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Neuropathology 2015; 35, 410–420
doi:10.1111/neup.12200
Or i gi na l Ar t i c l e
Clinicopathologic study of endolymphatic sac tumor (ELST) and differential diagnosis of papillary tumors located at the cerebellopontine angle Jiang Du,1 Junmei Wang,1 Yun Cui,1 Cuiping Zhang,2 Guilin Li,1 Jingyi Fang,1 Shenglin Yue1 and Li Xu1 Departments of 1Neuropathology and 2Electron Microscopy, Beijing Neurosurgical Institute, Capital Medical University, China National Clinical Research Center for Neurological Diseases, NCRC-ND, Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing Key Laboratory of Brian Tumor, Beijing, China
We investigated the clinicopathologic features and immunophenotypes of 10 cases of endolymphatic sac tumor (ELST) and compared them with other papillary tumors, including eight cases of choroid plexus papilloma (CPP), three cases of atypical choroid plexus papilloma (ACPP), two cases of papillary ependymoma (PE), three cases of papillary meningioma (PM) and two cases of metastatic carcinoma (MC) the at cerebellopontine angle (CPA). The age at onset of ELST ranged from 13 to 39 years. The male-to-female ratio was 1:1. The clinical presentations were primarily ear-related symptoms. The temporal bones showed extensive destruction. Histologically, the important characteristics for differential diagnosis with CPP, which is most similar to ELST, include the quantity of blood vessels, the nuclei location at apical surface of the papillary, clear cytoplasm cells sometimes with visible vacuoles, psammoma bodies and dura or bone invasion. Immunohistochemistry stains for AE1/AE3, cytokeratin CK)5/6, epithelial membrane antigen, CK8/18, S-100, and synaptophysin are helpful in diagnosis of ELST. In ELST, ultrastructure of uniform 2 μm vesicles in cytoplasm was seen, and gene analysis also showed missense mutation in
Correspondence: Guilin Li, MD, PHD, Department of Neuropathology, Beijing Neurosurgical Institute, Capital Medical University, China National Clinical Research Center for Neurological Diseases, NCRC-ND, Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing Key Laboratory of Brian Tumor, 6 Tiantan Xili, Dongcheng District, Beijing, 100050, China. Email: djlunwen@ sina.com Received 10 September 2014; revised 19 January 2015 and accepted 20 January 2015; published online 6 May 2015.
© 2015 Japanese Society of Neuropathology
exon 3. This study indicates that the above histological features combined with immunohistochemistry findings are important for making the correct diagnosis. Gene analysis should be used in patients without medical history to exclude von Hippel-Lindau disease. Key words: cerebellopontine angle, choroid plexus papilloma, endolymphatic sac tumor, immunohistochemistry, temporal bone.
INTRODUCTION Endolymphatic sac tumors (ELSTs) are rare tumors that destroy the temporal bones and exhibit a papillaryglandular appearance. They were originally confused with paraganglioma, choroid plexus papilloma (CPP), papillary ependymoma (PE), ceruminous adenoma and adenomatous tumors of the middle ear.1–4 In 1984, Hassard5 described the first case of ELST. In 1989, Heffner reviewed the clinical and histologic features of 20 cases of these neoplasms and concluded that these tumors were low-grade adenocarcinoma originated from the sac in the temporal bone.6 Features of this tumor include slow growth, widespread invasion of temporal bone and almost no metastasis. Misdiagnoses of these tumors based on radiologic or even pathologic exams have been seen.7–9 Common misdiagnoses based on radiologic exams include glomus jugulare tumors, meningiomas, aneurysmal bone cysts and bone tumors.10–13 It is even difficult for pathologists to differentiate ELST from some papillary tumors, especially CPP at the cerebellopontine angle (CPA) region.6,14–16 In this study, we collected 10 cases of ELST, and used a variety of CPA tumors as well as normal endolymphatic sac
Endolymphatic sac tumor as controls. We also reviewed the literature and summarized the clinicopathological characteristics and immunohistochemical (IHC) features to identify the key point for differential diagnosis of papillary tumors at the CPA.
MATERIALS AND METHODS Materials Patients’ data were reviewed retrospectively, and 10 cases of ELST were collected. Eight cases of CPP, three of atypical choroid plexus papilloma (ACPP), two of PE, three of papillary meningioma(PM), two of metastatic carcinoma (MC) and two cases of normal endolymphatic sac were included as controls for comparison of histological characteristics and IHC features. All samples were from the Neuropathology Department of Beijing Neurosurgical Institute. The 10 ELST cases were chosen from patients attending the hospital during January 1998 to December 2013, and the diagnosis of ELST was based on WHO criteria for head and neck tumors17 which describes that ELST has a variable papillary glandular appearance with wide invasion of the petrous bone. The control cases were chosen from patients during January 2003 to December 2011 who had complete clinical, radiological and pathological data and confirmed diagnosis by two pathologists. The two metastatic tumors were both from lung cancer.The two normal endolymphatic sac cases were 10% formalin-fixed normal cadaveric head samples without any intracranial tumors from the Neuroautopsy Department of Beijing Neurosurgical Institute. Five of the ELST cases have been described previously in a Chinese journal.18 Three patients of ELST had incomplete resection in other hospitals 6 months to 1 year before they were admitted to our institute. The complete data for these three patients were not available, so we could only analyze the data regarding the surgery performed at our center. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation of Capital Medical University, Beijing, China.
Methods Routine paraffin embedded tissue for HE and immunohistochemical staining by Envision were used to assess the histological features of each sample. The primary antibodies were pre-diluted monoclonal antibodies against vimentin, cytokeratin, pan (AE1/AE3), cytokeratin 8/18 (CK8/18), cytokeratin 5/6 (CK5/6), epithelial membrane antigen (EMA), GFAP, S-100 protein (S-100), synaptophysin (SYN), CEA, TTF-1, VEGF, D2-40, calponin and calretinin. All reagents were purchased from Invitrogen (Grand Island, NY, USA). SuperPictureTM 3rd © 2015 Japanese Society of Neuropathology
411 Gen IHC Detection Kit was used. For antigen retrieval, slides were boiled with EDTA buffer (pH 8.0) under high pressure for all the antigens. The counterstain used for the IHC was hematoxylin. Appropriate positive and negative controls were also performed. Positive results exhibit brown-yellow granules attached respectively to the cell membrane, cytoplasm or nucleus. Negative results are no coloring in cell membrane, cytoplasm or nucleus. Transmission electron microscopy was used to study two ELST samples and revealed thyroid-like structure and papillary-cystic glandular structure, respectively. VHL gene sequencing was performed for five cases (cases 1, 2, 4, 9, 10) from January 2010 to December 2013. Genomic DNA was isolated using Recover AllTM Total Necleic Acid Isolation Kit for FFPE (Life AM1975) from paraffin-embedded tissues, and exon 1, 2, 3 of VHL gene were amplified using Platinum® PCR super mix high fidelity (Life 12532-016) with the following primers: VHL-exon1-1-F 5′-CTGGATCGCGGAGGGAAT and VHL-exon1-1-R 5′-CGGCCTCCATCTCCTCCTC; VHLexon1-2-F 5′-AGTCCGGCCCGGAAGAGT and VHL-exon1-2-R 5′-GGCTTCAGACCGTGCTATCGT; VHL-exon2-F 5′-CGGTGTGGCTCTTTAACAACCT and VHL-exon2-R 5′-AACGTCAGTACCTGGCAGTG TG; VHL-exon3-F 5′-CTGAGACCCTAGTCTGCCAC TGA and VHL-exon3-R 5′-TGAGAATGAGACACT TTGAAACTA.
RESULTS Clinical data The clinical data are summarized in Table 1.The ages of the 10 ELST patients ranged from 13 to 39 years with a median age of 25.5 years. The male-to-female ratio was 1:1. The initial symptoms mostly correlated with ears. All 10 cases did not have other von Hippel-Lindau (VHL) disease or familial tumor syndrome that could lead to the clinical diagnosis of VHL disease. In the three patients with recurring tumors, radiotherapy (30 Gy) was used after the second surgery, but two of them still had recurrence later on. Two of the other six patients who did not receive radiotherapy had recurrence. The symptoms of tinnitus, headache and otorrhagia with effusion could be alleviated, but hearing loss and facial nerve paresis were irreversible. The control group included other tumors located in the CPA region. Eight CPPs were from 11- to 54-year-olds with a median age of 34 years and a male-to-female ratio of 1:1. Three ACPPs were from 47- to 62-year-olds with a median age of 54 years and a male-to-female ratio of 2:1. Two PEs were from 45- to 55-year-old men with a median age of 50 years. Three PMs were from 7- to 55-year-olds with a median age of 42 years and a male-to-female ratio of 1:2.
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Table 1 Clinical data of 10 cases of endolymphatic sac tumor (ELST) No.
Age (years) sex
History and symptoms prior to first surgery
1.
23 M
2.
24 M
Headache 6 months; L tinnitus and partial hearing loss 3 months L partial hearing loss 2 years
3.
25 F
Total R Hearing loss 4 years; R Ear bleeding and liquid flow 6 months
4.
35 F
5.
24 F
L Tinnitus 4 years; L otalgia 1 years; L Ear bleeding 6 months R partial Hearing loss 10 years; Headache and facial paralysis 5 years
6.
33 M
7.
30 F
8.
26 F
9
39 M
10
13 M
L tinnitus and Hearing loss onset 16 years; Total L Hearing loss 11 years; facial paralysis 8 years; walking lability 1 years L Hearing loss and facial paralysis 9 years R hearing loss 2 years; Ear bleeding and liquid flow 6 months R hearing loss and facial paralysis 5 years;
L hearing loss 7 years and dysphagia 2 months
Radiographic tumor characteristics
Clinical diagnosis
Previous surgery
Follow-up
L TBD, HV, 4 cm mass
Glomus jugulare tumor
N
Free of disease, 3 years
L TBD, HV, 5 cm mass, clear margins,
Hemangiopericytoma
Y
R TBD, CD, 4 cm mass, clear margins lobulated tumor Extensive L TBD; HV; 4 cm mass
Not given
Y
Glomus Jugulare Tumor
N
Recurred 1 years, partially removed, 1 years later totally removed, free of disease, 3 years Recurred 6 mo, partially removed, 6 months later totally removed, free of disease, 4 years Lost
Extensive R TBD HV, 5 cm mass
Teratomas
Y
L TBD, HV, 6.5 cm mass
glomus jugulare tumor or Angiomatous meningioma
Y
L TBD, HV, clear margins, 5 cm mass R TBD, HV, 5.5 cm mass
Glomus jugulare tumor or Osteogenic tumor Glomus jugulare tumor
N
Free of disease, 2 years
N
Free of disease, 10 months
R TBD, HV, CD 6.5 cm mass,
Glomus jugulare tumor
Y
L TBD, HV, CD and 6.3 cm mass
Bone tumors
N
Recurred 6 months, partially removed, 6 months later totally removed, Free of disease, 1 years Free of disease, 6 months
Recurred 5 years, partially removed, 1 years later totally removed, free of disease, 6 years Recurred 16 years, totally removed, free of disease, 2 years
CD, cystic degeneration; HV, highly vascular; TBD, temporal bone destruction; L, left; R, right
Two MCs were from 41- to 65-year-olds, one male and one female, with a median age of 53 years.
Radiological data Computed tomography and MRI scans showed that tumors were located in the CPA area with extensive destruction of petrous bone (Fig. 1). Digital subtraction angiography (DSA) was applied on three cases. The results showed that the blood supply was from branch vessels of arteriae auricularis posterior of external carotid artery in two tumors (nos. 7 and 8), and from ascending pharyngeal artery in one case (no. 3).
Macroscopy These groups of neoplasms were 4 × 5 × 5 cm to 6 × 6 × 6.5 cm in size, with white or reddish brown color. The tumors were lobulated and non-capsular, and the cut sur-
faces were solid, sometimes with cysts. Three cases were cystic lesions (nos. 3, 9, 10). The cysts were multilocular and the sizes were 0.5–1.5 cm. The thickness of the walls was 0.1–0.3 cm, and the walls had slight texture. The cysts were empty when we received the samples. Cases 3 and 9 which had prior surgery had some coagulated blood on the inner surface of the walls, while case 10 had a smooth wall with light yellow cysts.
Histopathology The most general character was papillary-cystic glandular structure. Another general character was thyroid-like pattern in which the adenoid cystic structure was expanded and the secretion of the glandular cavity resembled colloid, similar to the thyroid structure (case 5, 8, 9). Such areas may even dominate the histological pattern, but this structure was always accompanied by the papillary-cystic © 2015 Japanese Society of Neuropathology
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Fig. 1 Radiographic views of endolymphatic sac tumor (ELST). A: CT scan shows the tumor was located at the left cerebellopontine angel (CPA) region with extensive petrous bone destruction. B and C: MRI shows the clear boundary mass at CPA with the destruction of the left petrous bone. B: High signal intensity on T2-weighted images. C: T1 enhanced image exhibits irregular contrast hyperintensity mass. D: The lateral Digital subtraction angiography view of the left external carotid artery shows irregular mass with abnormal vascular staining in the left CPA and the petrous bone region; the blood supply is mainly through branch vessels of arteriae auricularis posterior of the external carotid artery.
glandular structure (Fig. 2A). Among this series, the main features of seven cases were papillary-cystic glandular structure, but the other three were mainly thyroid-like structure (7:3). The tumor cells were monolayered and covered the papillary structure. The nuclei of the tumor cells were at the same level, often in the middle or near the apical surface of the cavity or the papillary. The fibrous stroma was rich in vasculatures, with small vessels close to the epithelium lining the surface that sometimes looked like a double row of epithelial cells (Fig. 2B). In cases 4, 10 and the papillary region of case 9, we could see round vacuoles (Fig. 2C1) with clear boundaries and uniform sizes of about 2 μm in the cytoplasm. All 10 cases had dural or bone invasion (Fig. 2D). Mitotic activity was rarely seen. Two cases of autopsy in normal endolymphatic sacs showed glandular structure with cubic or columnar epithelial cells and visible eosinophilic colloid material in the © 2015 Japanese Society of Neuropathology
glandular cavity (Fig. 2E2). The nuclei were located in the middle or near the apical surface.
IHC staining Ten ELSTs (Fig. 3A, B): samples from all 10 cases were positively stained for AE1/AE3 and CK5/6 (100%). EMA was positive in nine cases (90%); CK8/18 was positive in eight cases (80%); SYN was focally weakly positive in one case (10%). The IHC staining of thyroid-like tumors resembled the papillary type, except that CK5/6 staining was faint in one case and calponin staining was always negative. Eight CPPs: all eight cases were positive for SYN (100%); S-100 was positive in seven cases (87.5%); eight cases were negative for EMA, CK5/6 (0%). Normal endolymphatic sac expresses AE1/AE3 but not S-100, GFAP or SYN. The results of all the tumors are shown in Table 2.
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Fig. 2 (A): The papillary-cystic glandular structure of endolymphatic sac tumor (ELST). Case 5 showed expanded glandular cavities and the secretion is similar to the thyroid structure. These areas may dominate the histological pattern, but are still accompanied by the papillary areas. HE ×100. (B) The papillary type area of ELST. Case 3 shows that the tumor cells are monolayer, the nuclei of the tumor cells are at the same level, often near the apical surface of the papillary. The fibrous stroma are rich in vasculatures with small vessels close to the epithelium lining the surface, which look like a double row of epithelial cells. HE ×200. (C1) Clear cytoplasm, visible vacuoles (black arrow) and abundant endothelial cells (red arrow) in ELST in case 10. Note the round shaped, uniform sized vacuoles with a clear boundary in the cytoplasm. HE ×400. (C2) Electron micrograph showing that the nuclei are atypical, and that the cytoplasm is filled with 2 μm visible vacuoles (black arrow). The endothelial cells of blood vessels always huddle together (red arrow). (D) Bone invasion of ELST in Case 4. HE ×100. (E1) The petrous bone showing the normal endolymphatic sac (white arrow), sinuses sigmoideus (black arrow) and internal auditory canal (red arrow). (E2) Normal endolymphatic sac shows glandular structure with cubic or columnar epithelial cells and visible eosinophilic colloid material in the glandular cavity. HE ×200.
© 2015 Japanese Society of Neuropathology
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Fig. 3 (A) Diffuse positive staining of CK5/6 in endolymphatic sac tumor (ELST). Immunohistochemistry (IHC) ×200. (B) Focal positive staining of GFAP in ELST. IHC ×200. (C) Electron micrograph of ELST. Imaging of case 9 showing scant microvilli (red arrow) and intercellular specialized junctions (green arrow). The basement membrane (black arrow) of this “thyroid-like” type is clear. (D) The sequence diagram of exon 3 showing 100T>C mutation that resulted in an amino acid 188 L/O.
Ultrastructure and gene analysis The electron micrographs of both papillary type and thyroid-like ELST areas showed that the tumor cells were atypical and had scant microvilli and intercellular specialized junctions. The cytoplasm contained filaments and electron-dense membrane-bound granules. The blood vessels were abundant and had crowded endothelial cells. It is worth noting that the cytoplasm of papillary type area also contained vesicles that resemble the normal endolymphatic sac epithelial cells that have intraluminal substance with similar density (Fig. 2C2). Interestingly, the cytoplasm of the thyroid-like region did not have the vesicular substance, and the basement membrane of thyroid-like type was clearer (Fig. 3C). Although the blood © 2015 Japanese Society of Neuropathology
vessels were abundant, the endothelial cells were more loosely arranged in the thyroid-like area. The five cases tested for VHL gene included four papillary type and one thyroid-like type. One papillary type tumor (case 2) exhibited VHL gene missense mutation. Exon 3 had 100T>C mutation (Fig. 3D), which resulted in amino acid 188 L/O.
DISCUSSION Because the malignant degree of ELST is low, the development of disease is slow, often lasting for months to years.19 This series of data showed that the important clinical characteristics to help doctors to differentiate ELST from other papillary tumors at CPA are long history and presentation of ear-related symptoms such as tinnitus,
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Table 2 Immunohistochemistry results of papillary tumors located at cerebellopontine angle No
CK
CK8/18
CK5/6
EMA
Calp
SYN
GFAP
VEGF
D2-40
calr
CEA
TTF-1
Vim
S100
1 2 3 4 5 6 7 8 9 10
+ + + + + + + + + +
+ + + + + − − + + +
f + + + f + + + + +
+ − + + + + + + + f
f f f − − f − f − f
− − − − fw − − − − −
− − f − − − − − − −
+ + + + + + + + + +
− − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
− + − + − − − + − +
− − f f − − + + − −
CPP
1 2 3 4 5 6 7 8
− − + − + − f +
− + + + − + + −
− − − − − − − −
− − − − − − − −
− − − − − − − −
+ + + + + + + +
− − − f − + − −
+ + + + + + + +
+ − − − − − − −
− − − − − − − −
− − − − − − − −
− − − − − − − −
f − + + − + − −
+ + − + fw + + +
ACPP
1 2 3
− − f
f + f
− − −
+ − f
− − −
f f +
− − −
+ + +
− − −
− − −
− − −
− − −
f − f
+ f +
PE
1 2
− −
− −
− −
− +
− −
+ −
− +
+ +
− −
− −
− −
− −
+ −
+ +
PM
1 2 3
− − f
− − −
− − −
+ + +
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
+ + +
f − −
MC
1 2
+ +
+ +
− −
+ +
− −
− −
− −
− −
− −
− −
+ +
+ +
− −
− −
ELST
−, negative; +, positive; ACPP, atypical choroid plexus papilloma; Calp, calponin; Calr, calretinin; CK, cytokeratin, pan (AE1/AE3); CK5/6, cytokeratin 5/6; CK8/18, cytokeratin 8/18; CPP, choroid plexus papilloma; ELST, endolymphatic sac tumors; EMA, epithelial membrane antigen; f, focally positive; fw, focally weakly positive; MC, metastatic carcinoma; PE, papillary ependymoma; PM, papillary meningioma; S100, S-100 protein; SYN, synaptophysin; Vim, vimentin.
earache, hearing loss and otorrhagia with effusion. Because the tumor has abundant blood supply, clinicians and radiologists could easily misdiagnose it as paraganglioma or hemangiopericytoma. It is worth mentioning that three cases had DSA examination, which showed that the blood supply came from the posterior auricular artery from the branch of external carotid artery or ascending pharyngeal artery. This result is consistent with the report by Nevoux16 that the feeding vessels often arise from branches of the external carotid artery (the posterior auricular artery) or ascending pharyngeal artery and sometimes from branches of the vertebral artery (the anterior inferior cerebellar artery). This finding can help us to distinguish ELST from CPP at the CPA region, the blood supply of which comes from the anterior inferior cerebellar artery.20,21 ELST arises in the epithelium of the endolymphatic sac (Fig. 2E1)/duct which is the normal structure of the inner ear, goes through the bone suture and is surrounded by the dura mater. All 10 cases of ELST had invasion of dura and bone, and this can be well explained by the normal anatomic location of the endolymphatic sac/duct. Some ELSTs are recently confirmed to be associated with VHL disease,
which is caused by germline mutations of VHL tumor suppressor gene located on chromosome 3p25-26.22 The most important neoplasm associated with VHL disease is hemangioblastoma. The most prominent features of VHLassociated tumors are abundant blood vessels and high expression of VEGF.23 However, ELST is not limited to VHL, and in the general population most of the reported ELSTs are sporadic.24,25 In this study, the gene analysis showed that one case had exon 3 missense mutation, although the medical history and follow-up of all ELSTs did not indicate VHL disease. This mutation case is papillary type tumor and did not have special histopathological features or uniform vesicles in the cytoplasm. Histopathologically, ELSTs have abundant vasculature in fibrous stroma.2 There are usually many capillaries, especially in stroma of papilla so that endothelial cells huddle together and are always packed in the stroma. This characteristic is the most important diagnostic clue that can help pathologists to distinguish ELST from other papillary tumors with similar histopathological features at the CPA. Immunohistochemically, CK5/6 was positive in some earlier case reports.26,27 These 10 ELST cases showed © 2015 Japanese Society of Neuropathology
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Fig. 4 (A) The papillae in endolymphatic sac tumor (ELST). Note that many of the endothelial cells of blood vessels are huddled together in the stroma. In addition, nuclei are near the apical surface. HE ×400. (B) The papillae in choroid plexus papilloma (CPP). Note that the small and tenuous blood vessels of CPP are located in the center of the papilla, and the nuclei of the tumor cells are in the basement. HE ×400. (C) Papillary ependymoma (PE). Finger-like projections are lined by multiple layers of cuboidal tumor cells with smooth contiguous surfaces. HE ×200. (D) Papillary meningioma (PM). Perivascular tumor cells of PM are “sheeting” and irregularly arranged without monolayer structures. HE ×200. (E) Metastatic carcinoma (MC). The tumor cells of MC are lined by a monolayer or multiple layers of columnar tumor cells. Bleeding, cellular pleomorphism, necrosis or karyokinesis is usually obvious. HE ×200.
positive AE1/AE3 and CK5/6 staining. Electron microscopy characteristically showed that some cases contained uniform 2 μm vesicles, which resemble the normal endolymphatic sac epithelium cells with intraluminal substance with similar density.28 These vesicles can also be seen in HE staining (Fig. 2C1). Our findings agree with Heffner’s conception that the neoplasm originates from the endolymphatic sac,6 and provide evidence for differential diagnosis with CPP, which may have clear cytoplasm cells but not these vesicles. Interestingly, the cytoplasm of thyroid-like region did not have this substance. Differential diagnosis of ELST should include1 CPP. CPP that locates at CPA is extremely rare, but it is the most common site for adult CPP, for which the direct extension of intraventricular tumor through the foramen of Luschka protruding at the CPA is common.21 CPP at CPA are more frequently benign and the age of onset is about 35.5.29 CPP have the most similar histopathological patterns with ELST and there is still a lack of criteria for the differential diagnosis. Fortunately, there are still some identifiable differences between them: (i) bone destruction: almost all © 2015 Japanese Society of Neuropathology
ELSTs at the CPA have bone destruction and are epidural located, while CPP are always located inside the brain without breaking the dura and bone; (ii) symptoms: the initial symptoms of ELST are often ear-related such as hearing loss, tinnitus or vertigo, whereas CPP is often associated with symptoms caused by increased cranial pressure such as headache, nausea or vomiting; (iii) histopathological pattern (Fig. 4A, B): first of all is the quantity of blood vessels, which is the most easily identifiable and is always not distorted by artificial factors. All of the 10 ELST cases, especially those of papillary type, had significantly more capillaries and the endothelial cells always huddled together in the stroma, whereas CPP had relatively small and tenuous blood vessels and the endothelial cells were scarce in the stroma. Second, the ELST nucleus was often in the middle or near the apical surface of the cavity or the papillary while the CPP nucleus was located near the basal epithelial cells. Third, ELST showed two cell types, clear cells and eosinophilic cytoplasm cells, and in some cases clear cell components were prominent. Vacuoles in the cytoplasm could be seen in some cases, and electron micro-
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Table 3 Immunohistochemistry profiles of CPA papillary tumors
ELST CPP ACPP PE PM MC
AE1/AE3
CK5/6
EMA
CK8/18
Vimentin
SYN
GFAP
S100
TTF-1
+ −/+ −/+ − −/+ +
+ − − − − −/+
+ − +/− +/− +/− +
+ + + − − +/−
+/− +/− +/− +/− + −
−/+ + + +/− − −
−/+ −/+ − +/− − −
−/+ + + + −/+ −
− − − − − +/−
−, negative; −/+, most tumors staining negative but few can be positive; +, most tumors staining positive; +/−, some tumors staining positive and some staining negative; ACPP, atypical choroid plexus papilloma; AE1/AE3, cytokeratin (pan); CK5/6, cytokeratin 5/6; CK8/18, cytokeratin 8/18; CPP, choroid plexus papilloma; ELST, endolymphatic sac tumor; EMA, epithelial membrane antigen; MC, metastatic carcinoma; PE, papillary ependymoma; PM, papillary meningioma; S-100, S-100 protein; SYN, synaptophysin.
graph also showed that there were abundant small vesicles in the cytoplasm, similar to the normal endolymphatic sac. In contrast, tumor cells of CPP usually had pale eosinophilic cytoplasm; clear cytoplasm cells were scarce and there were no small uniform vesicles in the cytoplasm. Fourth, psammoma bodies were not seen in ELST but often appeared in CPP (7/8 cases of CPP and 2/3 cases of ACPP had psammoma bodies). Moreover, thyroid-like structure was somewhat more predominant in ELST, while it seldom appeared in CPP. Last but not least, it was common to see dura and bone invasion in ELST but not in CPP. According to the histopathological characteristics and immunohistochemical staining of CK5/6, AE1/ AE3, EMA and SYN, ELST and CPP that are SYN positive and CK5/6, AE1/AE3, EMA negative can usually be distinguished. PE sometimes can be seen in the CPA region. Its papillae are always lined by a single layer or multiple layers of cuboidal tumor cells with smooth contiguous surfaces (Fig. 4C). Sometimes ependymal rosettes can be seen. The formation of perivascular pseudorosettes, which originate from tumor cells arranged radially around blood vessels with perivascular anuclear zones, is the key for diagnosis. This histological pattern always makes the layers of papilla seem to be multiple, and the nuclei are always arranged irregularly around the papilla. This classic feature in conjunction with the “salt and pepper” speckling of the chromatin of nuclei and positive GFAP, EMA and negative CK5/6, AE1/AE3 staining will provide the diagnostic clue to differentiate with other tumors. PM is a rare meningioma variant defined by the presence of a perivascular pseudo papillary pattern. This pattern always comprises the majority of the tumor, but there are still discernible regions that can be recognized to be meningioma because tumor cells are sheets or whorl structures and resemble those of the normal arachnoid cap cells. This entity is differentiable from ELST, because its perivascular tumor cells are “sheeting” without monolayer structures (Fig. 4D). Vimentin, EMA or focal AE1/AE3 positive IHC stain would be expected in PM. PM tends to
occur in young patients, and local invasion, recurrence or even metastasis can be seen. Some metastatic carcinoma exhibited papillary structure (Fig. 4E). Thus, due to its thyroid-like structure, ELST can be easily misdiagnosed as thyroid papillary carcinoma or teratoma. ELST is mainly located at CPA with invasion of dura or bone, but metastatic carcinoma is often located in the brain. Bleeding, necrosis, cellular pleomorphism or karyokinesis are usually obvious in metastatic carcinoma, and the primary tumor can often be found. The staining of AE1/AE3, CK8/18, CK5/6, EMA, thyroglobulin (TG), TTF-1 and CEA would be positive in these tumors. We summarized the important IHC results in Table 3, which would be useful in differentiating the tumors mentioned above.
Biological behavior and prognosis As a malignant tumor, ELST often shows bland tumor cell differentiation. Its morphological features often seem to be benign, but locally invasive characteristic and destruction of the temporal bone disclose the low grade malignant nature of the tumor. ELSTs have a tendency to recur,30–32 but only two cases of metastasis33,34 have been reported and there has been no death. In this group of 10 cases, all of them had temporal bone destruction; five cases had recurrence and one of these cases recurred three times. Multiple relapses are likely associated with the advanced stage of tumor along with extensive bone destruction and the difficulty for complete resection.
ACKNOWLEDGMENT Funding for this study was from 2013BAI09B03, National Key Technology Research and Development Program of the Ministry of Science and Technology of China, and BIBD-PXM2013_014226_07_000084 Beijing Institute for Brain Disorders. The authors thank Wang Fengmei for preparation of normal endolymphatic sac tissue sections. Conflict of interest: None Declared. © 2015 Japanese Society of Neuropathology
Endolymphatic sac tumor
REFERENCES 1. Eby TL, Makek MS, Fisch U. Adenomas of the temporal bone. Ann Otol Rhinol Laryngol 1988; 97 (6 Pt 1): 605–612. 2. Kempermann G, Neumann HP, Volk B. Endolymphatic sac tumours. Histopathology 1998; 33: 2–10. 3. Lavoie M, Morency RM. Low-grade papillary adenomatous tumors of the temporal bone: report of two cases and review of the literature. Mod Pathol 1995; 8: 603–608. 4. Li JC, Brackmann DE, Lo WW, Carberry JN, House JW. Reclassification of aggressive adenomatous mastoid neoplasms as endolymphatic sac tumors. Laryngoscope 1993; 103: 1342–1348. 5. Hassard AD, Boudreau SF, Cron CC. Adenoma of the endolymphatic sac. J Otolaryngol 1984; 13: 213–216. 6. Heffner DK. Low-grade adenocarcinoma of probable endolymphatic sac origin A clinicopathologic study of 20 cases. Cancer 1989; 64: 2292–2302. 7. Blamires TL, Friedmann I, Moffat DA. Von HippelLindau disease associated with an invasive choroid plexus tumour presenting as a middle ear mass. J Laryngol Otol 1992; 106: 429–435. 8. Panizza BJ, Jackson A, Ramsden RT, Lye RH. Choroid plexus papilloma of the cerebellopontine angle. Skull Base Surg 1992; 2: 155–160. 9. Pollak A, Bohmer A, Spycher M, Fisch U. Are papillary adenomas endolymphatic sac tumors? Ann Otol Rhinol Laryngol 1995; 104: 613–619. 10. Connor SE, Leung R, Natas S. Imaging of the petrous apex: a pictorial review. Br J Radiol 2008; 81 (965): 427–435. 11. Kang M, Khandelwal N, Singh P, Choudhary S, Mukherjee KK. Imaging features of endolymphatic sac tumours: case report with review of literature. Australas Radiol 2007; 51 (Suppl): B199–B201. 12. Patel NP, Wiggins R. H, 3rd, Shelton C. The radiologic diagnosis of endolymphatic sac tumors. Laryngoscope 2006; 116: 40–46. 13. Stendel R, Suess O, Prosenc N, Funk T, Brock M. Neoplasm of endolymphatic sac origin: clinical, radiological and pathological features. Acta Neurochir (Wien) 1998; 140: 1083–1087. 14. Bell D, Gidley P, Levine N, Fuller GN. Endolymphatic sac tumor (aggressive papillary tumor of middle ear and temporal bone): sine qua non radiology-pathology and the University of Texas MD Anderson Cancer Center experience. Ann Diagn Pathol 2011; 15: 117–123. 15. Megerian CA, Pilch BZ, Bhan AK, McKenna MJ. Differential expression of transthyretin in papillary tumors of the endolymphatic sac and choroid plexus. Laryngoscope 1997; 107: 216–221. © 2015 Japanese Society of Neuropathology
419 16. Nevoux J, Nowak C, Vellin JF et al. Management of endolymphatic sac tumors: sporadic cases and von Hippel-Lindau disease. Otol Neurotol 2014; 35: 899– 904. 17. Lyon. WHO classification of head and neck tumors: IARC; 2005. 18. Du J, Wang JM, Cui Y, Li GL. Clinicopathologic features of endolymphatic sac tumor at cerebellopontine angle. Zhonghua Bing Li Xue Za Zhi 2011; 40: 590– 594. 19. Manski TJ, Heffner DK, Glenn GM et al. Endolymphatic sac tumors. A source of morbid hearing loss in von Hippel-Lindau disease. JAMA 1997; 277: 1461– 1466. 20. Sharifi M, Ungier E, Ciszek B, Krajewski P. Microsurgical anatomy of the foramen of Luschka in the cerebellopontine angle, and its vascular supply. Surg Radiol Anat 2009; 31: 431–437. 21. Shi YZ, Wang ZQ, Xu YM, Lin YF. MR findings of primary choroid plexus papilloma of the cerebellopontine angle: report of three cases and literature reviews. Clin Neuroradiol 2014; 24: 263–267. 22. Latif F, Tory K, Gnarra J et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 1993; 260 (5112): 1317–1320. 23. Horiguchi H, Sano T, Toi H, Kageji T, Hirokawa M, Nagahiro S. Endolymphatic sac tumor associated with a von Hippel-Lindau disease patient: an immunohistochemical study. Mod Pathol 2001; 14: 727–732. 24. Bambakidis NC, Megerian CA, Ratcheson RA. Differential grading of endolymphatic sac tumor extension by virtue of von Hippel-Lindau disease status. Otol Neurotol 2004; 25: 773–781. 25. Luff DA, Simmons M, Malik T, Ramsden RT, Reid H. Endolymphatic sac tumours. J Laryngol Otol 2002; 116: 398–401. 26. Skalova A, Sima R, Bohus P, Curik R, Lukas J, Michal M. Endolymphatic sac tumor (aggressive papillary tumor of middle ear and temporal bone): report of two cases with analysis of the VHL gene. Pathol Res Pract 2008; 204: 599–606. 27. Sun YH, Wen W, Wu JH et al. Endolymphatic sac tumor: case report and review of the literature. Diagn Pathol 2012; 7: 36. doi: 10.1186/1746-1596-7-36. 28. Danckwardt-Lilliestrom N, Friberg U, Kinnefors A, Rask-Andersen H. Ultrastructural analysis of 20 intraosseous endolymphatic sacs from patients with cerebello-pontine angle tumours.A surgically obtained control material for histopathological studies. Auris Nasus Larynx 2000; 27: 311–321. 29. Wolff JE, Sajedi M, Brant R, Coppes MJ, Egeler RM. Choroid plexus tumours. Br J Cancer 2002; 87: 1086– 1091.
420 30. Asano K, Sekiya T, Hatayama T et al. A case of endolymphatic sac tumor with long-term survival. Brain Tumor Pathol 1999; 16: 69–76. 31. Bastier PL, de Mones E, Marro M et al. Endolymphatic sac tumors: experience of three cases. Eur Arch Otorhinolaryngol 2013; 270: 1551–1557. 32. Poletti AM, Dubey SP, Barbo R et al. Sporadic endolymphatic sac tumor: its clinical, radiological, and
J Du et al. histological features, management, and follow-up. Head Neck 2013; 35: 1043–1047. 33. Bambakidis NC, Rodrigue T, Megerian CA, Ratcheson RA. Endolymphatic sac tumor metastatic to the spine. Case report. J Neurosurg Spine 2005; 3: 68–70. 34. Tay KY, Yu E, Kassel E. Spinal metastasis from endolymphatic sac tumor. AJNR Am J Neuroradiol 2007; 28: 613–614.
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Neuropathology 2015; 35, 410–420
doi:10.1111/neup.12200
Or i gi na l Ar t i c l e
Clinicopathologic study of endolymphatic sac tumor (ELST) and differential diagnosis of papillary tumors located at the cerebellopontine angle Jiang Du,1 Junmei Wang,1 Yun Cui,1 Cuiping Zhang,2 Guilin Li,1 Jingyi Fang,1 Shenglin Yue1 and Li Xu1 Departments of 1Neuropathology and 2Electron Microscopy, Beijing Neurosurgical Institute, Capital Medical University, China National Clinical Research Center for Neurological Diseases, NCRC-ND, Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing Key Laboratory of Brian Tumor, Beijing, China
We investigated the clinicopathologic features and immunophenotypes of 10 cases of endolymphatic sac tumor (ELST) and compared them with other papillary tumors, including eight cases of choroid plexus papilloma (CPP), three cases of atypical choroid plexus papilloma (ACPP), two cases of papillary ependymoma (PE), three cases of papillary meningioma (PM) and two cases of metastatic carcinoma (MC) the at cerebellopontine angle (CPA). The age at onset of ELST ranged from 13 to 39 years. The male-to-female ratio was 1:1. The clinical presentations were primarily ear-related symptoms. The temporal bones showed extensive destruction. Histologically, the important characteristics for differential diagnosis with CPP, which is most similar to ELST, include the quantity of blood vessels, the nuclei location at apical surface of the papillary, clear cytoplasm cells sometimes with visible vacuoles, psammoma bodies and dura or bone invasion. Immunohistochemistry stains for AE1/AE3, cytokeratin CK)5/6, epithelial membrane antigen, CK8/18, S-100, and synaptophysin are helpful in diagnosis of ELST. In ELST, ultrastructure of uniform 2 μm vesicles in cytoplasm was seen, and gene analysis also showed missense mutation in
Correspondence: Guilin Li, MD, PHD, Department of Neuropathology, Beijing Neurosurgical Institute, Capital Medical University, China National Clinical Research Center for Neurological Diseases, NCRC-ND, Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing Key Laboratory of Brian Tumor, 6 Tiantan Xili, Dongcheng District, Beijing, 100050, China. Email: djlunwen@ sina.com Received 10 September 2014; revised 19 January 2015 and accepted 20 January 2015; published online 6 May 2015.
© 2015 Japanese Society of Neuropathology
exon 3. This study indicates that the above histological features combined with immunohistochemistry findings are important for making the correct diagnosis. Gene analysis should be used in patients without medical history to exclude von Hippel-Lindau disease. Key words: cerebellopontine angle, choroid plexus papilloma, endolymphatic sac tumor, immunohistochemistry, temporal bone.
INTRODUCTION Endolymphatic sac tumors (ELSTs) are rare tumors that destroy the temporal bones and exhibit a papillaryglandular appearance. They were originally confused with paraganglioma, choroid plexus papilloma (CPP), papillary ependymoma (PE), ceruminous adenoma and adenomatous tumors of the middle ear.1–4 In 1984, Hassard5 described the first case of ELST. In 1989, Heffner reviewed the clinical and histologic features of 20 cases of these neoplasms and concluded that these tumors were low-grade adenocarcinoma originated from the sac in the temporal bone.6 Features of this tumor include slow growth, widespread invasion of temporal bone and almost no metastasis. Misdiagnoses of these tumors based on radiologic or even pathologic exams have been seen.7–9 Common misdiagnoses based on radiologic exams include glomus jugulare tumors, meningiomas, aneurysmal bone cysts and bone tumors.10–13 It is even difficult for pathologists to differentiate ELST from some papillary tumors, especially CPP at the cerebellopontine angle (CPA) region.6,14–16 In this study, we collected 10 cases of ELST, and used a variety of CPA tumors as well as normal endolymphatic sac
Endolymphatic sac tumor as controls. We also reviewed the literature and summarized the clinicopathological characteristics and immunohistochemical (IHC) features to identify the key point for differential diagnosis of papillary tumors at the CPA.
MATERIALS AND METHODS Materials Patients’ data were reviewed retrospectively, and 10 cases of ELST were collected. Eight cases of CPP, three of atypical choroid plexus papilloma (ACPP), two of PE, three of papillary meningioma(PM), two of metastatic carcinoma (MC) and two cases of normal endolymphatic sac were included as controls for comparison of histological characteristics and IHC features. All samples were from the Neuropathology Department of Beijing Neurosurgical Institute. The 10 ELST cases were chosen from patients attending the hospital during January 1998 to December 2013, and the diagnosis of ELST was based on WHO criteria for head and neck tumors17 which describes that ELST has a variable papillary glandular appearance with wide invasion of the petrous bone. The control cases were chosen from patients during January 2003 to December 2011 who had complete clinical, radiological and pathological data and confirmed diagnosis by two pathologists. The two metastatic tumors were both from lung cancer.The two normal endolymphatic sac cases were 10% formalin-fixed normal cadaveric head samples without any intracranial tumors from the Neuroautopsy Department of Beijing Neurosurgical Institute. Five of the ELST cases have been described previously in a Chinese journal.18 Three patients of ELST had incomplete resection in other hospitals 6 months to 1 year before they were admitted to our institute. The complete data for these three patients were not available, so we could only analyze the data regarding the surgery performed at our center. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation of Capital Medical University, Beijing, China.
Methods Routine paraffin embedded tissue for HE and immunohistochemical staining by Envision were used to assess the histological features of each sample. The primary antibodies were pre-diluted monoclonal antibodies against vimentin, cytokeratin, pan (AE1/AE3), cytokeratin 8/18 (CK8/18), cytokeratin 5/6 (CK5/6), epithelial membrane antigen (EMA), GFAP, S-100 protein (S-100), synaptophysin (SYN), CEA, TTF-1, VEGF, D2-40, calponin and calretinin. All reagents were purchased from Invitrogen (Grand Island, NY, USA). SuperPictureTM 3rd © 2015 Japanese Society of Neuropathology
411 Gen IHC Detection Kit was used. For antigen retrieval, slides were boiled with EDTA buffer (pH 8.0) under high pressure for all the antigens. The counterstain used for the IHC was hematoxylin. Appropriate positive and negative controls were also performed. Positive results exhibit brown-yellow granules attached respectively to the cell membrane, cytoplasm or nucleus. Negative results are no coloring in cell membrane, cytoplasm or nucleus. Transmission electron microscopy was used to study two ELST samples and revealed thyroid-like structure and papillary-cystic glandular structure, respectively. VHL gene sequencing was performed for five cases (cases 1, 2, 4, 9, 10) from January 2010 to December 2013. Genomic DNA was isolated using Recover AllTM Total Necleic Acid Isolation Kit for FFPE (Life AM1975) from paraffin-embedded tissues, and exon 1, 2, 3 of VHL gene were amplified using Platinum® PCR super mix high fidelity (Life 12532-016) with the following primers: VHL-exon1-1-F 5′-CTGGATCGCGGAGGGAAT and VHL-exon1-1-R 5′-CGGCCTCCATCTCCTCCTC; VHLexon1-2-F 5′-AGTCCGGCCCGGAAGAGT and VHL-exon1-2-R 5′-GGCTTCAGACCGTGCTATCGT; VHL-exon2-F 5′-CGGTGTGGCTCTTTAACAACCT and VHL-exon2-R 5′-AACGTCAGTACCTGGCAGTG TG; VHL-exon3-F 5′-CTGAGACCCTAGTCTGCCAC TGA and VHL-exon3-R 5′-TGAGAATGAGACACT TTGAAACTA.
RESULTS Clinical data The clinical data are summarized in Table 1.The ages of the 10 ELST patients ranged from 13 to 39 years with a median age of 25.5 years. The male-to-female ratio was 1:1. The initial symptoms mostly correlated with ears. All 10 cases did not have other von Hippel-Lindau (VHL) disease or familial tumor syndrome that could lead to the clinical diagnosis of VHL disease. In the three patients with recurring tumors, radiotherapy (30 Gy) was used after the second surgery, but two of them still had recurrence later on. Two of the other six patients who did not receive radiotherapy had recurrence. The symptoms of tinnitus, headache and otorrhagia with effusion could be alleviated, but hearing loss and facial nerve paresis were irreversible. The control group included other tumors located in the CPA region. Eight CPPs were from 11- to 54-year-olds with a median age of 34 years and a male-to-female ratio of 1:1. Three ACPPs were from 47- to 62-year-olds with a median age of 54 years and a male-to-female ratio of 2:1. Two PEs were from 45- to 55-year-old men with a median age of 50 years. Three PMs were from 7- to 55-year-olds with a median age of 42 years and a male-to-female ratio of 1:2.
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Table 1 Clinical data of 10 cases of endolymphatic sac tumor (ELST) No.
Age (years) sex
History and symptoms prior to first surgery
1.
23 M
2.
24 M
Headache 6 months; L tinnitus and partial hearing loss 3 months L partial hearing loss 2 years
3.
25 F
Total R Hearing loss 4 years; R Ear bleeding and liquid flow 6 months
4.
35 F
5.
24 F
L Tinnitus 4 years; L otalgia 1 years; L Ear bleeding 6 months R partial Hearing loss 10 years; Headache and facial paralysis 5 years
6.
33 M
7.
30 F
8.
26 F
9
39 M
10
13 M
L tinnitus and Hearing loss onset 16 years; Total L Hearing loss 11 years; facial paralysis 8 years; walking lability 1 years L Hearing loss and facial paralysis 9 years R hearing loss 2 years; Ear bleeding and liquid flow 6 months R hearing loss and facial paralysis 5 years;
L hearing loss 7 years and dysphagia 2 months
Radiographic tumor characteristics
Clinical diagnosis
Previous surgery
Follow-up
L TBD, HV, 4 cm mass
Glomus jugulare tumor
N
Free of disease, 3 years
L TBD, HV, 5 cm mass, clear margins,
Hemangiopericytoma
Y
R TBD, CD, 4 cm mass, clear margins lobulated tumor Extensive L TBD; HV; 4 cm mass
Not given
Y
Glomus Jugulare Tumor
N
Recurred 1 years, partially removed, 1 years later totally removed, free of disease, 3 years Recurred 6 mo, partially removed, 6 months later totally removed, free of disease, 4 years Lost
Extensive R TBD HV, 5 cm mass
Teratomas
Y
L TBD, HV, 6.5 cm mass
glomus jugulare tumor or Angiomatous meningioma
Y
L TBD, HV, clear margins, 5 cm mass R TBD, HV, 5.5 cm mass
Glomus jugulare tumor or Osteogenic tumor Glomus jugulare tumor
N
Free of disease, 2 years
N
Free of disease, 10 months
R TBD, HV, CD 6.5 cm mass,
Glomus jugulare tumor
Y
L TBD, HV, CD and 6.3 cm mass
Bone tumors
N
Recurred 6 months, partially removed, 6 months later totally removed, Free of disease, 1 years Free of disease, 6 months
Recurred 5 years, partially removed, 1 years later totally removed, free of disease, 6 years Recurred 16 years, totally removed, free of disease, 2 years
CD, cystic degeneration; HV, highly vascular; TBD, temporal bone destruction; L, left; R, right
Two MCs were from 41- to 65-year-olds, one male and one female, with a median age of 53 years.
Radiological data Computed tomography and MRI scans showed that tumors were located in the CPA area with extensive destruction of petrous bone (Fig. 1). Digital subtraction angiography (DSA) was applied on three cases. The results showed that the blood supply was from branch vessels of arteriae auricularis posterior of external carotid artery in two tumors (nos. 7 and 8), and from ascending pharyngeal artery in one case (no. 3).
Macroscopy These groups of neoplasms were 4 × 5 × 5 cm to 6 × 6 × 6.5 cm in size, with white or reddish brown color. The tumors were lobulated and non-capsular, and the cut sur-
faces were solid, sometimes with cysts. Three cases were cystic lesions (nos. 3, 9, 10). The cysts were multilocular and the sizes were 0.5–1.5 cm. The thickness of the walls was 0.1–0.3 cm, and the walls had slight texture. The cysts were empty when we received the samples. Cases 3 and 9 which had prior surgery had some coagulated blood on the inner surface of the walls, while case 10 had a smooth wall with light yellow cysts.
Histopathology The most general character was papillary-cystic glandular structure. Another general character was thyroid-like pattern in which the adenoid cystic structure was expanded and the secretion of the glandular cavity resembled colloid, similar to the thyroid structure (case 5, 8, 9). Such areas may even dominate the histological pattern, but this structure was always accompanied by the papillary-cystic © 2015 Japanese Society of Neuropathology
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Fig. 1 Radiographic views of endolymphatic sac tumor (ELST). A: CT scan shows the tumor was located at the left cerebellopontine angel (CPA) region with extensive petrous bone destruction. B and C: MRI shows the clear boundary mass at CPA with the destruction of the left petrous bone. B: High signal intensity on T2-weighted images. C: T1 enhanced image exhibits irregular contrast hyperintensity mass. D: The lateral Digital subtraction angiography view of the left external carotid artery shows irregular mass with abnormal vascular staining in the left CPA and the petrous bone region; the blood supply is mainly through branch vessels of arteriae auricularis posterior of the external carotid artery.
glandular structure (Fig. 2A). Among this series, the main features of seven cases were papillary-cystic glandular structure, but the other three were mainly thyroid-like structure (7:3). The tumor cells were monolayered and covered the papillary structure. The nuclei of the tumor cells were at the same level, often in the middle or near the apical surface of the cavity or the papillary. The fibrous stroma was rich in vasculatures, with small vessels close to the epithelium lining the surface that sometimes looked like a double row of epithelial cells (Fig. 2B). In cases 4, 10 and the papillary region of case 9, we could see round vacuoles (Fig. 2C1) with clear boundaries and uniform sizes of about 2 μm in the cytoplasm. All 10 cases had dural or bone invasion (Fig. 2D). Mitotic activity was rarely seen. Two cases of autopsy in normal endolymphatic sacs showed glandular structure with cubic or columnar epithelial cells and visible eosinophilic colloid material in the © 2015 Japanese Society of Neuropathology
glandular cavity (Fig. 2E2). The nuclei were located in the middle or near the apical surface.
IHC staining Ten ELSTs (Fig. 3A, B): samples from all 10 cases were positively stained for AE1/AE3 and CK5/6 (100%). EMA was positive in nine cases (90%); CK8/18 was positive in eight cases (80%); SYN was focally weakly positive in one case (10%). The IHC staining of thyroid-like tumors resembled the papillary type, except that CK5/6 staining was faint in one case and calponin staining was always negative. Eight CPPs: all eight cases were positive for SYN (100%); S-100 was positive in seven cases (87.5%); eight cases were negative for EMA, CK5/6 (0%). Normal endolymphatic sac expresses AE1/AE3 but not S-100, GFAP or SYN. The results of all the tumors are shown in Table 2.
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Fig. 2 (A): The papillary-cystic glandular structure of endolymphatic sac tumor (ELST). Case 5 showed expanded glandular cavities and the secretion is similar to the thyroid structure. These areas may dominate the histological pattern, but are still accompanied by the papillary areas. HE ×100. (B) The papillary type area of ELST. Case 3 shows that the tumor cells are monolayer, the nuclei of the tumor cells are at the same level, often near the apical surface of the papillary. The fibrous stroma are rich in vasculatures with small vessels close to the epithelium lining the surface, which look like a double row of epithelial cells. HE ×200. (C1) Clear cytoplasm, visible vacuoles (black arrow) and abundant endothelial cells (red arrow) in ELST in case 10. Note the round shaped, uniform sized vacuoles with a clear boundary in the cytoplasm. HE ×400. (C2) Electron micrograph showing that the nuclei are atypical, and that the cytoplasm is filled with 2 μm visible vacuoles (black arrow). The endothelial cells of blood vessels always huddle together (red arrow). (D) Bone invasion of ELST in Case 4. HE ×100. (E1) The petrous bone showing the normal endolymphatic sac (white arrow), sinuses sigmoideus (black arrow) and internal auditory canal (red arrow). (E2) Normal endolymphatic sac shows glandular structure with cubic or columnar epithelial cells and visible eosinophilic colloid material in the glandular cavity. HE ×200.
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Fig. 3 (A) Diffuse positive staining of CK5/6 in endolymphatic sac tumor (ELST). Immunohistochemistry (IHC) ×200. (B) Focal positive staining of GFAP in ELST. IHC ×200. (C) Electron micrograph of ELST. Imaging of case 9 showing scant microvilli (red arrow) and intercellular specialized junctions (green arrow). The basement membrane (black arrow) of this “thyroid-like” type is clear. (D) The sequence diagram of exon 3 showing 100T>C mutation that resulted in an amino acid 188 L/O.
Ultrastructure and gene analysis The electron micrographs of both papillary type and thyroid-like ELST areas showed that the tumor cells were atypical and had scant microvilli and intercellular specialized junctions. The cytoplasm contained filaments and electron-dense membrane-bound granules. The blood vessels were abundant and had crowded endothelial cells. It is worth noting that the cytoplasm of papillary type area also contained vesicles that resemble the normal endolymphatic sac epithelial cells that have intraluminal substance with similar density (Fig. 2C2). Interestingly, the cytoplasm of the thyroid-like region did not have the vesicular substance, and the basement membrane of thyroid-like type was clearer (Fig. 3C). Although the blood © 2015 Japanese Society of Neuropathology
vessels were abundant, the endothelial cells were more loosely arranged in the thyroid-like area. The five cases tested for VHL gene included four papillary type and one thyroid-like type. One papillary type tumor (case 2) exhibited VHL gene missense mutation. Exon 3 had 100T>C mutation (Fig. 3D), which resulted in amino acid 188 L/O.
DISCUSSION Because the malignant degree of ELST is low, the development of disease is slow, often lasting for months to years.19 This series of data showed that the important clinical characteristics to help doctors to differentiate ELST from other papillary tumors at CPA are long history and presentation of ear-related symptoms such as tinnitus,
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Table 2 Immunohistochemistry results of papillary tumors located at cerebellopontine angle No
CK
CK8/18
CK5/6
EMA
Calp
SYN
GFAP
VEGF
D2-40
calr
CEA
TTF-1
Vim
S100
1 2 3 4 5 6 7 8 9 10
+ + + + + + + + + +
+ + + + + − − + + +
f + + + f + + + + +
+ − + + + + + + + f
f f f − − f − f − f
− − − − fw − − − − −
− − f − − − − − − −
+ + + + + + + + + +
− − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
− + − + − − − + − +
− − f f − − + + − −
CPP
1 2 3 4 5 6 7 8
− − + − + − f +
− + + + − + + −
− − − − − − − −
− − − − − − − −
− − − − − − − −
+ + + + + + + +
− − − f − + − −
+ + + + + + + +
+ − − − − − − −
− − − − − − − −
− − − − − − − −
− − − − − − − −
f − + + − + − −
+ + − + fw + + +
ACPP
1 2 3
− − f
f + f
− − −
+ − f
− − −
f f +
− − −
+ + +
− − −
− − −
− − −
− − −
f − f
+ f +
PE
1 2
− −
− −
− −
− +
− −
+ −
− +
+ +
− −
− −
− −
− −
+ −
+ +
PM
1 2 3
− − f
− − −
− − −
+ + +
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
+ + +
f − −
MC
1 2
+ +
+ +
− −
+ +
− −
− −
− −
− −
− −
− −
+ +
+ +
− −
− −
ELST
−, negative; +, positive; ACPP, atypical choroid plexus papilloma; Calp, calponin; Calr, calretinin; CK, cytokeratin, pan (AE1/AE3); CK5/6, cytokeratin 5/6; CK8/18, cytokeratin 8/18; CPP, choroid plexus papilloma; ELST, endolymphatic sac tumors; EMA, epithelial membrane antigen; f, focally positive; fw, focally weakly positive; MC, metastatic carcinoma; PE, papillary ependymoma; PM, papillary meningioma; S100, S-100 protein; SYN, synaptophysin; Vim, vimentin.
earache, hearing loss and otorrhagia with effusion. Because the tumor has abundant blood supply, clinicians and radiologists could easily misdiagnose it as paraganglioma or hemangiopericytoma. It is worth mentioning that three cases had DSA examination, which showed that the blood supply came from the posterior auricular artery from the branch of external carotid artery or ascending pharyngeal artery. This result is consistent with the report by Nevoux16 that the feeding vessels often arise from branches of the external carotid artery (the posterior auricular artery) or ascending pharyngeal artery and sometimes from branches of the vertebral artery (the anterior inferior cerebellar artery). This finding can help us to distinguish ELST from CPP at the CPA region, the blood supply of which comes from the anterior inferior cerebellar artery.20,21 ELST arises in the epithelium of the endolymphatic sac (Fig. 2E1)/duct which is the normal structure of the inner ear, goes through the bone suture and is surrounded by the dura mater. All 10 cases of ELST had invasion of dura and bone, and this can be well explained by the normal anatomic location of the endolymphatic sac/duct. Some ELSTs are recently confirmed to be associated with VHL disease,
which is caused by germline mutations of VHL tumor suppressor gene located on chromosome 3p25-26.22 The most important neoplasm associated with VHL disease is hemangioblastoma. The most prominent features of VHLassociated tumors are abundant blood vessels and high expression of VEGF.23 However, ELST is not limited to VHL, and in the general population most of the reported ELSTs are sporadic.24,25 In this study, the gene analysis showed that one case had exon 3 missense mutation, although the medical history and follow-up of all ELSTs did not indicate VHL disease. This mutation case is papillary type tumor and did not have special histopathological features or uniform vesicles in the cytoplasm. Histopathologically, ELSTs have abundant vasculature in fibrous stroma.2 There are usually many capillaries, especially in stroma of papilla so that endothelial cells huddle together and are always packed in the stroma. This characteristic is the most important diagnostic clue that can help pathologists to distinguish ELST from other papillary tumors with similar histopathological features at the CPA. Immunohistochemically, CK5/6 was positive in some earlier case reports.26,27 These 10 ELST cases showed © 2015 Japanese Society of Neuropathology
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Fig. 4 (A) The papillae in endolymphatic sac tumor (ELST). Note that many of the endothelial cells of blood vessels are huddled together in the stroma. In addition, nuclei are near the apical surface. HE ×400. (B) The papillae in choroid plexus papilloma (CPP). Note that the small and tenuous blood vessels of CPP are located in the center of the papilla, and the nuclei of the tumor cells are in the basement. HE ×400. (C) Papillary ependymoma (PE). Finger-like projections are lined by multiple layers of cuboidal tumor cells with smooth contiguous surfaces. HE ×200. (D) Papillary meningioma (PM). Perivascular tumor cells of PM are “sheeting” and irregularly arranged without monolayer structures. HE ×200. (E) Metastatic carcinoma (MC). The tumor cells of MC are lined by a monolayer or multiple layers of columnar tumor cells. Bleeding, cellular pleomorphism, necrosis or karyokinesis is usually obvious. HE ×200.
positive AE1/AE3 and CK5/6 staining. Electron microscopy characteristically showed that some cases contained uniform 2 μm vesicles, which resemble the normal endolymphatic sac epithelium cells with intraluminal substance with similar density.28 These vesicles can also be seen in HE staining (Fig. 2C1). Our findings agree with Heffner’s conception that the neoplasm originates from the endolymphatic sac,6 and provide evidence for differential diagnosis with CPP, which may have clear cytoplasm cells but not these vesicles. Interestingly, the cytoplasm of thyroid-like region did not have this substance. Differential diagnosis of ELST should include1 CPP. CPP that locates at CPA is extremely rare, but it is the most common site for adult CPP, for which the direct extension of intraventricular tumor through the foramen of Luschka protruding at the CPA is common.21 CPP at CPA are more frequently benign and the age of onset is about 35.5.29 CPP have the most similar histopathological patterns with ELST and there is still a lack of criteria for the differential diagnosis. Fortunately, there are still some identifiable differences between them: (i) bone destruction: almost all © 2015 Japanese Society of Neuropathology
ELSTs at the CPA have bone destruction and are epidural located, while CPP are always located inside the brain without breaking the dura and bone; (ii) symptoms: the initial symptoms of ELST are often ear-related such as hearing loss, tinnitus or vertigo, whereas CPP is often associated with symptoms caused by increased cranial pressure such as headache, nausea or vomiting; (iii) histopathological pattern (Fig. 4A, B): first of all is the quantity of blood vessels, which is the most easily identifiable and is always not distorted by artificial factors. All of the 10 ELST cases, especially those of papillary type, had significantly more capillaries and the endothelial cells always huddled together in the stroma, whereas CPP had relatively small and tenuous blood vessels and the endothelial cells were scarce in the stroma. Second, the ELST nucleus was often in the middle or near the apical surface of the cavity or the papillary while the CPP nucleus was located near the basal epithelial cells. Third, ELST showed two cell types, clear cells and eosinophilic cytoplasm cells, and in some cases clear cell components were prominent. Vacuoles in the cytoplasm could be seen in some cases, and electron micro-
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Table 3 Immunohistochemistry profiles of CPA papillary tumors
ELST CPP ACPP PE PM MC
AE1/AE3
CK5/6
EMA
CK8/18
Vimentin
SYN
GFAP
S100
TTF-1
+ −/+ −/+ − −/+ +
+ − − − − −/+
+ − +/− +/− +/− +
+ + + − − +/−
+/− +/− +/− +/− + −
−/+ + + +/− − −
−/+ −/+ − +/− − −
−/+ + + + −/+ −
− − − − − +/−
−, negative; −/+, most tumors staining negative but few can be positive; +, most tumors staining positive; +/−, some tumors staining positive and some staining negative; ACPP, atypical choroid plexus papilloma; AE1/AE3, cytokeratin (pan); CK5/6, cytokeratin 5/6; CK8/18, cytokeratin 8/18; CPP, choroid plexus papilloma; ELST, endolymphatic sac tumor; EMA, epithelial membrane antigen; MC, metastatic carcinoma; PE, papillary ependymoma; PM, papillary meningioma; S-100, S-100 protein; SYN, synaptophysin.
graph also showed that there were abundant small vesicles in the cytoplasm, similar to the normal endolymphatic sac. In contrast, tumor cells of CPP usually had pale eosinophilic cytoplasm; clear cytoplasm cells were scarce and there were no small uniform vesicles in the cytoplasm. Fourth, psammoma bodies were not seen in ELST but often appeared in CPP (7/8 cases of CPP and 2/3 cases of ACPP had psammoma bodies). Moreover, thyroid-like structure was somewhat more predominant in ELST, while it seldom appeared in CPP. Last but not least, it was common to see dura and bone invasion in ELST but not in CPP. According to the histopathological characteristics and immunohistochemical staining of CK5/6, AE1/ AE3, EMA and SYN, ELST and CPP that are SYN positive and CK5/6, AE1/AE3, EMA negative can usually be distinguished. PE sometimes can be seen in the CPA region. Its papillae are always lined by a single layer or multiple layers of cuboidal tumor cells with smooth contiguous surfaces (Fig. 4C). Sometimes ependymal rosettes can be seen. The formation of perivascular pseudorosettes, which originate from tumor cells arranged radially around blood vessels with perivascular anuclear zones, is the key for diagnosis. This histological pattern always makes the layers of papilla seem to be multiple, and the nuclei are always arranged irregularly around the papilla. This classic feature in conjunction with the “salt and pepper” speckling of the chromatin of nuclei and positive GFAP, EMA and negative CK5/6, AE1/AE3 staining will provide the diagnostic clue to differentiate with other tumors. PM is a rare meningioma variant defined by the presence of a perivascular pseudo papillary pattern. This pattern always comprises the majority of the tumor, but there are still discernible regions that can be recognized to be meningioma because tumor cells are sheets or whorl structures and resemble those of the normal arachnoid cap cells. This entity is differentiable from ELST, because its perivascular tumor cells are “sheeting” without monolayer structures (Fig. 4D). Vimentin, EMA or focal AE1/AE3 positive IHC stain would be expected in PM. PM tends to
occur in young patients, and local invasion, recurrence or even metastasis can be seen. Some metastatic carcinoma exhibited papillary structure (Fig. 4E). Thus, due to its thyroid-like structure, ELST can be easily misdiagnosed as thyroid papillary carcinoma or teratoma. ELST is mainly located at CPA with invasion of dura or bone, but metastatic carcinoma is often located in the brain. Bleeding, necrosis, cellular pleomorphism or karyokinesis are usually obvious in metastatic carcinoma, and the primary tumor can often be found. The staining of AE1/AE3, CK8/18, CK5/6, EMA, thyroglobulin (TG), TTF-1 and CEA would be positive in these tumors. We summarized the important IHC results in Table 3, which would be useful in differentiating the tumors mentioned above.
Biological behavior and prognosis As a malignant tumor, ELST often shows bland tumor cell differentiation. Its morphological features often seem to be benign, but locally invasive characteristic and destruction of the temporal bone disclose the low grade malignant nature of the tumor. ELSTs have a tendency to recur,30–32 but only two cases of metastasis33,34 have been reported and there has been no death. In this group of 10 cases, all of them had temporal bone destruction; five cases had recurrence and one of these cases recurred three times. Multiple relapses are likely associated with the advanced stage of tumor along with extensive bone destruction and the difficulty for complete resection.
ACKNOWLEDGMENT Funding for this study was from 2013BAI09B03, National Key Technology Research and Development Program of the Ministry of Science and Technology of China, and BIBD-PXM2013_014226_07_000084 Beijing Institute for Brain Disorders. The authors thank Wang Fengmei for preparation of normal endolymphatic sac tissue sections. Conflict of interest: None Declared. © 2015 Japanese Society of Neuropathology
Endolymphatic sac tumor
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