23/26 (88)
7/7 (100)
7/7 (100)
0/4 (0)
0/5 (0)
0/4 (0)
0/12 (0)
0/12 (0)
0/3 (0)
0/15 (0)
0/25 (0)
2/25 (8)
0/5 (0)
2/5 (40)
16/16 (100) 13/18 (72) 16/19 (84)
0/19 (0) 0/25 (0) 0/25 (0)
31/31 (100) 37/37 (100) 31/38 (82)
1/31 (3) 37/37 (100) 31/38 (82)
9/9 (100) 1/10 (10) 1/10 (10)
43/49 (88) 4/55 (7) 2/56 (4)
27/29 (93) 2/43 (5) 2/44 (5)
26/29 (90) 33/43 (77) 39/44 (89)
7/7 (100) 0/4 (0) 0/4 (0)
7*/7 (100) 3/4 (75) 4/4 (100)
Markers of sex-cord differentiation/steroidogenesis Calretinin CRT 15/16 (94) Inhibin alpha INH 0/15 (0) Steroidogenic factor 1 SF1 10/15 (67) Markers of sex-steroid enzymes Aldo–keto reductase AKR1C3 family 1, member C3 Cytochrome P450 CYP19 Aromatase
Mesothelium Pos/tot (%)
Markers of sex-steroid hormone receptors Androgen receptor AR 0/15 (0) Oestrogen receptor ER 10/16 (63) Progesterone receptor PR 11/17 (65)
Pos, total positive cases; Tot, total cases analysed; OSE, ovarian surface epithelium. PAX2, PAX8, WT1, SF1, AR, ER and PR are nuclear markers. CEA is a cytoplasmic and extracytoplasmic staining. On the other hand, INH is cytoplasmic, whereas CRT, AKR1C3 and CYP19 are both nuclear and cytoplasmic. Usually all the markers, when positive, are diffusely positive in the majority (>80%) of the cells, except for these markers that are variably positive in the stromal component (>10% of cells) with typical condensation around the transitional-like epithelial nests.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
Markers of Mu¨llerian-lineage Paired box 2 PAX2 Paired box 8 PAX8 Wilms tumour 1 WT1
Ovary
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3843
Fig. 1. Representative images showing the histological appearance of ovarian surface epithelium (OSE), mesothelium, fallopian tubal epithelium (FTE), transitional cell metaplasia (TM) and Walthard cell nest (haematoxylin–eosin staining, HE), and the expression of calretinin (CRT), WT1, PAX2, PAX8, androgen receptor (AR), oestrogen receptor (ER), progesterone receptor (PR) and AKR1C3 in each epithelium.
displaying a longitudinal groove and showing transitional-type differentiation. They may be solid or cystic. PAX2 was present in 1/37 (3%), PAX8 in 2/27 (7%) and WT1 in 32/48 (67%) cases. Calretinin, inhibin and SF1 were not detected. ER was expressed in 4/55 (7%) and PR in 2/56 (4%) whereas AR in 43/49 (88%) cases.
All cases analysed showed moderate expression of AKR1C3 and negativity for CYP19. 3.1.6. Benign Brenner tumours Forty-two BTs measuring between 1.1 and 21 cm (average 7.3 cm), and seven microscopic tumours (range 0.3–0.7 cm, average 0.5 cm) were examined.
3844
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
Fig. 2. Histological appearance of benign Brenner tumour (haematoxylin–eosin, HE) and immunohistochemical staining. The tumour is composed of nests of epithelial cells resembling urothelium, surrounded by a prominent fibromatous stroma (HE). The female sex-steroid receptors, oestrogen receptor (ER) and progesterone receptor (PR), show nuclear positivity in the stromal component, but are completely negative in the transitional nests. Androgen receptor (AR) is strongly positive in both epithelium and stroma. CYP19 (aromatase) is negative in the epithelial nests and stroma. WT1 is weakly positive in the epithelial and stromal component. Alpha-inhibin (INH), calretinin (CRT) and steroidogenic factor 1 (SF1) (markers of sex-cord differentiation and steroidogenesis) are positive in the stromal component. Notably, CRT, INH and SF1 show strong immunopositivity in the stromal cells that are in close proximity to the epithelial nests, which are negative for these markers. AKR1C3 (type 5,17-b-hydroxysteroid dehydrogenase which locally converts androstenedione to testosterone) shows strong positivity in the nuclei and cytoplasm in the epithelial cells component and weak positivity in the stroma.
3.1.6.1. Epithelial component. These tumours contained nests of cells identical to those in TM and WCs. In addition, cells with mucinous epithelium were frequently found in the centre of the nests.
Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 4/41 (10%) cases. Calretinin, inhibin and SF1 were not detected. ER was expressed in 2/43 (5%) and PR in 2/44 (5%) whereas AR in 27/29 (93%) cases.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3845
Fig. 3. High magnification of an atypical proliferative Brenner tumour, showing a papillary frond of transitional epithelium with a delicate stromal stalk (haematoxylin–eosin, HE). The tumour epithelium shows diffuse, moderate expression of androgen receptor (AR), but is completely negative for oestrogen receptor (ER), progesterone receptor (PR), alpha-inhibin (INH) and calretinin (CRT) (the latter two are markers of sex-cord differentiation and steroidogenesis). INH and CRT, as well as WT1, are weakly positive in the tumour stroma. In contrast AKR1C3 is intensely positive in the epithelium and weakly positive in the stroma.
Intense AKR1C3 was detected in 26/26 (100%) cases but CYP19 was not detectable in any case. 3.1.6.2. Stromal component. It is very similar to ovarian stroma, but stromal cells that are closely applied to the epithelial nests are plumper, suggesting that they are luteinised. Stroma, distant from the nests, are more fibrotic and frequently contain spiculated calcification (29/50 cases, 58%). Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 13/41 (32%) cases. Calretinin was positive in 35/42 (83%), inhibin in 34/42 (81%) and SF1 in 39/41 (95%) cases. ER was expressed in 33/43 (77%), PR in 39/ 44 (89%) whereas AR in 26/29 (90%) cases. AKR1C3 was present in 23/26 (88%) and CYP19 in 2/25 (8%) cases.
3.1.7. Atypical proliferative Brenner tumours Six (86%) of seven atypical proliferative BTs were macroscopically solid and cystic, and the remaining was solid. The average size was 11.9 cm (range 6–22 cm). 3.1.7.1. Epithelial component. It was composed of large, irregularly elongated nests, sometimes forming thick papillae. The epithelial cells had a transitional-type appearance, similar to benign BTs and TM. Welldeveloped cilia were multifocally identified in 6/7 (86%) cases. Diffuse mucinous metaplasia was prominent in 5/7 (71%) cases and focal squamous metaplasia was present in 4/7 (57%) cases. A minor component of benign BT was also present in 5/7 (71%) cases, which directly abutted the atypical proliferative component.
3846
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
The only positive markers were AKR1C3 and AR, which were positive in 7/7 (100%) cases. 3.1.7.2. Stromal component. The stromal cells that were closely apposed to the epithelial nests were plumper and had more rounded vesicular nuclei with an occasional single small nucleolus compared to the more fibromatous stromal cells that were further located from the nests. Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 3/3 (100%) cases. Calretinin and inhibin were positive in 4/4 (100%) cases, and SF1 in the only case analysed. ER and PR were expressed in 3/4 (75%) and 4/4 (100%) cases respectively, whereas AR and AKR1C3 in 7/7 (100%) cases analysed. CYP19 was detected in 2/5 (40%) cases. Table 3 Presence of cilia differentiation based on double-colour immunofluorescence staining for a-tubulin and c-tubulin. Lesion
Total
Cilia sketches/ ciliated vacuoles
Welldeveloped cilia
n (%)
n (%)
Transitional metaplasia Walthard cell nest Benign Brenner tumour Atypical proliferative Brenner tumour
17 35 17 6
8 (47) 9 (26) 17 (100) 1 (17)
0 0 0 5
(0) (0) (0) (83)
3.2. Immunofluorescence findings Based on our morphologic findings of cilia in the majority of atypical proliferative BTs we applied immunofluorescent staining for a-tubulin (green) and c-tubulin (red) in an effort to detect cilia and basal bodies with greater sensitivity than by haematoxylin–eosin morphology (Table 3). We observed multiple cilia, characterised by superficial filamentous structures of a-tubulin rising from basal bodies of c-tubulin, in all 35 tubes analysed (Fig. 4). Secretory cells did not display diffuse apical cilia formation and multiple basal bodies, but did show a single centrosome in the apical portion of the cell and occasionally stretches of cilia or vacuoles containing cilia (Fig. 4). At the tuboperitoneal junction, features of progressive attenuation of cilia formation from tubal ciliary epithelium to TM were occasionally identifiable (Fig. 5). Very small groups of cilia, rising from apical centrosome structures (Fig. 5), were detected in 8/17 (47%) TMs, and in 9/35 (26%) WCs. In addition, all 17 benign BTs showed multifocal stretches of cilia and/or ciliated vacuoles (Fig. 5). Finally, 5/6 (83%) atypical proliferative BTs showed well-developed cilia (Fig. 5), with the remaining 1/6 (17%) showing only stretches of cilia. 4. Discussion Benign BTs are biphasic neoplasms composed of nests of transitional-type epithelium surrounded by a
Fig. 4. Representative images of tubulin distribution in normal-appearing tubal epithelium, using a double immunofluorescence staining for a- and c-tubulin. (A) Tubal ciliary epithelium shows multiple cilia as apical filamentous structures (a-tubulin in green) arising from a continuous line of basal bodies (c-tubulin in red). (B) Higher magnification. (C) Secretory cells show the absence of multiple apical cilia and multiple basal bodies, but apical presence of a single centrosome. (D) Occasionally, in the secretory cells, there are stretches of cilia appearing as short, single or sometimes a few filamentous a-tubulin tufts emerging from apical centrosomes (white arrow), or basal ciliated vacuoles of concentrically located cilia appearing as basal rounded structures (yellow arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3847
Fig. 5. Representative images of tubulin distribution in the epithelium of transitional metaplasia, Walthard cell nest (WC), Brenner tumour (BT) and atypical proliferative BT using a double immunofluorescence staining for a-tubulin (green) and c-tubulin (red). (A) At the tuboperitoneal junction transitional metaplasia on left side contains attenuated cilia formation (arrow) in contrast to the obvious tubal cilia on the right. (B) Transitional metaplasia and (C) WCs present very focal evidence of cilia (green), rising from an apical single centrosome (red) (arrows). (D and E) Benign BT shows multifocal cilia stretches (white arrow) and/or ciliated vacuoles (yellow arrow). (F) Atypical proliferative BT shows welldeveloped cilia as luminal microtubules tufts (green) arising from a continuous line of basal bodies (red). (G) Higher magnification. Insert: mitotic figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
densely fibromatous stroma. A variety of theories have been proposed for their origin but the most widely accepted is derivation from OSE that has undergone transitional-type metaplasia. Recently, we drew attention to the junction between the epithelium of the fimbria and the mesothelial serosa of the tube, the so-called ‘tuboperitoneal junction’, and suggested that, like other junctions between different types of epithelia, this might be a ‘hot spot’ for carcinogenesis. We also noted the frequent presence of transitional-type epithelium in close proximity to the junction. Foci of TM are typically quite small but when they are large they are termed ‘Walthard cell nests’. In other words, TM and WCs are one and the same, the latter just larger. Accordingly, in this discussion we use the term ‘transitional metaplasia’ to include both the small foci of TM and WCs. Morphologically, TM and the epithelium of BTs are virtually identical. BTs differ by their presence in the
ovary, their associated fibromatous stroma and focal mucinous changes. Our IHC analysis was undertaken to determine whether the origin of BTs is mesothelial or Mu¨llerian. Our findings are somewhat puzzling. For example, although TM and the epithelial component of BTs did not express calretinin, unlike peritoneal mesothelium and OSE, they both failed to express the Mu¨llerian markers PAX2 and PAX8. Notably, these markers are also absent in tubal ciliated cells. On the other hand, our studies demonstrated cilia formation in FTE and BTs, suggesting a close association with Mu¨llerian epithelium as opposed to mesothelium. In line with our findings, Tang and Kang have identified the presence of desmosome-tonofilament complex and cilia at an ultrastructural level in malignant BTs, and concluded that they are derived from Mu¨llerian epithelium that has differentiated into squamous epithelium similar to vaginocervical epithelium [15]. We also found that when cilia were present in the OSE, an immunopheno-
3848
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
type identical to FTE was also present, suggesting that ciliated epithelium may have resulted from implantation of tubal epithelium on the ovarian surface [16]. Therefore, we propose that BTs are derived from FTE which, following trauma and regeneration, loses its Mu¨llerian phenotype as it transdifferentiates into transitional cell epithelium [15,17]. When metaplastic transitional epithelium is implanted into the ovary, hormonal stimulation may lead to the reacquisition of cilia [18,19]. Although the possibility that BTs derive from OSE undergone transitional-like metaplastic change cannot be entirely dismissed. By IHC we identified steroid hormone-producing and hormone-responsive cells using antibodies to inhibin, calretinin and SF1, hormone receptors (AR, ER and PR), and enzymes involved in hormone steroidogenesis, AKR1C3 and CYP19, suggesting hormonal biosynthesis of the epithelial and stromal components of BTs [20,21]. AKR1C3 is of particular interest as it has been demonstrated that it regulates local production of androgens, oestrogens and progestins in the prostate and breast tissues by binding to their respective nuclear receptors [22]. In fact, AKR1C3 is expressed in higher levels in normal prostate epithelial cells than in the stroma implying that reductive androgen metabolism occurs preferentially in epithelial cells. Furthermore, in the breast AKR1C3 converts androstenedione to testosterone, which is then aromatised by CYP19 to estradiol. AKR1C3 is highly expressed in intraductal and invasive ductal carcinomas of the breast. Thus, the findings in the present study of high expression of AKR1C3 in FTE, TM and BTs indicate that they have the capacity to synthesise androgens. This provides further evidence that TM and BTs are linked to FTE as both OSE and peritoneal mesothelium do not express AKR1C3. In addition, among the various hormone receptors, AR was consistently more highly expressed than ER and PR in secretory cells, TM and BTs. These findings and the absence of CYP19 indicate a more important role for androgens than oestrogens in the development of BTs. Taken together, our findings suggest that following implantation of transitional metaplastic epithelium into the ovary, the ovarian stroma may become activated to produce steroid hormones, which may then be converted by both epithelium and stroma to androgens, and further stimulate epithelium-stroma growth and lead to the development of a BT. In conclusion, this study provides morphologic and immunohistochemical evidence suggesting that, like serous ovarian tumours, BTs may arise from FTE that has undergone TM and has seeded the ovary. Furthermore, our findings confirm the role of the ovarian stroma in tumour development which, by synthesising steroid hormones locally, can stimulate growth of the implanted epithelium, a proposal put forth by Scully 50 years ago [20]. This is an intriguing tumourigenic mechanism that
appears to apply to other ovarian tumours (unpublished data) and may, therefore, provide a novel therapeutic target using antiandrogenic drugs [20,23]. Conflict of interest statement None declared. Acknowledgements The authors thank Ms. Asli Bahadirli-Talbott and Ms. Nichelle Gray for their excellent technical assistance. This study was supported by OSB1 grant from HERA foundation, and CDMRP Grant (No. OC100517) from the US Department of Defense. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2013.08.011. References [1] Brenner F. Das Oophoroma Folliculare. Frankfurt Z Pathol 1907;1:150. [2] Kuhn E, Kurman RJ, Shih IM. Ovarian cancer is an imported disease: fact or fiction? Curr Obstet Gynecol Rep 2012;1:1–9. [3] Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory. Am J Surg Pathol 2010;34:433–43. [4] Seidman JD, Yemelyanova A, Zaino RJ, et al. The fallopian tubeperitoneal junction: a potential site of carcinogenesis. Int J Gynecol Pathol 2011;30:4–11. [5] Rabban JT, Crawford B, Chen LM, et al. Transitional cell metaplasia of fallopian tube fimbriae: a potential mimic of early tubal carcinoma in risk reduction salpingo-oophorectomies from women With BRCA mutations. Am J Surg Pathol 2009;33:111–9. [6] Seidman JD, Khedmati F. Exploring the histogenesis of ovarian mucinous and transitional cell (Brenner) neoplasms and their relationship with Walthard cell nests: a study of 120 tumors. Arch Pathol Lab Med 2008;132:1753–60. [7] Kuhn E, Kurman RJ, Sehdev AS, et al. Ki-67 labeling index as an adjunct in the diagnosis of serous tubal intraepithelial carcinoma. Int J Gynecol Pathol 2012;31:416–22. [8] Kuhn E, Kurman RJ, Vang R, et al. TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma-evidence supporting the clonal relationship of the two lesions. J Pathol 2012;226:421–6. [9] Kuhn E, Kurman RJ, Soslow RA, et al. The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma. Am J Surg Pathol 2012;36:1826–34. [10] Tong GX, Chiriboga L, Hamele-Bena D, et al. Expression of PAX2 in papillary serous carcinoma of the ovary: immunohistochemical evidence of fallopian tube or secondary Mullerian system origin? Mod Pathol 2007;20:856–63. [11] Li J, Abushahin N, Pang S, et al. Tubal origin of ‘ovarian’ lowgrade serous carcinoma. Mod Pathol 2011;24:1488–99. [12] Barcena C, Oliva E. WT1 expression in the female genital tract. Adv Anat Pathol 2011;18:454–65. [13] Lin SX, Shi R, Qiu W, et al. Structural basis of the multispecificity demonstrated by 17beta-hydroxysteroid dehydrogenase types 1 and 5. Mol Cell Endocrinol 2006;248:38–46.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849 [14] Hanukoglu I. Steroidogenic enzymes: structure, function, and role in regulation of steroid hormone biosynthesis. J Steroid Biochem Mol Biol 1992;43:779–804. [15] Tang SE, Kang YQ. Ultrastructure of malignant Brenner tumor of the ovary. Chin Med J (Engl) 1990;103:759–62. [16] Auersperg N. Ovarian surface epithelium as a source of ovarian cancers: Unwarranted speculation or evidence-based hypothesis? Gynecol Oncol 2013;130:246–51. [17] Slack JM. Metaplasia and transdifferentiation: from pure biology to the clinic. Nat Rev Mol Cell Biol 2007;8:369–78. [18] Hagiwara H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–41.
3849
[19] Lei ZM, Zou W, Mishra S, et al. Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy. Biol Reprod 2003;68:888–95. [20] Scully RE, Cohen RB. Oxidative-enzyme activity in normal and pathologic human ovaries. Obstet Gynecol 1964;24:667–81. [21] Song Z. Ovarian enzymatically active stromal cells can be a promoter of ovarian surface epithelial tumor. Med Hypotheses 2011;77:356–8. [22] Penning TM, Byrns MC. Steroid hormone transforming aldo– keto reductases and cancer. Ann N Y Acad Sci 2009;1155:33–42. [23] Papadatos-Pastos D, Dedes KJ, de Bono JS, et al. Revisiting the role of antiandrogen strategies in ovarian cancer. Oncologist 2011;16:1413–21.
Available at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Ovarian Brenner tumour: A morphologic and immunohistochemical analysis suggesting an origin from fallopian tube epithelium Elisabetta Kuhn a,⇑, Ayse Ayhan d, Ie-Ming Shih a,b,c, Jeffrey D. Seidman e, Robert J. Kurman a,b,c a
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA Department of Gynecology and Obstetrics, The Johns Hopkins Medical Institutions, Baltimore, MD, USA c Department of Oncology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA d Department of Pathology, Seirei Mikatahara Hospital, Hamamatsu, Japan e Department of Pathology and Laboratory Medicine, Washington Hospital Center, Washington, DC, USA b
Available online 5 September 2013
KEYWORDS Brenner tumour Pathogenesis Histogenesis Ovarian tumour Metaplasia Transitional Tuboperitoneal junction
Abstract Background: Brenner tumours (BTs), like other epithelial ovarian tumours, are thought to develop from the ovarian surface epithelium. Aim and Methods: We hypothesised that BTs arise from transitional metaplasia near the tuboperitoneal junction which, when embedded in the ovary as Walthard cell nests, may progress to BTs. The aim of this study was to validate this hypothesis by a morphologic and immunohistochemical (IHC) analysis. Results: The IHC analysis revealed that fallopian tube secretory cells, transitional metaplasia, Walthard cell nests and the epithelial component of BTs shared a similar IHC profile, consistently expressing AKR1C3 (an enzyme involved in androgen biosynthesis) and androgen receptor, but not calretinin. The tumour stromal cells that immediately surrounded the epithelial nests showed strong expression of calretinin, inhibin and steroidogenic factor 1 (markers of steroidogenic cells) in the majority of BTs. Using a highly sensitive immunofluorescent staining method, we detected small groups of cilia in transitional metaplasia and Walthard cell nests, multifocal stretches of cilia and/or ciliated vacuoles in benign BTs and well-developed cilia in atypical proliferative BTs. Conclusions: Our findings suggest a tubal origin of BTs through transitional metaplasia and Walthard cell nests, based on their anatomic proximity, similar IHC profile and the presence of cilia. In addition, we hypothesise a role of androgenic stimulation in the pathogenesis of BT, based on the IHC staining pattern of calretinin, inhibin and steroidogenic factor 1 expressed in the luteinised stromal cells surrounding the epithelial nests of the tumours, and
⇑ Corresponding author: Address: Department of Pathology, Johns Hopkins University, 1550 Orleans Street, CRBII, Room 376, Baltimore, MD 21231, USA. Tel.: +1 410 502 7774; fax: +1 410 502 7943. E-mail address: [email protected] (E. Kuhn).
0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.08.011
3840
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
AKR1C3 and androgen receptor expressed in both the epithelial and stromal components. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Described by Brenner in 1907 as oophoroma folliculare ovarii [1], Brenner tumours (BTs) account for 5% of benign ovarian tumours. They are composed of nests of epithelial cells resembling urothelium, which are surrounded by dense fibromatous stroma and are believed to arise from the ovarian surface epithelium (OSE) that undergoes transitional-type metaplasia. In recent years the origin of ovarian epithelial tumours, as being primarily derived from the OSE, has been challenged. It is now believed that most serous tumours are derived from fallopian tube epithelium (FTE), and that endometrioid and clear cell tumours, which are derived from endometriosis, ultimately originate from endometrial tissue by retrograde menstruation [2,3]. This new model of ovarian carcinogenesis is consistent with the Mu¨llerian-type appearance of serous, endometrioid and clear cell neoplasms. However, the epithelium of BTs does not display a Mu¨llerian phenotype and therefore their origin remains enigmatic. We previously described the junction between the FTE and the mesothelium of the tubal serosa, the socalled ‘tuboperitoneal junction’, and reported that transitional cell metaplasia (TM) is frequently found in this location [4–6]. We hypothesised that BTs arise from TM which, when it invaginates into the paratubal or ovarian tissue, is termed a ‘Walthard cell nest’ (WC). When embedded in the ovary these nests develop into a BT. In keeping with this hypothesis, Seidman et al. found an association of WCs with BT and identified that WCs are 79% more common in women with BT compared with controls (50% and 28%, respectively) [6]. However, this association was partially mitigated by the fact that the identification of WCs was correlated with increased tissue sampling [6]. The aim of the present study was to evaluate the morphologic and immunohistochemical features of tuboperitoneal junction, TM, WCs, BTs and atypical proliferative BTs, in an attempt to more clearly understand their relationship.
available slides were reviewed and clinicopathological data collected. This study was approved by the Institutional Review Board of the Johns Hopkins Medical Institutions. 2.2. Immunohistochemistry Immunohistochemistry (IHC) was performed using the primary antibodies and conditions shown in Table 1. Formalin-fixed, paraffin-embedded sections were immunostained using protocols described previously [7–9] and detailed in the Supporting information, Supplementary methods. 2.3. Description of the selected antibodies PAX2, PAX8 and WT1 are markers of Mu¨llerianlineage expressed by secretory cells of the fallopian tube [10–12]. CEA is a sialomucin present in mucin-producing cells, that has been identified in BTs but never studied in TM or WCs. Expression of the sex-steroid receptors androgen receptor (AR), oestrogen receptor (ER) and progesterone receptor (PR) was examined because they are the main effectors of sex-steroid hormone action. Calretinin, inhibin and steroidogenic factor 1 (SF1) were selected as markers of sex-cord differentiation and steroidogenesis. Calretinin is also a reliable marker for mesothelium. In order to gain insight into which cells are capable of sex-steroid hormone production, we immunostained for CYP19 (aromatase) and AKR1C3 (HSD17 b5), key enzymes in the production of oestrogens and androgens respectively [13,14]. 2.4. Immunofluorescence Cilia were visualised with double-colour immunofluorescent staining with a-tubulin (green) for microtubules and c-tubulin (red) for basal bodies using a protocol described in the Supporting information, Supplementary methods. 3. Results
2. Materials and methods 3.1. Morphologic and IHC findings 2.1. Case selection A total of 17 ovaries, 38 fallopian tubes, 25 peritoneal mesothelium, 17 TMs, 56 WCs, 50 BTs and seven atypical proliferative BTs were retrieved from the pathology files of the Johns Hopkins Hospital (Baltimore, MD), Washington Hospital Center (Washington, DC) and Seirei Mikatahara Hospital (Hamamatsu, Japan). All
The detailed correlation of the morphologic and IHC findings is shown in Table 2 and representative images in Figs. 1–3. 3.1.1. Ovary 3.1.1.1. Ovarian stroma. It is composed of densely packed spindle cells with scant cytoplasm except
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3841
Table 1 Primary antibodies, sources and conditions used in this study. Antibody
Symbol
Clone
Vendor
Address
Cat#
Dilution
Stain type
Instrument
Aldo–keto reductase family 1, member C3 Androgen receptor
AKR1C3
NP6.G6.A6
St. Louis, MO
LS-B2295
1:10000
Manual
NA
AR
ER179
Sigma– Aldrich Epitomics
3184-1
1:400
Manual
NA
Calretinin
CRT
Polyclonal
Cell Marque
232A-78
Predilute
Automated
Carcinoembryonic antigen
CEA
TF-3H8-1
Ventana
Burlingame, CA Hot Springs, AK Tucson, AZ
760-2507
Predilute
Automated
Carcinoembryonic antigen
CEA
Polyclonal
Dako
A0115
1:500
Automated
Cytochrome P450 Aromatase Oestrogen receptor
CYP19 ER
H4 6F11
Manual Automated
INH PAX2 PAX8 PR
Polyclonal Polyclonal 3G11 16
1:25 1:100 1:100 Predilute
Automated Automated Automated Automated
Steroidogenic factor 1 Wilms tumour 1
SF1 WT1
N1665 6F-H2
MCA2077S RTU-ER6F11 0100-0549 71-6000 10336-1-ap RTUPGR-312 434200 348M-98
1:800 Predilute
Alpha-inhibin Paired box 2 Paired box 8 Progesterone receptor
AbD Serotec Leica Microsystems AbD Serotec Invitrogen Invitrogen Leica Microsystems Invitrogen Cell Marque
1:100 Predilute
Automated Automated
BenchMark XT BenchMark XT BenchMark XT NA BenchMark XT Bond Max Bond Max Bond Max BenchMark XT Bond Max BenchMark XT
immediately surrounding follicles where the theca cells are larger and contain more abundant cytoplasm. In non-specific ovarian stroma WT1 was positive in 5/17 (29%), calretinin in 11/17 (65%), inhibin in 10/16 (63%) and SF1 in 15/15 (100%) cases. ER was expressed in 13/18 (72%), PR in 16/19 (84%), whereas AR in 16/16 (100%) cases. AKR1C3 was present in 6/9 (67%) cases but CYP19 was not detected. CYP19 was consistently strongly positive in granulosa-lutein cells of the corpus luteum, granulosa cells, theca interna cells and AKR1C3 in theca cells and luteinised stromal cells. 3.1.1.2. Ovarian surface epithelium. It was composed of flattened cells with elongated bland nuclei which, in foci, were cuboidal. Occasionally short stretches of ciliated epithelium were found between the predominant population. WT1 was positive in 16/16 (100%), calretinin in 15/16 (94%), ER in 10/16 (63%), PR in 11/17 (65%) and AR in 0/15 cases. Inhibin was negative in all cases but SF1 was positive in 10/15 (67%) cases. AKR1C3 and CYP19 were negative in all cases. 3.1.2. Peritoneal mesothelium Peritoneal mesothelium was composed of flattened epithelium; ciliated cells were not identified. The only positive markers were WT1 in 23/24 (96%) and calretinin in 23/23 (100%) cases. 3.1.3. Fallopian tube epithelium 3.1.3.1. Secretory cells. These cells were columnar with bland nuclei arranged perpendicular to the basement membrane and were interspersed with ciliated cells.
Carpinteria, CA Raleigh, NC Bannockburn, IL Raleigh, NC Carlsbad, CA Carlsbad, CA Bannockburn, IL Carlsbad, CA Hot Springs, AK
All cells were positive for PAX2, PAX8 and WT1. Except for a single case (3%) that was positive for calretinin, all the other cases were negative for calretinin, inhibin and SF1. ER was expressed in 37/37 (100%), PR in 31/38 (82%) and AR in 31/31 (100%) cases. AKR1C3 was present in 16/16 (100%) cases, but CYP19 was not detected. 3.1.3.2. Ciliated cells. These cells, like secretory cells, are columnar and arranged perpendicularly to the basement membrane. They contain large oval to round nuclei and multiple apical cilia. PAX2 was not expressed whereas PAX8 was expressed in 2/26 (8%) and WT1 in 30/35 (86%) cases. Calretinin, inhibin and SF1 were not detected. ER was detected in 37/37 (100%), PR in 31/38 (82%) and AR in 1/31 (3%) cases. AKR1C3 was present in 16/16 (100%) cases but CYP19 was not detected. 3.1.4. Transitional metaplasia The cells are identical to those that comprise WCs (see below). These small foci of cells are located on the tubal serosa and are generally smaller than WCs. They are frequently found near the tuboperitoneal junction. PAX2 was present in 1/9 (11%), PAX8 in 2/5 (40%) and WT1 in all nine (100%) cases. Calretinin, inhibin and SF1 were not detected. ER and PR were expressed in 1/10 (10%), whereas AR in 9/9 (100%) cases. Moderate AKR1C3 was present in 4/4 (100%) cases but CYP19 was not detected. 3.1.5. Walthard cell nests As noted above the cells in WCs are identical to those that comprise TM with bland nuclei, often
3842
Table 2 Immunohistochemical results. Antibody
Gene Symbol
Markers of mucinous differentiation Carcinoembryonic CEA antigen monoclonal Carcinoembryonic CEA antigen polyclonal
OSE Pos/tot (%)
Stroma Pos/tot (%)
3/12 (25) 3/16 (19) 16/16 (100)
0/14 (0) 0/16 (0) 5/17 (29)
0/6 (0) 0/6 (0)
Fallopian tube epithelium
Transitional metaplasia Pos/tot (%)
Walthard cell nest Pos/tot (%)
Benign Brenner tumour
Atypical proliferative Brenner tumour
Epithelium Pos/tot (%)
Stroma Pos/tot (%)
Epithelium Pos/tot (%)
Stroma Pos/tot (%)
Secretory cells Pos/tot (%)
Ciliary cells Pos/tot (%)
0/23 (0) 3/15 (20) 23/24 (96)
34/34 (100) 26/26 (100) 35/35 (100)
0/34 (0) 2/26 (8) 30/35 (86)
1/9 (11) 2/5 (40) 9/9 (100)
1/37 (3) 2/27 (7) 32/48 (67)
0/38 (0) 0/40 (0) 4/41 (10)
0/38 (0) 0/40 (0) 13/41 (32)
0/3 (0) 0/3 (0) 0/3 (0)
0/3 (0) 0/3 (0) 3/3 (100)
0/6 (0)
0/4 (0)
0/7 (0)
0/7 (0)
NA
NA
1/16 (6)
0/16 (0)
NA
NA
0/6 (0)
0/4 (0)
0/7 (0)
0/7 (0)
1/1
3/4 (75)
17/17 (100)
0/17 (0)
NA
NA
11/17 (65) 10/16 (63) 15/15 (100)
23/23 (100) 0/24 (0) 0/20 (0)
1/30 (3) 0/30 (0) 0/25 (0)
0/30 (0) 0/30 (0) 0/25 (0)
0/7 (0) 0/7 (0) 0/6 (0)
0/38 (0) 0/41 (0) 0/33 (0)
0/42 (0) 0/42 (0) 0/41 (0)
35*/42 (83) 34*/42 (81) 39*/41 (95)
0/4 (0) 0/4 (0) 0/1 (0)
4*/4 (100) 4*/4 (100) 1*/1 (100)
0/7 (0)
6/9 (67)
0/6 (0)
16/16 (100)
16/16 (100)
4/4 (100)
21/21 (100)
26/26 (100)
23/26 (88)
7/7 (100)
7/7 (100)
0/4 (0)
0/5 (0)
0/4 (0)
0/12 (0)
0/12 (0)
0/3 (0)
0/15 (0)
0/25 (0)
2/25 (8)
0/5 (0)
2/5 (40)
16/16 (100) 13/18 (72) 16/19 (84)
0/19 (0) 0/25 (0) 0/25 (0)
31/31 (100) 37/37 (100) 31/38 (82)
1/31 (3) 37/37 (100) 31/38 (82)
9/9 (100) 1/10 (10) 1/10 (10)
43/49 (88) 4/55 (7) 2/56 (4)
27/29 (93) 2/43 (5) 2/44 (5)
26/29 (90) 33/43 (77) 39/44 (89)
7/7 (100) 0/4 (0) 0/4 (0)
7*/7 (100) 3/4 (75) 4/4 (100)
Markers of sex-cord differentiation/steroidogenesis Calretinin CRT 15/16 (94) Inhibin alpha INH 0/15 (0) Steroidogenic factor 1 SF1 10/15 (67) Markers of sex-steroid enzymes Aldo–keto reductase AKR1C3 family 1, member C3 Cytochrome P450 CYP19 Aromatase
Mesothelium Pos/tot (%)
Markers of sex-steroid hormone receptors Androgen receptor AR 0/15 (0) Oestrogen receptor ER 10/16 (63) Progesterone receptor PR 11/17 (65)
Pos, total positive cases; Tot, total cases analysed; OSE, ovarian surface epithelium. PAX2, PAX8, WT1, SF1, AR, ER and PR are nuclear markers. CEA is a cytoplasmic and extracytoplasmic staining. On the other hand, INH is cytoplasmic, whereas CRT, AKR1C3 and CYP19 are both nuclear and cytoplasmic. Usually all the markers, when positive, are diffusely positive in the majority (>80%) of the cells, except for these markers that are variably positive in the stromal component (>10% of cells) with typical condensation around the transitional-like epithelial nests.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
Markers of Mu¨llerian-lineage Paired box 2 PAX2 Paired box 8 PAX8 Wilms tumour 1 WT1
Ovary
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3843
Fig. 1. Representative images showing the histological appearance of ovarian surface epithelium (OSE), mesothelium, fallopian tubal epithelium (FTE), transitional cell metaplasia (TM) and Walthard cell nest (haematoxylin–eosin staining, HE), and the expression of calretinin (CRT), WT1, PAX2, PAX8, androgen receptor (AR), oestrogen receptor (ER), progesterone receptor (PR) and AKR1C3 in each epithelium.
displaying a longitudinal groove and showing transitional-type differentiation. They may be solid or cystic. PAX2 was present in 1/37 (3%), PAX8 in 2/27 (7%) and WT1 in 32/48 (67%) cases. Calretinin, inhibin and SF1 were not detected. ER was expressed in 4/55 (7%) and PR in 2/56 (4%) whereas AR in 43/49 (88%) cases.
All cases analysed showed moderate expression of AKR1C3 and negativity for CYP19. 3.1.6. Benign Brenner tumours Forty-two BTs measuring between 1.1 and 21 cm (average 7.3 cm), and seven microscopic tumours (range 0.3–0.7 cm, average 0.5 cm) were examined.
3844
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
Fig. 2. Histological appearance of benign Brenner tumour (haematoxylin–eosin, HE) and immunohistochemical staining. The tumour is composed of nests of epithelial cells resembling urothelium, surrounded by a prominent fibromatous stroma (HE). The female sex-steroid receptors, oestrogen receptor (ER) and progesterone receptor (PR), show nuclear positivity in the stromal component, but are completely negative in the transitional nests. Androgen receptor (AR) is strongly positive in both epithelium and stroma. CYP19 (aromatase) is negative in the epithelial nests and stroma. WT1 is weakly positive in the epithelial and stromal component. Alpha-inhibin (INH), calretinin (CRT) and steroidogenic factor 1 (SF1) (markers of sex-cord differentiation and steroidogenesis) are positive in the stromal component. Notably, CRT, INH and SF1 show strong immunopositivity in the stromal cells that are in close proximity to the epithelial nests, which are negative for these markers. AKR1C3 (type 5,17-b-hydroxysteroid dehydrogenase which locally converts androstenedione to testosterone) shows strong positivity in the nuclei and cytoplasm in the epithelial cells component and weak positivity in the stroma.
3.1.6.1. Epithelial component. These tumours contained nests of cells identical to those in TM and WCs. In addition, cells with mucinous epithelium were frequently found in the centre of the nests.
Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 4/41 (10%) cases. Calretinin, inhibin and SF1 were not detected. ER was expressed in 2/43 (5%) and PR in 2/44 (5%) whereas AR in 27/29 (93%) cases.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3845
Fig. 3. High magnification of an atypical proliferative Brenner tumour, showing a papillary frond of transitional epithelium with a delicate stromal stalk (haematoxylin–eosin, HE). The tumour epithelium shows diffuse, moderate expression of androgen receptor (AR), but is completely negative for oestrogen receptor (ER), progesterone receptor (PR), alpha-inhibin (INH) and calretinin (CRT) (the latter two are markers of sex-cord differentiation and steroidogenesis). INH and CRT, as well as WT1, are weakly positive in the tumour stroma. In contrast AKR1C3 is intensely positive in the epithelium and weakly positive in the stroma.
Intense AKR1C3 was detected in 26/26 (100%) cases but CYP19 was not detectable in any case. 3.1.6.2. Stromal component. It is very similar to ovarian stroma, but stromal cells that are closely applied to the epithelial nests are plumper, suggesting that they are luteinised. Stroma, distant from the nests, are more fibrotic and frequently contain spiculated calcification (29/50 cases, 58%). Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 13/41 (32%) cases. Calretinin was positive in 35/42 (83%), inhibin in 34/42 (81%) and SF1 in 39/41 (95%) cases. ER was expressed in 33/43 (77%), PR in 39/ 44 (89%) whereas AR in 26/29 (90%) cases. AKR1C3 was present in 23/26 (88%) and CYP19 in 2/25 (8%) cases.
3.1.7. Atypical proliferative Brenner tumours Six (86%) of seven atypical proliferative BTs were macroscopically solid and cystic, and the remaining was solid. The average size was 11.9 cm (range 6–22 cm). 3.1.7.1. Epithelial component. It was composed of large, irregularly elongated nests, sometimes forming thick papillae. The epithelial cells had a transitional-type appearance, similar to benign BTs and TM. Welldeveloped cilia were multifocally identified in 6/7 (86%) cases. Diffuse mucinous metaplasia was prominent in 5/7 (71%) cases and focal squamous metaplasia was present in 4/7 (57%) cases. A minor component of benign BT was also present in 5/7 (71%) cases, which directly abutted the atypical proliferative component.
3846
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
The only positive markers were AKR1C3 and AR, which were positive in 7/7 (100%) cases. 3.1.7.2. Stromal component. The stromal cells that were closely apposed to the epithelial nests were plumper and had more rounded vesicular nuclei with an occasional single small nucleolus compared to the more fibromatous stromal cells that were further located from the nests. Neither PAX2 nor PAX8 was detected, whereas WT1 was found in 3/3 (100%) cases. Calretinin and inhibin were positive in 4/4 (100%) cases, and SF1 in the only case analysed. ER and PR were expressed in 3/4 (75%) and 4/4 (100%) cases respectively, whereas AR and AKR1C3 in 7/7 (100%) cases analysed. CYP19 was detected in 2/5 (40%) cases. Table 3 Presence of cilia differentiation based on double-colour immunofluorescence staining for a-tubulin and c-tubulin. Lesion
Total
Cilia sketches/ ciliated vacuoles
Welldeveloped cilia
n (%)
n (%)
Transitional metaplasia Walthard cell nest Benign Brenner tumour Atypical proliferative Brenner tumour
17 35 17 6
8 (47) 9 (26) 17 (100) 1 (17)
0 0 0 5
(0) (0) (0) (83)
3.2. Immunofluorescence findings Based on our morphologic findings of cilia in the majority of atypical proliferative BTs we applied immunofluorescent staining for a-tubulin (green) and c-tubulin (red) in an effort to detect cilia and basal bodies with greater sensitivity than by haematoxylin–eosin morphology (Table 3). We observed multiple cilia, characterised by superficial filamentous structures of a-tubulin rising from basal bodies of c-tubulin, in all 35 tubes analysed (Fig. 4). Secretory cells did not display diffuse apical cilia formation and multiple basal bodies, but did show a single centrosome in the apical portion of the cell and occasionally stretches of cilia or vacuoles containing cilia (Fig. 4). At the tuboperitoneal junction, features of progressive attenuation of cilia formation from tubal ciliary epithelium to TM were occasionally identifiable (Fig. 5). Very small groups of cilia, rising from apical centrosome structures (Fig. 5), were detected in 8/17 (47%) TMs, and in 9/35 (26%) WCs. In addition, all 17 benign BTs showed multifocal stretches of cilia and/or ciliated vacuoles (Fig. 5). Finally, 5/6 (83%) atypical proliferative BTs showed well-developed cilia (Fig. 5), with the remaining 1/6 (17%) showing only stretches of cilia. 4. Discussion Benign BTs are biphasic neoplasms composed of nests of transitional-type epithelium surrounded by a
Fig. 4. Representative images of tubulin distribution in normal-appearing tubal epithelium, using a double immunofluorescence staining for a- and c-tubulin. (A) Tubal ciliary epithelium shows multiple cilia as apical filamentous structures (a-tubulin in green) arising from a continuous line of basal bodies (c-tubulin in red). (B) Higher magnification. (C) Secretory cells show the absence of multiple apical cilia and multiple basal bodies, but apical presence of a single centrosome. (D) Occasionally, in the secretory cells, there are stretches of cilia appearing as short, single or sometimes a few filamentous a-tubulin tufts emerging from apical centrosomes (white arrow), or basal ciliated vacuoles of concentrically located cilia appearing as basal rounded structures (yellow arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
3847
Fig. 5. Representative images of tubulin distribution in the epithelium of transitional metaplasia, Walthard cell nest (WC), Brenner tumour (BT) and atypical proliferative BT using a double immunofluorescence staining for a-tubulin (green) and c-tubulin (red). (A) At the tuboperitoneal junction transitional metaplasia on left side contains attenuated cilia formation (arrow) in contrast to the obvious tubal cilia on the right. (B) Transitional metaplasia and (C) WCs present very focal evidence of cilia (green), rising from an apical single centrosome (red) (arrows). (D and E) Benign BT shows multifocal cilia stretches (white arrow) and/or ciliated vacuoles (yellow arrow). (F) Atypical proliferative BT shows welldeveloped cilia as luminal microtubules tufts (green) arising from a continuous line of basal bodies (red). (G) Higher magnification. Insert: mitotic figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
densely fibromatous stroma. A variety of theories have been proposed for their origin but the most widely accepted is derivation from OSE that has undergone transitional-type metaplasia. Recently, we drew attention to the junction between the epithelium of the fimbria and the mesothelial serosa of the tube, the so-called ‘tuboperitoneal junction’, and suggested that, like other junctions between different types of epithelia, this might be a ‘hot spot’ for carcinogenesis. We also noted the frequent presence of transitional-type epithelium in close proximity to the junction. Foci of TM are typically quite small but when they are large they are termed ‘Walthard cell nests’. In other words, TM and WCs are one and the same, the latter just larger. Accordingly, in this discussion we use the term ‘transitional metaplasia’ to include both the small foci of TM and WCs. Morphologically, TM and the epithelium of BTs are virtually identical. BTs differ by their presence in the
ovary, their associated fibromatous stroma and focal mucinous changes. Our IHC analysis was undertaken to determine whether the origin of BTs is mesothelial or Mu¨llerian. Our findings are somewhat puzzling. For example, although TM and the epithelial component of BTs did not express calretinin, unlike peritoneal mesothelium and OSE, they both failed to express the Mu¨llerian markers PAX2 and PAX8. Notably, these markers are also absent in tubal ciliated cells. On the other hand, our studies demonstrated cilia formation in FTE and BTs, suggesting a close association with Mu¨llerian epithelium as opposed to mesothelium. In line with our findings, Tang and Kang have identified the presence of desmosome-tonofilament complex and cilia at an ultrastructural level in malignant BTs, and concluded that they are derived from Mu¨llerian epithelium that has differentiated into squamous epithelium similar to vaginocervical epithelium [15]. We also found that when cilia were present in the OSE, an immunopheno-
3848
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849
type identical to FTE was also present, suggesting that ciliated epithelium may have resulted from implantation of tubal epithelium on the ovarian surface [16]. Therefore, we propose that BTs are derived from FTE which, following trauma and regeneration, loses its Mu¨llerian phenotype as it transdifferentiates into transitional cell epithelium [15,17]. When metaplastic transitional epithelium is implanted into the ovary, hormonal stimulation may lead to the reacquisition of cilia [18,19]. Although the possibility that BTs derive from OSE undergone transitional-like metaplastic change cannot be entirely dismissed. By IHC we identified steroid hormone-producing and hormone-responsive cells using antibodies to inhibin, calretinin and SF1, hormone receptors (AR, ER and PR), and enzymes involved in hormone steroidogenesis, AKR1C3 and CYP19, suggesting hormonal biosynthesis of the epithelial and stromal components of BTs [20,21]. AKR1C3 is of particular interest as it has been demonstrated that it regulates local production of androgens, oestrogens and progestins in the prostate and breast tissues by binding to their respective nuclear receptors [22]. In fact, AKR1C3 is expressed in higher levels in normal prostate epithelial cells than in the stroma implying that reductive androgen metabolism occurs preferentially in epithelial cells. Furthermore, in the breast AKR1C3 converts androstenedione to testosterone, which is then aromatised by CYP19 to estradiol. AKR1C3 is highly expressed in intraductal and invasive ductal carcinomas of the breast. Thus, the findings in the present study of high expression of AKR1C3 in FTE, TM and BTs indicate that they have the capacity to synthesise androgens. This provides further evidence that TM and BTs are linked to FTE as both OSE and peritoneal mesothelium do not express AKR1C3. In addition, among the various hormone receptors, AR was consistently more highly expressed than ER and PR in secretory cells, TM and BTs. These findings and the absence of CYP19 indicate a more important role for androgens than oestrogens in the development of BTs. Taken together, our findings suggest that following implantation of transitional metaplastic epithelium into the ovary, the ovarian stroma may become activated to produce steroid hormones, which may then be converted by both epithelium and stroma to androgens, and further stimulate epithelium-stroma growth and lead to the development of a BT. In conclusion, this study provides morphologic and immunohistochemical evidence suggesting that, like serous ovarian tumours, BTs may arise from FTE that has undergone TM and has seeded the ovary. Furthermore, our findings confirm the role of the ovarian stroma in tumour development which, by synthesising steroid hormones locally, can stimulate growth of the implanted epithelium, a proposal put forth by Scully 50 years ago [20]. This is an intriguing tumourigenic mechanism that
appears to apply to other ovarian tumours (unpublished data) and may, therefore, provide a novel therapeutic target using antiandrogenic drugs [20,23]. Conflict of interest statement None declared. Acknowledgements The authors thank Ms. Asli Bahadirli-Talbott and Ms. Nichelle Gray for their excellent technical assistance. This study was supported by OSB1 grant from HERA foundation, and CDMRP Grant (No. OC100517) from the US Department of Defense. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2013.08.011. References [1] Brenner F. Das Oophoroma Folliculare. Frankfurt Z Pathol 1907;1:150. [2] Kuhn E, Kurman RJ, Shih IM. Ovarian cancer is an imported disease: fact or fiction? Curr Obstet Gynecol Rep 2012;1:1–9. [3] Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory. Am J Surg Pathol 2010;34:433–43. [4] Seidman JD, Yemelyanova A, Zaino RJ, et al. The fallopian tubeperitoneal junction: a potential site of carcinogenesis. Int J Gynecol Pathol 2011;30:4–11. [5] Rabban JT, Crawford B, Chen LM, et al. Transitional cell metaplasia of fallopian tube fimbriae: a potential mimic of early tubal carcinoma in risk reduction salpingo-oophorectomies from women With BRCA mutations. Am J Surg Pathol 2009;33:111–9. [6] Seidman JD, Khedmati F. Exploring the histogenesis of ovarian mucinous and transitional cell (Brenner) neoplasms and their relationship with Walthard cell nests: a study of 120 tumors. Arch Pathol Lab Med 2008;132:1753–60. [7] Kuhn E, Kurman RJ, Sehdev AS, et al. Ki-67 labeling index as an adjunct in the diagnosis of serous tubal intraepithelial carcinoma. Int J Gynecol Pathol 2012;31:416–22. [8] Kuhn E, Kurman RJ, Vang R, et al. TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma-evidence supporting the clonal relationship of the two lesions. J Pathol 2012;226:421–6. [9] Kuhn E, Kurman RJ, Soslow RA, et al. The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma. Am J Surg Pathol 2012;36:1826–34. [10] Tong GX, Chiriboga L, Hamele-Bena D, et al. Expression of PAX2 in papillary serous carcinoma of the ovary: immunohistochemical evidence of fallopian tube or secondary Mullerian system origin? Mod Pathol 2007;20:856–63. [11] Li J, Abushahin N, Pang S, et al. Tubal origin of ‘ovarian’ lowgrade serous carcinoma. Mod Pathol 2011;24:1488–99. [12] Barcena C, Oliva E. WT1 expression in the female genital tract. Adv Anat Pathol 2011;18:454–65. [13] Lin SX, Shi R, Qiu W, et al. Structural basis of the multispecificity demonstrated by 17beta-hydroxysteroid dehydrogenase types 1 and 5. Mol Cell Endocrinol 2006;248:38–46.
E. Kuhn et al. / European Journal of Cancer 49 (2013) 3839–3849 [14] Hanukoglu I. Steroidogenic enzymes: structure, function, and role in regulation of steroid hormone biosynthesis. J Steroid Biochem Mol Biol 1992;43:779–804. [15] Tang SE, Kang YQ. Ultrastructure of malignant Brenner tumor of the ovary. Chin Med J (Engl) 1990;103:759–62. [16] Auersperg N. Ovarian surface epithelium as a source of ovarian cancers: Unwarranted speculation or evidence-based hypothesis? Gynecol Oncol 2013;130:246–51. [17] Slack JM. Metaplasia and transdifferentiation: from pure biology to the clinic. Nat Rev Mol Cell Biol 2007;8:369–78. [18] Hagiwara H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–41.
3849
[19] Lei ZM, Zou W, Mishra S, et al. Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy. Biol Reprod 2003;68:888–95. [20] Scully RE, Cohen RB. Oxidative-enzyme activity in normal and pathologic human ovaries. Obstet Gynecol 1964;24:667–81. [21] Song Z. Ovarian enzymatically active stromal cells can be a promoter of ovarian surface epithelial tumor. Med Hypotheses 2011;77:356–8. [22] Penning TM, Byrns MC. Steroid hormone transforming aldo– keto reductases and cancer. Ann N Y Acad Sci 2009;1155:33–42. [23] Papadatos-Pastos D, Dedes KJ, de Bono JS, et al. Revisiting the role of antiandrogen strategies in ovarian cancer. Oncologist 2011;16:1413–21.