Immunohistochemical Detection of CD34,E-cadherin,Claudin-1,Glucose Transporter 1,Laminin,and Protein Gene Product 9.5 in 28 Canine and 8 Feline Meningiomas

Abstract

The variation in histologic pattern of meningiomas can make their diagnosis challenging. The immunohistochemical profile of 28 canine and 8 feline meningiomas was examined. Tumor types included anaplastic (6 dogs), angiomatoid (1 cat), fibroblastic (3 dogs, 1 cat), meningothelial (1 dog), microcystic (2 dogs), myxoid (3 dogs), psammomatous (4 cats), and transitional (13 dogs, 2 cats). The authors compared the expression of novel markers (CD34, E-cadherin, claudin-1, glucose transporter 1 [GLUT-1], laminin, and protein gene product [PGP] 9.5) with published markers (cytokeratins, glial fibrillary acidic protein [GFAP], progesterone receptor, S100, and vimentin). Neoplastic cells were immunohistochemically positive for vimentin in 100% of the meningiomas; CD34, 94%; GLUT-1, 86%; E-cadherin, 81%; S100, 75%; laminin, 72%; claudin-1, 60%; PGP 9.5, 55%; progesterone receptor, 44%; pancytokeratins, 39%; cytokeratins 8/18, 17%, and GFAP in 9%. Ki67 index did not correlate well with mitotic index. Based on these results and those in the human literature, immunohistochemistry for vimentin, CD34, and E-cadherin is proposed to support a diagnosis of meningioma. Immunohistochemistry for claudin-1, albeit of only moderate to low sensitivity in canine and feline meningiomas, may help to distinguish meningioma from some mesenchymal neoplasms involving the brain and associated structures, such as schwannomas, which in humans express claudin-1 poorly or not at all. Further studies with CD34, E-cadherin, and claudin-1 in canine and feline tumors that may mimic meningiomas are needed to determine the adequacy of this approach.

Keywords

E-cadherin cat CD34 claudin-1 dog glucose transporter 1 immunohistochemistry laminin meningioma neoplasia protein gene product 9.5

Meningiomas are tumors arising from cells of the arachnoid membrane and pia mater of the nervous system.²² They are one of the most common primary neoplasms of the central nervous system in dogs and cats but are rare in other species.^{30,40,41,54,57,61} Meningiomas in domestic species are mostly intracranial and less commonly spinal, paranasal, or cutaneous.^22,40 In dogs, meningiomas are typically solitary, whereas in cats, meningiomas can be single or multiple masses.^22,25

The histologic diversity of meningiomas, with mesenchymal and epithelial patterns, reflects mesodermal and neural crest contributions to their formation.³⁷ The current World Health Organization (WHO) classification of nervous system tumors of domestic animals includes 9 types of meningioma: meningotheliomatous, fibrous (fibroblastic), transitional (mixed), psammomatous, angiomatous (angioblastic), papillary, granular cell, myxoid, and anaplastic (malignant).²¹ An additional type, microcystic, used in the WHO classification of human meningiomas, has been reported in the dog.^22,33,57 Because of this diversity in pattern, meningiomas may resemble other tumors, such as astrocytomas, oligondendrogliomas, metastatic carcinomas, germ cell tumors, and peripheral nerve sheath tumors.^11,22,31

The number of immunohistochemical markers used in human meningiomas is large, ranging from generic markers (such as vimentin and cytokeratins) to more specialized markers (such as CD34, epithelial membrane antigen [EMA], E-cadherin, and claudin-1).^{6,15,31,43,52,53} To our knowledge, antibodies to EMA that cross-react with formalin-fixed, paraffin-embedded (FFPE) canine or feline tissues are not available. An antibody to CD34 that reacts with canine endothelial cells on FFPE tissues has been recently reported.⁵⁵ The main focus of immunohistochemistry in human meningioma is to distinguish this tumor from its mimics (eg, hemangiopericytoma, solitary fibrous tumor, schwannoma, metastatic carcinoma) with a secondary interest in prognostic markers, such as proliferation markers.¹¹ Markers used in meningiomas of domestic species include cytokeratins, estrogen receptor, glial fibrillary acidic protein (GFAP), Ki67, neuron-specific enolase, progesterone receptor, proliferating cell nuclear antigen, S100, synaptophysin, h-telomerase, and vimentin.^{1,3,27,28,42,59} Combining the results of 2 large series of canine meningiomas, vimentin was expressed in all (37 of 37), followed by neuron-specific enolase (29 of 37), S100 (28 of 37), GFAP (6 of 37), and cytokeratins (11 of 37). Synaptophysin was not reactive in any case (0 of 15).^3,33 Although the number of cases and markers studied in cats is fewer than that in dogs, results are similar (eg, consistent labeling for vimentin and S100 and no labeling for cytokeratins).²⁵

There are few reported immunohistochemical studies of large series of canine or feline meningiomas. The purpose of this study was to evaluate the immunohistochemical reactivity of canine and feline meningiomas with novel markers, such as CD34, E-cadherins, claudin-1, glucose transporter 1 (GLUT-1), laminin, and protein gene product (PGP) 9.5, in addition to classic markers (cytokeratins 8 and 18, GFAP, Ki67, pancytokeratins, progesterone receptor, S100, and vimentin) on FFPE tissues.

Methods

The databases of the Animal Disease Diagnostic Laboratory at Purdue University and the Diagnostic Center for Population and Animal Health at Michigan State University were reviewed for canine and feline meningioma cases from 1998 to May 2009. Of a total of 54 cases with a diagnosis of meningioma, 36 were selected for this study on the basis of the following case definition: A superficial mass in the central nervous system adhered to the leptomeninges or dura mater; it did not involve a nerve; and it comprised histologic features of one of the described subtypes of meningioma.²¹ In addition, selected cases needed to have enough relevant tissue for immunohistochemistry. In some cases, the exact location of the tumor was not included in the pathology report or the clinical history; however, histologic evaluation determined that the mass was superficial and adhered to meninges. Leptomeninges from the cerebrum of 2 cats and 1 dog with no neurologic disease were included in this study. All tissues were fixed in 10% neutral buffered formalin for an undetermined period, estimated at fewer than 2 weeks for all the samples, and stored in paraffin blocks at room temperature for 1 week to 11 years. In our experience, immunoreactivity for all the markers examined is not significantly affected by formalin fixation up to 1 month.⁶² For CD34, immunoreactivity is not reduced in tissues fixed for at least 2 weeks (personal observations). When multiple slides of the same tumor were available, the most representative sample was selected by 1 author (J.A.R.). Cases were reviewed and classified by 2 pathologists in each laboratory without knowledge of the classification in the pathology report. Any discrepancies between the authors in the same laboratory were resolved by consensus among the pathologists reviewing the cases. Classification in some cases was challenging because of the rather common presence of several patterns within the same tumor.³ A final classification was produced by consensus between 2 pathologists (J.A.R, M.A.M) following the current WHO classification for domestic animal meningiomas.²¹ Key descriptions for each tumor type follow: anaplastic (lack of distinct growth pattern, increased mitotic index, and/or extensive necrosis and parenchymal invasion), angiomatous (numerous and variably sized vessels among aggregates of meningeal cells), fibroblastic (spindle cells forming long interlacing fascicles), meningothelial (solid whorls of polygonal cells), microcystic (intercellular pale vacuoles progressing to cysts, admixed with spindloid cells), myxoid (neoplastic cells separated by myxomatous matrix), psammomatous (whorls of meningothelial cells around hyaline or mineralized concretions), transitional (including features of meningothelial and fibroblastic types).

Table 1 summarizes the 13 primary antibodies used in this study and the immunohistochemical protocol. Immunoreactivity for CD34, claudin-1, E-cadherin, GLUT-1, laminin, S100, and vimentin was examined in most tumors. In addition, antibodies to pancytokeratins, cytokeratins 8 and 18, GFAP, Ki67, PGP 9.5, and progesterone receptor were evaluated in a subset of cases. Immunohistochemistry was performed with a Dako autostainer; all the incubations were at room temperature. Immunoperoxidase procedures with diaminobenzidine as chromogen have been published.^38,45,46 Positive tissue controls from each species were used simultaneously. The negative reagent control was rabbit immunoglobulins (for polyclonal antibodies) or mouse immunoglobulins (for monoclonal antibodies). The expected staining pattern for each antibody was as follows: CD34 (cytoplasmic), claudin-1 (cytoplasmic and/or membrane), cytokeratins 8 and 18 and pancytokeratins (cytoplasmic), E-cadherins (cytoplasmic and/or membrane), GFAP (cytoplasmic), GLUT-1 (cytoplasmic), Ki67 (nuclear), laminin (cytoplasmic, interstitial), PGP 9.5 (nuclear and/or cytoplasmic), progesterone receptor (nuclear), S100 (nuclear and/or cytoplasmic), vimentin (cytoplasmic). After the immunohistochemical procedure, slides were counterstained with Mayer’s hematoxylin. Immunoreactivity was scored as follows: 3 (high), > 60% positive cells; 2 (moderate), 30 to 59% positive cells; 1 (low), 10 to 29% positive cells; or 0 (negative), < 10% positive cells.⁴⁶ Immunoreactivity in normal leptomeninges was graded as positive or negative. The sensitivity of a given marker was the percentage of positive cases relative to all meningiomas examined for the given marker. We did not attempt to determine the specificity of the examined markers (ie, the reactivity of meningioma-like tumors or meningioma mimics). Mitotic index (average number of mitotic figures in 10 fields [400×]) and Ki67 index (average number of positive nuclei in 10 fields [400×]) were also recorded.

Table 1.

Antibody Reagents, Antigen Retrieval, and Detection Systems Used in Immunohistochemistry

Antibody (clone)	Dilution	Incubation Time	Pretreatment^a	Detection System	Source of Antibody
CD34 (goat polyclonal)	1:80	120 minutes	HIER citrate buffer, pH 6.0	LSAB+	Santa Cruz Biotechnology, Santa Cruz, CA
Claudin-1 (rabbit polyclonal)	1:25	60 minutes	HIER citrate buffer, pH 6.0	EnVision+	Abcam, Cambridge, MA
Cytokeratins (AE1/AE3)	1:100	60 minutes	HIER citrate buffer, pH 6.0	LSAB+	Dako, Carpenteria, CA
Cytokeratins 8/18 (5D3)	1:25	90 minutes	Proteinase K, 4 minutes	EnVision+	Vector Laboratories, Burlingame, CA
E-cadherin (36)	1:100	60 minutes	HIER citrate buffer, pH 6.0	LSAB+	BD Transduction, Franklin Lakes, NJ
Glial fibrillary acidic protein (rabbit polyclonal)	1:1000	30 minutes	None	LSAB2	Dako, Carpenteria, CA
Glut-1 (rabbit polyclonal)	1:25	60 minutes	HIER citrate buffer, pH 6.0	EnVision+	Dako, Carpenteria, CA
Ki67 (7B11)	1:200	60 minutes	HIER citrate buffer, pH 6.0	LSAB 2	Zymed, Carlsbad, CA
Laminin (rabbit polyclonal)	1:100	60 minutes	Proteinase K, 4 minutes	EnVision+	Dako, Carpenteria, CA
Progesterone receptor (SP21)	1:20	90 minutes	HIER citrate buffer, pH 6.0	LSAB+	LabVision, Freemont, CA
Protein gene product 9.5 (rabbit polyclonal)	1:50	60 minutes	None	EnVision+	Dako, Carpenteria, CA
S100 (rabbit polyclonal)	1:800	30 minutes	HIER citrate buffer, pH 6.0	LSAB 2	Dako, Carpenteria, CA
Vimentin (SP20)	1:100	30 minutes	Proteinase K, 4 minutes	LSAB 2	LabVision, Freemont, CA

^a HIER, heat-induced epitope retrieval with decloaker, 20 to 24 psi, 125C° for 30 seconds.

Results

In sum, 8 feline and 28 canine meningiomas were included in this study (Table 2 ). There were 12 male dogs, 13 female dogs, and 3 of unknown sex. Cats were equally distributed in terms of sex, with 2 unknown. The average age at diagnosis for meningiomas was 8.1 years for dogs (range, 4 to 14 years) and 13.3 years for cats (range, 6 to 17 years). A variety of canine breeds was represented, mostly medium size to large. The breeds of 3 dogs were unknown. All cats with recorded breed were domestic shorthair, except for 1 Maine coon cat. Thirty-two meningiomas were intracranial (24 dogs, 8 cats); 4 were spinal (all dogs). The meningiomas were classified as follows: anaplastic (6 dogs), angiomatoid (1 cat), fibroblastic (3 dogs, 1 cat), meningothelial (1 dog), microcystic (2 dogs), myxoid (3 dogs), psammomatous (4 cats), transitional (13 dogs, 2 cats) (Figs. 1–8). Except for 3 anaplastic meningiomas with 32, 19, and 9 mitotic figures per field (400×), mitotic index was 3 per field or fewer, with 15 tumors having no identified mitotic figures. Table 3 summarizes the immunohistochemical results.

Table 2.

Signalment, Site, and Histologic Type of Canine and Feline Meningiomas Examined

No.	Species	Breed	Age, years	Organ	Location	Type
1	Cat	Domestic shorthair	17	Cerebrum	Thalamus	Psammomatous
2	Cat	Unknown	Unknown	Brain	Unknown	Angiomatoid
3	Cat	Domestic shorthair	15	Brain	Piriform lobe	Psammomatous
4	Cat	Domestic shorthair	13	Cerebrum	Parietal lobe	Transitional
5	Cat	Domestic shorthair	16	Cerebrum	Longitudinal fissure	Transitional
6	Cat	Maine coon	12	Cerebrum	Left hemisphere	Psammomatous
7	Cat	Domestic shorthair	14	Cerebrum	Unknown	Psammomatous
8	Cat	Domestic shorthair	6	Cerebrum	Piriform lobe	Fibroblastic
9	Dog	Unknown	Unknown	Cerebrum	Unknown	Transitional
10	Dog	Unknown	Unknown	Cerebrum	Unknown	Transitional
11	Dog	Unknown	Unknown	Cerebrum	Unknown	Myxoid
12	Dog	Mixed breed	9	Cerebrum	Brainstem	Transitional
13	Dog	Labrador Retriever	11	Cerebrum	Thalamus	Microcystic
14	Dog	Mixed breed	Unknown	Brain	Unknown	Anaplastic
15	Dog	German Shepherd	7	Cerebrum	Olfactory bulb	Transitional
16	Dog	Golden Retriever	12.5	Cerebellum	Ventral	Transitional
17	Dog	Mixed breed	8	Cerebrum	Parietal lobe	Microcystic
18	Dog	Labrador Retriever	7	Brainstem	Brainstem	Anaplastic
19	Dog	American Eskimo	5	Spinal cord	Dorsal C1-C2	Fibroblastic
20	Dog	Mixed breed	6	Cerebrum	Unknown	Meningothelial
21	Dog	Boxer	11	Spinal cord	Dorsal T13-L1	Myxoid
22	Dog	Bernese	9	Cerebrum	Parietal lobe	Transitional
23	Dog	Maltese	17	Cerebellum	Cerebellum	Fibroblastic
24	Dog	Boxer	9	Cerebellum	Cerebellum	Anaplastic
25	Dog	Mixed breed	6	Spinal cord	Dorsal C1-C2	Myxoid
26	Dog	Schnauzer	12	Brain	Unknown	Transitional
27	Dog	Schnauzer	12	Brain	Unknown	Transitional
28	Dog	Labrador Retriever	6	Cerebrum	Occipital lobe	Fibroblastic
29	Dog	Labrador Retriever	4	Cerebrum	Parietal lobe	Anaplastic
30	Dog	Labrador Retriever	7	Spinal cord	C1	Anaplastic
31	Dog	Shetland Sheepdog	11	Cerebrum	Occipital lobe	Transitional
32	Dog	American Pit Bull Terrier	6	Cerebrum	Frontal lobe	Transitional
33	Dog	Mixed breed	14	Cerebrum	Frontal and olfactory bulbs	Transitional
34	Dog	West Highland Terrier	12	Cerebrum	Frontal lobe	Transitional
35	Dog	Dachshund	13	Cerebrum	Olfactory lobe	Anaplastic
36	Dog	Dachshund	9	Cerebrum	Frontal lobe	Transitional
37	Dog	Maltese	17	Brainstem	Cerebrum	Normal leptomeninges
38	Cat	Domestic shorthair	1	Cerebrum	Cerebrum	Normal leptomeninges
39	Cat	Domestic shorthair	1	Cerebrum	Cerebrum	Normal leptomeninges

Figure 1. Cerebrum; dog No. 14. Anaplastic meningioma. Mitosis (arrowheads). HE.

Figure 2. Cerebrum; cat No. 2. Angiomatoid meningioma. HE.

Figure 3. Spinal cord; dog No. 19. Fibroblastic meningioma. HE.

Figure 4. Cerebrum; dog No. 20. Meningothelial meningioma. HE.

Figure 5. Spinal cord; dog No. 17. Microcystic meningoma. Variably sized cystic spaces (asterisks) between meningeal cells. HE.

Figure 6. Cerebrum; dog No. 25. Myxoid meningioma. Myxomatous extracellular matrix (asterisks). HE.

Figure 7. Cerebrum; cat No. 3. Psammomatous meningioma. Psammoma bodies (asterisks). HE.

Figure 8. Cerebrum; dog No. 27. Transitional meningioma. Whorls of meningothelial cells (asterisks) surrounded by bundles of spindle cells. HE.

Figure 9. Cerebellum; dog No. 24. Anaplastic meningioma. Infiltrative behavior (arrowheads). Immunoperoxidase labeling for E-cadherin.

Figure 10. Spinal cord; dog No. 19. Fibroblastic meningioma. Spinal cord (S). Immunoperoxidase labeling for E-cadherin.

Figure 11. Cerebrum; dog No. 9. Transitional meningioma. Immunoperoxidase labeling for E-cadherin.

Figure 12. Cerebrum; dog No. 17. Microcystic meningioma. Immunoperoxidase labeling for E-cadherin. Inset: detail of the labeling.

Figure 13. Spinal cord; dog No. 21. Myxoid meningioma. Immunoperoxidase labeling for E-cadherin. Inset: note the granular pattern of the labeling in the cytoplasmic membrane (arrowhead).

Figure 14. Cerebrum; cat No. 4. Transitional meningioma. Immunoperoxidase labeling for E-cadherin. Inset: detail of the granular pattern of labeling (arrowhead).

Figure 15. Spinal cord; dog No. 30. Anaplastic meningioma. Immunoperoxidase labeling for claudin-1.

Figure 16. Cerebrum; cat No. 6. Psammomatous meningioma. Immunoperoxidase labeling for claudin-1.

Figure 17. Cerebrum; cat No. 6. Psammomatous meningioma. Detail of immunoperoxidase labeling for claudin-1.

Figure 18. Cerebrum; dog No. 13. Microcystic meningioma. Only the adjacent normal choroid plexus epithelium is positive (arrowheads). Immunoperoxidase labeling for claudin-1.

Figure 19. Spinal cord; dog No. 22. Transitional meningioma. Immunoperoxidase labeling for claudin-1.

Figure 20. Cerebrum; cat No. 4. Transitional meningioma. Immunoperoxidase labeling for claudin-1.

Figure 21. Cerebrum; dog No. 9. Transitional meningioma. Immunoperoxidase labeling for cytokeratins 8 and 18.

Figure 22. Cerebellum; dog No. 16. Transitional meningioma. Immunoperoxidase labeling for cytokeratins.

Figure 23. Cerebrum; dog No. 29. Anaplastic meningioma. Moderate cytoplasmic labeling of numerous neoplastic cells. Strong labeling of endothelial cells (arrowheads). Mitosis (arrows). Immunoperoxidase labeling for CD34.

Figure 24. Cerebrum; cat No. 8. Fibroblastic meningioma. Strong labeling of most neoplastic cells. Immunoperoxidase labeling for CD34.

Figure 25. Cerebrum; cat No. 4. Transitional meningioma. Meningothelial and spindloid cells are strongly labeled. Immunoperoxidase labeling for CD34.

Figure 26. Cerebrum; dog No. 17. Microcystic meningioma. Immunoperoxidase labeling for CD34.

Figure 27. Cerebrum; cat No. 2. Angiomatoid meningioma. Inset: Cerebellum; dog No.23. Fibroblastic meningioma. Immunoperoxidase labeling for glial fibrillary acidic protein.

Figure 28. Cerebrum; dog No. 9. Transitional meningioma. Immunoperoxidase labeling for glucose transporter 1.

Figure 29. Cerebrum; dog No. 17. Microcystic meningioma. Immunoperoxidase labeling for laminin. Inset: cerebrum, dog. Anaplastic meningioma. Laminin in this case is detected as globular precipitates of 3,3′-diaminobenzidine.

Figure 30. Cerebrum; dog No. 17. Microcystic meningioma. Immunoperoxidase labeling for progesterone receptor.

Figure 31. Cerebrum; cat No. 4. Transitional meningioma. Immunoperoxidase labeling for protein gene product 9.5.

Figure 32. Cerebrum; dog No. 22. Transitional meningioma. Immunoperoxidase labeling for S100.

Table 3.

Canine and Feline Meningiomas Examined: Immunohistochemical Results^a

No.	Type	Mitosis^b	CD34	Claudins	AE1/AE3^c	CK 8/18^d	EC^e	GFAP^f	Ki67	GLUT-1^g	Laminin	PR	PGP^h	S100	Vimentin
1	Psammomatous	0	3+	Neg	Neg	Neg	3+ M/C	Neg	ND	2+	3+ C	2+	3+	2+ C	3+
2	Angiomatoid	0	1+	1+ M/C	Neg	Neg	Neg	2+	1.9	3+	Neg	3+	3+	3+ N/C	3+
3	Psammomatous	0	3+	Neg	Neg	Neg	3+ M/C	Neg	2.9	Neg	3+ C	2+	3+	3+ N/C	3+
4	Transitional	0	3+	3+ C	Neg	Neg	3+ M/C	Neg	0.8	3+	3+ C	Neg	3+	Neg	3+
5	Transitional	0	ND	3+ C	ND	ND	3+ M/C	ND	0.4	1+	3+ C	ND	ND	2+ N/C	3+
6	Psammomatous	0	1+	3+ C	ND	ND	3+ M/C	Neg	1.8	1+	1+ C	ND	ND	3+ N/C	3+
7	Psammomatous	1	3+	3+ C	ND	ND	3+ M/C	Neg	1.1	1+	2+ C	ND	ND	1+ N/C	3+
8	Fibroblastic	0	3+	1+ C	ND	ND	3+ M/C	Neg	0	Neg	2+ C	ND	ND	Neg	3+
9	Transitional	1	2+	Neg	2+	2+	3+ M/C	Neg	52	3+	3+ C	Neg	1+	1+ C	3+
10	Transitional	0	3+	Neg	Neg	1+	3+ M/C	Neg	47	3+	3+ C	Neg	Neg	3+ N/C	2+
11	Myxoid	1	1+	Neg	Neg	Neg	Neg	3+	ND	Neg	Neg	2+	3+	3+ N/C	3+
12	Transitional	1	ND	Neg	Neg	Neg	3+ M/C	ND	ND	3+	Neg	Neg	Neg	1+ C	2+
13	Microcystic	0	1+	Neg	1+	Neg	3+ M/C	Neg	20	3+	2+ C	Neg	Neg	1+ C	3+
14	Anaplastic	32	2+	2+ M/C	Neg	Neg	Neg	Neg	250	2+	2+ C	2-3+	1+	3+ N	3+
15	Transitional	1	1+	2+ M/C	1+	Neg	3+ M/C	Neg	3.5	3+	3+ C	Neg	Neg	3+ C	2+
16	Transitional	0	1+	1+ M/C	1+	1+	3+ M/C	Neg	20	1+	Neg	Neg	1+	3+ N/C	3+
17	Microcystic	1	3+	Neg	1+	Neg	3+ M/C	Neg	24.5	3+	3+ C	3+	Neg	Neg	3+
18	Anaplastic	74	1+	2+ C	ND	ND	Neg	Neg	570	2+	2+ C	ND	3+	Neg	3+
19	Fibroblastic	0	2+	Neg	Neg	Neg	3+ M/C	Neg	8.9	3+	3+ C	Neg	Neg	3+ N	1+
20	Meningothelial	0	1+	ND	Neg	Neg	3+ M/C	Neg	ND	ND	Neg	Neg	Neg	Neg	3+
21	Myxoid	1	1+	Neg	1+	Neg	3+ M/C	Neg	31	2+	3+ C	3+	Neg	3+ N/C	3+
22	Transitional	1	3+	1+ C	1+	Neg	3+ M/C	Neg	60	3+	2+ C	2+	Neg	3+ N/C	3+
23	Fibroblastic	3	3+	3+ C/M	ND	ND	3+ M/C	3+	42	3+	3+ C/M	ND	ND	2+	3+
24	Anaplastic	3	Neg	1+ M/C	Neg	Neg	3+ M/C	Neg	36	2+	Neg	Neg	Neg	3+ C	2+
25	Myxoid	0	ND	Neg	ND	ND	3+ C	ND	13	Neg	Neg	ND	ND	2+ N/C	2+
26	Transitional	2	3+	Neg	ND	ND	3+ M/C	Neg	ND	1+	Neg	ND	ND	1+ N/C	1+
27	Transitional	1	3+	Neg	ND	ND	3+ M/C	Neg	45	2+	Neg	ND	ND	1+ N/C	2+
28	Fibroblastic	2	Neg	Neg	ND	ND	Neg	Neg	117	1+	3+ C	ND	ND	Neg	1+
29	Anaplastic	19	3+	3+ N/C	ND	ND	Neg	Neg	109	1+	3+ C/P	ND	ND	Neg	3+
30	Anaplastic	9	1+	3+ C	ND	ND	3+ M/C	Neg	140	1-2+	3+ C	ND	ND	Neg	2+
31	Transitional	0	3+	2+ C	ND	ND	3+ M/C	Neg	23	3+	Neg	ND	ND	1+ C	1+
32	Transitional	3	1+	2+ C	ND	ND	Neg	Neg	117	Neg	3+ C	ND	ND	Neg	2+
33	Transitional	3	3+	3+ C	ND	ND	3+ M/C	Neg	47	2+	2+ C	ND	ND	3+ C	2+
34	Transitional	3	3+	3+ C	ND	ND	3+ C	Neg	43	3+	2+ C	ND	3+	3+ C	3+
35	Anaplastic	2	2+	3+ C	ND	ND	3+ C	Neg	43	3+	3+ C	ND	1+	3+ C/N	3+
36	Transitional		3+	3+ C	ND	ND	3+ M/C	Neg	15	2+	2+ C	ND	1+	1+ C	3+
37	Normal lepto	0	+	+	Neg	Neg	+	Neg	0	+	+	ND	+	+	+
38	Normal lepto	0	+	+	Neg	Neg	+	Neg	0	+	+	ND	+	+	+
39	Normal lepto	0	+	+	Neg	Neg	Neg	Neg	0	+	+	ND	+	+	+
Total positive ^j (%)			94	60	39	17	81	9	NA	86	72	47	55	75	100

^a M, membrane; C, cytoplasmic; N, nuclear; P, paranuclear; ND, not done; NA, not applicable.

^b Average per high-power field of a total of 10 fields.

^c Pancytokeratins AE1/AE3.

^d Cytokeratins 8/18.

^e E-cadherin.

^f Glial fibrillary acidic protein.

^g Glucose transporter 1.

^h Progesterone receptor.

ⁱ Protein gene product 9.5.

^j Only meningiomas (case Nos. 1–36) were used for this calculation.

Normal Meninges

Immunoreactivity of normal canine meninges resembled that of normal feline meninges. CD34 antibody strongly labeled the external limiting layer of the leptomeninges and scattered cells in the deeper layers. Endothelial cells were also labeled. For E-cadherin, the reactivity was intense in the outer layer and patchy and less intense in cells in the inner aspect of the leptomeninges. Claudin-1 was not detected in the outer leptomeninges and only in some cells in the inner aspect. There was mild GLUT-1 granular cytoplasmic labeling of some cells in the external limiting layer and fewer labeled cells in the deeper aspect of the leptomeninges. Expression of laminin in normal leptomeninges was mostly located in cells of the outer layer with less reactivity in cells in deeper aspect. The leptomeninges were highly and diffusely labeled for PGP 9.5 and S100. Vimentin expression was moderate throughout. There was no labeling of normal leptomeninges with antibodies to pancytokeratins, cytokeratins 8 and 18, and GFAP.

Meningiomas

Antibody to E-cadherin decorated > 60% of neoplastic cells in 29 of 36 (81%) meningiomas (Figs. 9–14). The labeling was strong and present in the cytoplasmic membrane and the cytoplasm, with the cytoplasmic membrane more intensely labeled and with a granular pattern (see insets of Figs. 13, 14). Seven meningiomas did not react with the antibody to E-cadherin, including 3 anaplastic, 1 myxoid, 1 angiomatoid, 1 transitional, and 1 fibroblastic. Of 35 meningiomas, 21 (60%) expressed claudin-1. The reactivity for claudin-1 was cytoplasmic in all cases, with cytoplasmic membrane reactivity in 5 cases (Figs. 15–20). Membrane staining was diffuse or granular and more intense than cytoplasmic labeling. For claudin-1, 4 anaplastic meningiomas had a moderate or high reactivity, and 1 had low reactivity. The staining was high in 11 cases and moderate or low and patchy in the rest.

Pancytokeratins and cytokeratins 8 and 18 were detected in 7 of 18 meningiomas (39%; 4 transitional, 2 microcystic, 1 myxoid) and 3 of 18 meningiomas (17%; 3 transitional), respectively (Figs. 21, 22). Neither of the 2 anaplastic meningiomas examined for cytokeratins was positive. None of the tumors that expressed cytokeratins had strong immunoreactivity.

Of 33 meningiomas, 31 (94%) expressed CD34 with a diffuse cytoplasmic or granular pattern in most neoplastic cells or multifocally (Figs. 23–26). All feline meningiomas and all but 2 canine meningiomas (1 fibroblastic, 1 anaplastic) were positive. One anaplastic meningioma had a punctate pattern. Twenty meningiomas had moderate to high reactivity. Transitional meningiomas usually had a strong and diffuse reactivity, although in some instances the meningothelial cells were more strongly labeled than spindloid cells.

Of 33 meningiomas, 3 (9%) were reactive to antibody to GFAP: 1 feline angiomatoid, 1 canine myxoid, and 1 canine fibroblastic meningioma had moderate to strong cytoplasmic labeling (Fig. 27). Two GFAP-positive meningiomas were negative for E-cadherin and weakly positive for CD34, whereas the third tumor was strongly positive for these 2 markers.

Of 35 meningiomas, 22 (63%) had moderate to high reactivity to GLUT-1; 8 (23%) had weak reactivity; and 5 had no detectable expression. GLUT-1 expression did not correlate with type of meningioma (Fig. 28 ). The cytoplasm of GLUT-1 positive cells was diffusely labeled. GLUT-1 internal positive control was perivascular mesenchymal structures.

Although tumors with the highest mitotic index had high Ki67 index, in most cases there was little correlation between these 2 indices. Of 5 anaplastic meningiomas, 4 had a Ki67 index > 100. Other types with a Ki67 index > 100 were transitional (1 case) and fibroblastic (1 case). Tissue from 5 meningiomas was not available for Ki67 evaluation. One fibroblastic meningioma had a Ki67 index of 0. Interestingly, of 5 meningiomas with a Ki67 index > 100, 4 did not express E-cadherin, whereas 3 of 4 meningiomas with a Ki67 index > 100 expressed claudin-1.

Laminin labeled the cytoplasm and, much less commonly, the cytoplasmic membrane of neoplastic cells with moderate to high scores (25 cases, 69%) or a low score (1 case, 3%) (Fig. 29 ). Ten meningiomas were negative for laminin, including 5 of 13 canine transitional types. The laminin internal control was vascular basement membrane.

Progesterone receptor antibody decorated the nucleus of neoplastic cells in some tumors. Of 18 cases, 8 (44%) had moderate to high labeling; 10 cases (56%) were negative (Fig. 30 ).

PGP 9.5 usually labeled both the nucleus and the cytoplasm of neoplastic cells: Of 22 tumors, 7 (32%) had moderate to high labeling and 5 (23%) had low labeling (Fig. 31 ).

Of 36 meningiomas, 19 (53%) had moderate to high labeling with antibody to S100 protein; 8 (22%) had low scores; and 9 (25%) were negative. Labeling was nuclear and cytoplasmic in 13 cases, cytoplasmic in 9, and nuclear in 2 (Fig. 32). Two fibroblastic meningiomas were positive and 2 were negative. Other S-100-negative cases were evenly distributed among other meningioma types.

There was diffuse cytoplasmic vimentin labeling of all meningiomas in this study. The immunoreactivity was moderate to high in 29 cases and low in 4.

Discussion

In this study, we examined the immunoreactivity of 28 canine and 8 feline meningiomas using novel markers (CD34, E-cadherin, claudin-1, GLUT-1, laminin, and PGP 9.5) and previously reported markers (cytokeratins, GFAP, progesterone receptor, S100, and vimentin). Vimentin, CD34, GLUT-1, E-cadherin, and S100 were expressed in at least 75% of meningiomas examined.

With some exceptions, transitional meningioma is the most commonly reported meningioma type in dogs and cats.^{1,3,22,27,30,33,41} In our series, transitional meningioma was the most common type in dogs, but psammomatous meningioma was the most common type in cats. Meningiomas in cats tend to occur at a later age than do those in dogs.²² Similar results were observed in our series.

Two epithelial markers, EMA and cytokeratins, have been used in the immunohistochemical characterization of human meningiomas. EMA is expressed in 75% to 100% of meningiomas.^24,31,58 Although EMA is more consistently expressed in carcinomas than meningiomas, it is used to rule out some meningioma mimics, such as schwannomas, hemangiopericytomas, and solitary fibrous tumors in which this marker is not expressed.^11,24,31 Unfortunately, to our knowledge, no antibodies to EMA cross-react with canine and feline tissues.²² However, we examined the cytokeratin immunoreactivity in this group of meningiomas using pancytokeratins (clones AE1 and AE3) and a monoclonal antibody to cytokeratins 8 and 18. In human pathology, cytokeratins are detected in 6% to 80% of meningiomas.^{2,24,31,32,58} Interestingly, several studies comparing benign and malignant human meningiomas demonstrated high cytokeratin expression in malignant tumors, whereas cytokeratin expression in benign meningiomas was low or undetectable.^20,24 The expression of cytokeratins seemed to be inversely correlated with that of EMA in meningiomas, with increased cytokeratin expression and decreased EMA expression as tumor grade increased.^20,31 In a comprehensive study of cytokeratin expression in human meningiomas, cytokeratin 18 was the most commonly expressed cytokeratin in most meningioma types, followed by clone AE1.³² The 2 cytokeratin antibodies used in our series labeled few meningiomas and, in positive cases, usually a low or (rarely) moderate number of cells. One explanation could be that the use of antibodies that recognize multiple cytokeratins (eg, AE1 and AE3, cytokeratins 8 and 18) reduced the chances of detecting the cytokeratin more commonly expressed in meningiomas (eg, clone AE1, cytokeratin 18). Our results with cytokeratin immunohistochemistry of meningiomas are somewhat contradictory to those in human studies; none of the anaplastic meningiomas in our series were positive for cytokeratins, whereas human malignant or anaplastic meningiomas tend to have strong cytokeratin expression.^20,24 Studies in canine meningiomas have reported higher immunoreactivity for cytokeratins than that in this series, ranging from 18% to 73%, although the number of positive cells is usually low.^3,33 Based on our and others' results, cytokeratins are not a reliable marker for meningiomas, although the patchy reactivity typically observed in meningiomas can be used to rule out carcinoma, which usually expresses these proteins strongly and in most neoplastic cells.

To our knowledge, CD34 expression in meningiomas of domestic species has not been reported. CD34 was the second most common marker expressed in our series, after vimentin. The expression of CD34 in human meningiomas varies from 0% to 40%.^11,15,53 Contrary to CD34 expression in human meningiomas, which is typically observed in the fibroblastic type only, all types of canine and feline meningiomas strongly expressed CD34. This result is not surprising considering that, in our study, normal canine and feline leptomeninges strongly expressed CD34. CD34 is a protein in hematopoietic cells of the bone marrow and in endothelial cells.^13,23,44 This protein was recently reported in vascular endothelial cells of canine FFPE.⁵⁵ Using the same antibody, we detected CD34 expression in endothelial cells in a various canine and feline tissues. Interestingly, this marker is not expressed in human peripheral nerve sheath tumors (eg, neurofibromas, schwannomas).^11,15 Additional studies are needed to characterize CD34 reactivity in canine and feline peripheral nerve sheath tumors and other meningioma mimics to determine its specificity for meningiomas.

E-cadherin, a glycoprotein of the adherens junction, is expressed in most epithelial cells.^14,51 E-cadherin is consistently expressed in most human meningiomas^12,18,51,52 and has been used to distinguish human meningiomas from their mimics. E-cadherin is not expressed in human hemangiopericytoma, although it is consistently detected in schwannomas.^17,52 In addition, E-cadherin has not been detected in other human brain tumors except choroid plexus papilloma.⁵¹ Though not reported in meningiomas of domestic species, E-cadherin was detected in most meningomas in our series. Some studies of human meningiomas indicate a tendency to stronger expression of this protein in meningotheliomatous versus fibroblastic areas^12,51 or even lack of expression in the fibroblastic meningioma.⁶⁰ E-cadherin expression did not correlate with meningioma types in our series, although 2 of 4 anaplastic meningiomas were negative. The significance of expression (or lack thereof) of E-cadherin in human malignant meningiomas is controversial. Some reports indicated that many malignant meningiomas do not express this protein whereas all benign types do; tumors undergoing malignant transformation lack E-cadherin expression.⁵¹ However, differences in E-cadherin expression among different meningioma tumor grades have not been found in other studies.^6,12,52 In our series, all but 1 E-cadherin-positive tumor had both cytoplasmic membrane and cytoplasmic expression. The cytoplasmic expression was distinguished from the cytoplasmic membrane expression by its typical granular pattern. In a recent study, 34% of human meningiomas had cytoplasmic expression of E-cadherin without cytoplasmic membrane expression. The lack of cytoplasmic membrane expression did not correlate with tumor grade.⁶ Based on the consistent expression of E-cadherin in epithelial cells and their tumors, this marker is not useful as a single marker to distinguish meningiomas from metastatic carcinomas.

To our knowledge, the expression of claudin-1 in meningiomas of domestic species has not been reported. Claudins are tight junction–associated proteins. There are more than 15 types of claudins, and each is associated with certain cells and tissues.¹⁴ Claudin-1 is variably expressed in 50% to 100% of human meningiomas.^4,15,43,48 Of 33 meningiomas, 19 (58%) in our series reacted with antibody to claudin-1. Claudin-1 seems to be an excellent marker to distinguish human meningiomas from schwannomas, given that the latter do not express this protein.^4,15 Zero to 13% of human hemangiopericytomas, another possible mimic of meningiomas, have low expression of claudin-1^15,43; however, it is expressed in most perineuriomas.²⁶ Based on our results with meningiomas and on results in the human literature with meningiomas and their mimics, claudin-1 immunohistochemistry has high specificity (if epithelial neoplasms are excluded) and low to moderate sensitivity for meningiomas. Additional studies with canine and feline mesenchymal neoplasms are necessary to evaluate the specificity of claudin-1 for meningioma.

GFAP expression was moderate to high in 3 meningiomas: 1 feline angiomatoid, 1 canine myxoid, and 1 canine fibroblastic. GFAP immunoreactivity in meningiomas has been examined in 3 studies totaling 56 canine meningiomas, with only 3 cases being reactive: 2 fibroblastic and 1 anaplastic.^3,33,42 GFAP immunoreactivity in human meningiomas, albeit rare, has been reported.^7,56 We used the same antibody and a similar immunohistochemical protocol as in previous studies.^3,7,33,56 GFAP is considered to be a specific marker for glial cells and their tumors; however, based on our results and published studies, some meningiomas can be strongly immunoreactive to GFAP, when a differential diagnosis includes glioma or peripheral nerve sheath tumor.

GLUT-1 is expressed in erythrocytes, blood–brain barrier, liver, placenta, perineurial cells, germinal centers of lymphoid follicles, and renal tubules.^50,53 Human perineurial tumors commonly express GLUT-1 and may mimic meningiomas, particularly, meningothelial type.^16,50,53 This marker is expressed in 100% of human meningiomas, albeit with a wide range in the number of positive cells in a given tumor.⁵³ In our series, of 32 meningiomas, 27 (85%) were positive for GLUT-1. All anaplastic meningiomas expressed this protein to various degrees. Although GLUT-1 is a moderately to highly sensitive marker for meningiomas, it is expressed in some human and bovine meningioma mimics and should therefore be used in conjunction with other markers.³⁴

Ki67 immunohistochemistry is used to determine the growth fraction of a tumor.⁵ It has been used in human meningiomas as a prognostic marker, with conflicting results.¹¹ However, it appears that Ki67 index is associated with tumor behavior and recurrences.^11,20 Ki67 and proliferating cell nuclear antigen, a cell cycle–specific marker, were inversely correlated with progesterone receptor expression in canine meningiomas.^27,59 The apparent lack of correlation between the mitotic and Ki67 indices in this series of meningiomas might be explained by differences in the duration of the cycling phases of tumor cells or by difficulties in distinguishing mitotic cells from pyknotic nuclei.⁵

Laminin is a protein that is commonly present in basement membranes and the extracellular matrix. It is produced by a variety of mesenchymal and epithelial cell types and their tumors. The expression of laminin and other extracellular matrix proteins has been studied in human meningiomas^8,35,36,49 but, to our knowledge, not in meningiomas of domestic animals. This marker was included in our series to determine whether it could be used to distinguish meningiomas from some mimics, such as schwannomas, which typically express this marker.^29,39 In our series, 67% of meningiomas expressed laminin with no significant differences among histologic subtypes, although 50% of the transitional meningiomas did not express this marker. In an ultrastructural study of human meningiomas, the meningothelial type did not express laminin except in vascular structures, whereas in the fibroblastic type, laminin was present among the tumor cells but not within their cytoplasm.³⁶ In our series, all fibroblastic meningiomas had strong or moderate cytoplasmic expression and, less common, cytoplasmic membrane expression. Based on the presence of laminin in human schwannomas (and its expected expression in animal schwannomas), the expression of laminin in many canine and feline meningiomas makes it unsuitable to distinguish between meningioma and schwannoma.

Progesterone receptor is expressed in 70% to 100% of canine and feline meningiomas.^27,59 We examined progesterone receptor expression in only 19 meningiomas, and less than 50% were positive (3 of 4 feline and 5 of 19 canine meningiomas). All meningioma types in our series had at least one positive case except for the sole meningothelial type. In human meningiomas, lack of progesterone receptor expression is linked to an aggressive behavior.¹⁹ The significance regarding the loss of progesterone receptor expression in domestic animals is unknown.

PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase-1 originally reported to be expressed only in neurons and neuroendocrine cells.⁹ It is currently accepted that PGP 9.5 is expressed in a variety of tissue and cell types, including nerve sheath cells and their tumors.^9,46 We have observed PGP 9.5 immunoreactivity in canine peripheral nerve sheath tumors (unpublished observations). PGP 9.5 has not been used in the characterization of meningiomas. Our interest in using PGP 9.5 was to determine if it could be used to distinguish meningiomas from nerve sheath tumors. Of 22 meningiomas, 12 (55%) were positive for PGP 9.5 in our series, including most histologic types. Based on these results, PGP 9.5 is not a good marker to distinguish between nerve sheath tumors and meningiomas.

S100 protein is one of the most commonly used markers in human and animal meningiomas. It is expressed in human meningiomas but less commonly than other markers, such as vimentin, EMA, E-cadherin, and claudin-1; however, up to 80% of fibrous meningiomas express S100 protein.^2,11,31,47 In our series, S100 protein was detected in 24 of 32 meningiomas (75%), with more frequent nuclear and cytoplasmic labeling, rather than just cytoplasmic. In 2 series of canine meningiomas, most tumors expressed S100 protein^3,33; however, the number of positive cells varied, and the pattern was typically multifocal with labeling being nuclear, cytoplasmic, or both.³ Montoliu et al³³ reported a stronger labeling of fibroblastic than meningothelial cells. In many meningiomas in our series, there were no differences among cellular phenotypes or patterns, although in a limited number of cases, bundles of fibroblastic cells had stronger reactivity than that of adjacent whorls of meningothelial cells. S100 was a moderately sensitive marker in our series. However, this marker is expressed in multiple mesenchymal tumors of domestic animals, including some meningioma mimics (eg, schwannoma).¹⁰

Vimentin, a generic marker of mesenchymal neoplasms, is consistently expressed in human and animal meningiomas. In dogs and cats, vimentin is expressed in all histologic subtypes of meningiomas, and its reactivity is usually higher than with S100.^3,25,30,33 Our results are consistent with those in the literature. All meningiomas in this series were positive for vimentin, and most had a strong and diffuse reactivity. The use of vimentin in the characterization of meningiomas is still valuable when a differential diagnosis includes metastatic carcinoma, but it should be used in conjunction with other markers.

In summary, the markers used in this study labeled meningiomas in the following descending order based on the percentage of tumors that were positive: vimentin (100%), CD34 (94%), GLUT-1 (86%), E-cadherin (81%), S100 (75%), laminin (72%), claudin-1 (60%), PGP 9.5 (55%), progesterone receptor (44%), pancytokeratins (39%), cytokeratins 8 and 18 (17%), and GFAP (9%). Given these results and observations on human meningiomas and their mimics, we propose a basic immunohistochemical panel of vimentin, CD34, and E-cadherin for the characterization of canine and feline meningiomas. The additional use of claudin-1, albeit of moderate to low sensitivity in meningiomas of small animals, can help to rule out some mesenchymal neoplasms involving the brain and associated structures, such as schwannomas. CD34 was consistently expressed in canine and feline meningiomas; as such, it may be used to distinguish them from peripheral nerve sheath tumors. However, validation of these and other markers for meningiomas will require their evaluation in canine and feline tumors that could resemble meningiomas.

Footnotes

Acknowledgements

We very much appreciate the technical expertise of Dee DuSold in performing histologic preparations and immunohistochemical testing.

The authors declared that they had no conflicts of interest with respect to their authorship or the publication of this article.

The authors declared that they received no financial support for their research and/or authorship of this article.

References

Adamo

Cantile

Steinberg

: Evaluation of progesterone and estrogen receptor expression in 15 meningiomas of dogs and cats. Am J Vet Res 64:1310–1318, 2003.

Artlich

Scmidt

: Immunohistochemical profile of meningiomas and their histological subtypes. Hum Pathol 21:843–849, 1990.

Barnhart

Wojcieszyn

Storts

: Immunohistochemical staining patterns of canine meningiomas and correlation with published immunophenotypes. Vet Pathol 39:311–321, 2002.

Bhattacharjee

Adesina

Goodman

Powell

: Claudin-1 expression in meningiomas and schwannomas: Possible role in differential diagnosis [abstract]. J Neuropathol Exp Neurol 62:581, 2003.

Brugal

: Interpretation of proliferation markers. Comp Cytol Histol Lab 234–239, 1994.

Brunner

Romeike

BFM

Jung

Comtesse

Meese

: Altered expression of β-catenin/E-cadherin in meningiomas. Histopathology 49:178–187, 2006.

Budka

: Non-glial specificities of immunocytochemistry for the glial fibrillary acidic protein (GFAP): triple expression of GFAP, vimentin and cytokeratins in papillary meningioma and metastasizing renal carcinoma. Acta Neuropathol 72:43–54, 1986.

Caffo

Caruso

Galatioto

Meli

Cacciola

Germanò

Alafaci

Tomasello

: Immunohistochemical study of the extracellular matrix proteins laminin, fibronectin and type IV collagen in secretory meningiomas. J Clin Neurosci 15:806–811, 2008.

Campbell

Thomas

Lamps

Smoller

Folpe

: Protein gene product 9.5 (PGP 9.5) is not a specific marker of neural and nerve sheath tumors: An immunohistochemical study of 95 mesenchymal tumors. Mod Pathol 16:963–969, 2003.

10.

Cerilli

Wick

: Immunohistology of soft tissue and osseous neoplasms. In: Diagnostic Immunohistochemistry, ed. Dabbs

, 2nd ed., pp. 65–120. Churchill Livingstone, Philadelphia, PA, 2006.

11.

Commins

Pinsky

Hinton

: Meningiomas. In: Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, ed. Taylor

Cote

, 3rd ed., pp. 353–358. WB Saunders, Philadelphia, PA, 2006.

12.

Figarella-Branger

Pellisier

Bouillot

Bianco

Mayan

Grisoli

Rougon

: Expression of neural cell-adhesion molecule isoforms and epithelial cadherin adhesion molecules in 47 human meningiomas: correlation with clinical and morphological data. Mod Pathol 7:752–761, 1994.

13.

Fina

Molgaard

Robertson

Bradley

Monaghan

Delia

Sutherland

Baker

Greaves

: Expression of the CD34 gene in vascular endothelial cells. Blood 75:2417–2426

14.

Folpe

Billings

McKenney

Walsh

Nusrat

Weiss

: Expression of Claudin-1, a recently described tight junction-associated protein, distinguishes soft tissue perineuroma from potential mimics. Am J Surg Pathol 26:1620–1626, 2002.

15.

Hahn

Bundock

Hornick

: Immunohistochemical staining for Claudin-1 can help distinguish meningiomas from histologic mimics. Am J Clin Pathol 125:203–208, 2006.

16.

Hamazaki

Fujiwara

Okada

: Intraneural perineurioma involving the ulnar nerve. Pathol Int 54:371–375, 2004.

17.

Hasegawa

Muramatsu

Tohma

Fukaya

Fusijawa

Hayashi

Tachibana

Kida

Yamashita

Saito

: Expression of E-cadherin-catenin complex in human benign schwannomas. Histol Histopathol 17:39–44, 2002.

18.

Howng

S-L

C-H

Cheng

T-S

W-D

Lin

P-C

Wang

Hing

Y-R

: Differential expression of Wnt genes, β-catenin and E-cadherin in human brain tumors. Cancer Lett 183:95–101, 2002.

19.

Hsu

Efird

Hedley-Whyte

: Progesterone and estrogen receptors in meningiomas: prognostic considerations. J Neurosurg 86:113–120, 1997.

20.

Ikeda

Yoshimoto

: Immunohistochemical study of anaplastic meningioma with special reference to the phenotypic change of intermediate filament protein. Ann Diagn Pathol 7:214–222, 2003.

21.

Koestner

Bilzer

Fatzer

Schulman

Summers

Van Winkle

: Tumors of the meninges. In: Histological Classification of Tumors of the Nervous System of Domestic Animals, ed. Koestner

Bilzer

Fatzer

Schulman

Summers

Van Winkle

, 2nd ed., pp. 27–30. AFIP, Washington, DC, 1999.

22.

Koestner

Higgins

: Meningiomas. In: Tumors in Domestic Animals, ed. Meuten

, 4th ed., pp. 717–723. Iowa State Press, Ames, IA, 2002.

23.

Krause

Fackler

Civin

Stratford-May

: CD34: structure, biology, and clinical utility. Blood 87:1–13, 1996.

24.

Liu

Sturgis

Bunker

Saad

Tung

Raab

Silverman

: Expression of cytokeratin by malignant meningiomas: diagnostic pitfall of cytokeratin to separate malignant meningiomas from metastatic carcinoma. Mod Pathol 17:1129–1133, 2004.

25.

Pocknell

Lamb

Targett

: Concurrent benign and malignant multiple meningiomas in a cat: clinical, MRI and pathological findings. Vet Rec 152:780–782, 2003.

26.

Macarenco

Ellinger

Oliveira

: Perineurioma: a distinctive and underrecognized peripheral nerve sheath neoplasm. Arch Pathol Lab Med 131:625–636, 2007.

27.

Mandara

Ricci

Rinaldi

Sarli

Vitellozzi

: Immunohistochemical identification and image analysis quantification of oestrogen and progesterone receptors in canine and feline meningioma. J Comp Pathol 127:214–218, 2002.

28.

Mandrioli

Panarese

Cesari

Mandara

Marcato

Bettini

: Immunohisitochemical expression of h-telomerase reverse transcriptase in canine and feline meningiomas. J Vet Sci 8:111–115, 2007.

29.

Matsuo

Notohara

: Choroidal schwannoma: immunohistochemical and electron-microscopic study. Ophthalmologica 214:156–160, 2000.

30.

Mauldin

Deehr

Hetzke

Dubielzig

: Canine orbital menigiomas: a review of 22 cases. Vet Ophthalmol 3:11–16, 2000.

31.

McKeever

: Meningeal and related tumors. In: Diagnostic Immunohistochemistry, ed. Dabbs

, 2nd ed., pp. 787–793. Churchill Livingstone, Philadelphia, PA, 2006.

32.

Miettinen

Paetau

: Mapping of the keratin polypeptides in meningiomas of different types: an immunohistochemical analysis of 463 cases. Hum Pathol 33:590–598, 2002.

33.

Montoliu

Añor

Vidal

Pumarola

: Histological and immunohistochemical study of 30 cases of canine meningioma. J Comp Pathol 135:200–207, 2006.

34.

Murcia

Delhon

González

Vilas

Ramos-Vara

De las Heras

Nordhausen

Uzal

: Cluster of cases of malignant schwannoma in cattle. Vet Rec 163:331–335, 2008.

35.

Wong

ATC

: Expression of epithelial and extracellular matrix protein markers in meningiomas. Histopathology 22:113–126, 1993.

36.

Nitta

Yamashima

Yamashita

Kubota

: An ultrastructural and immunohistochemical study of extracellular matrix in meningiomas. Histol Histopathol 5:267–274, 1990.

37.

O’Rahilly

Muller

: The meninges in human development. J Neuropathol Exp Neurol 45:588–608, 1986.

38.

Owston

Ramos-Vara

: Histologic and immunohistochemical characterization of a testicular mixed germ cell sex cord-stromal tumor and a Leydig cell tumor in a dog. Vet Pathol 44:936–943, 2007.

39.

Palm

Furcht

: Production of laminin and fibronectin by Schwannoma cells: cell-protein interactions in vitro and protein localization in peripheral nerve in vivo. J Cell Biol 96:1218–1226, 1983.

40.

Patnaik

Jay

Hurvitz

: Intracranial meningioma: A comparative pathologic study of 28 dogs. Vet Pathol 23:369–373, 1986.

41.

Patnaik

Lieberman

Erlandson

Shaker

Hurvitz

: Paranasal meningioma in the dog: A clinicopathologic study of ten cases. Vet Pathol 23:362–368, 1986.

42.

Pérez

Vidal

González

Benavides

Ferreras

Villagrasa

Pumarola

: Orbital meningioma with a granular cell component in a dog, with extracranial metastasis. J Comp Pathol 133:212–217, 2005.

43.

Rajaram

Brat

Perry

: Anaplastic meningioma versus meningeal hemangiopericytoma: Immunohistochemical and genetic markers. Hum Pathol 35:1413–1418, 2004.

44.

Ramani

Bradley

Fletcher

CDM

: QBEND/10, a new monoclonal antibody to endothelium: assessment of its diagnostic utility in paraffin sections. Histopathology 17:237–242, 1990.

45.

Ramos-Vara

Beissenherz

: Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed, paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest 12:307–311, 2000.

46.

Ramos-Vara

Miller

: Immunohistochemical detection of protein gene product 9.5 (PGP 9.5) in canine epitheliotropic T-cell lymphoma (mycosis fungoides). Vet Pathol 44:74–79, 2007.

47.

Rishi

Coffey

Javed

Powell

Takei

: Immunohistochemical comparison of spindle cell lesions: schwannoma versus fibroblastic meningioma [abstract]. J Neuropathol Exp Neurol 66:439, 2007.

48.

Rivera

Takei

Bhattacharjee

Adesina

Goodman

Powell

: Claudin-1 versus EMA staining in low and high grade meningiomas. J Neuropathol Exp Neurol 66:439, 2007.

49.

Rutka

Giblin

Dougherty

McCullogh

DeArmond

Rosenblum

: An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures. J Neuropathol Exp Neurol 45:285–303, 1986.

50.

Salla

Johann

ACBR

Lana

AMA

do Carmo

MAV

Nunes

Mesquita

: Immunohistochemical study of GLUT-1 in oral peripheral nerve sheath tumors. Oral Dis 14:510–513, 2008.

51.

Schwechheimer

Zhou

Birchmeier

: E-cadherin in human brain tumors: Loss of immunoreactivity in malignant meningiomas. Virchows Arch 432:163–167, 1998.

52.

Shimada

Ishizawa

Hirose

: Expression of E-cadherin and catenins in meningioma: Ubiquitous expression and its irrelevance to malignancy. Pathol Int 55:1–7, 2005.

53.

Smith

MEF

Awasthi

O’Shaughnessy

Fisher

: Evaluation of perineurial differentiation in epithelioid sarcoma. Histopathology 47:575–581, 2005.

54.

Snyder

Shofer

Van Winkle

Massicotte

: Canine intracranial primary neoplasia: 173 cases (1986–2003). J Vet Intern Med 20:669–675, 2006.

55.

Stoica

Lungu

Martini-Stoica

Waghela

Levine

Smith

: Identification of cancer stem cells in dog glioblastoma. Vet Pathol 46:391–406, 2009.

56.

Ono

Tanaka

Takahashi

: An unusual meningioma variant with glial fibrillary acidic protein expression. Acta Neuropathol 94:499–503, 1997.

57.

Summers

Cummings

de LaHunta

: Meningeal tumors. In: Veterinary Neuropathology, ed. Summers

Cummings

de LaHunta

, pp. 355–362. Mosby, St. Louis, MO, 1995.

58.

Theaker

Gatter

Esiri

Fleming

: Epithelial membrane antigen and cytokeratin expression by meningiomas: an immunohistochemical study. J Clin Pathol 39:435–439, 1986.

59.

Théon

Mecouteur

Carr

Griffey

: Influence of tumor cell proliferation and sex-hormone receptors on effectiveness of radiation therapy for dogs with incompletely resected meningiomas. J Vet Med Assoc 216:701–707, 2000.

60.

Tohma

Yamashima

Yamashita

: Immunohistochemical localization of cell adhesion molecule epithelial cadherin in human arachnoid villi and meningiomas. Cancer Res 52:1981–1987, 1992.

61.

Troxel

Vite

Van Winkle

Newton

Tiches

Dayrell-Hart

Kapatkin

Shofer

Steinberg

: Feline intracranial neoplasia: retrospective review of 160 cases (1985–2001). J Vet Intern Med 17:850–859, 2003.

62.

Webster

Miller

DuSold

Ramos-Vara

: Effects of prolonged formalin-fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem 57:753–761, 2009.