OLIG2 immunolabeling in feline ependymoma

Abstract

Ependymoma, one of the most common gliomas in cats, occurs most often in the lateral and third ventricles and has variable histologic patterns that often form rosettes and pseudorosettes. Oligodendrocyte transcription factor (OLIG2) is expressed in oligodendrocyte precursor cells and mature oligodendrocytes. Although widely used as a diagnostic marker for most gliomas, OLIG2 is reported to have minimal immunolabeling in ependymomas. Here we characterize the OLIG2 immunolabeling pattern in 19 cases of feline ependymoma, which occurred predominantly in the lateral and third ventricles. Immunohistochemistry for GFAP was variable in 14 cases and was typically localized in the cytoplasmic processes of the neoplastic ependymal cells, especially in the rosettes and pseudorosettes. Nuclear OLIG2 immunolabeling was present in 17 cases and varied in intensity from weak (4 cases) to strong (13 cases). The distribution of OLIG2 immunolabeling within the neoplasms included none (2 cases), <25% (7 cases), 25–50% (6 cases), 51–75% (2 cases), and >75% (3 cases). OLIG2 immunolabeling intensity and distribution is widespread in feline ependymoma, in contrast to ependymomas in other species, and should not be relied upon as a specific marker for feline oligodendroglioma.

Ependymomas are uncommon tumors in domestic animals; however, they are observed more frequently in cats than in other species, including dogs. ¹² In the cat, these tumors typically arise in association with the lateral or third ventricles but can occur in extraventricular locations and in the spinal cord. ¹³ Clinical signs are widely variable and dependent on the location of the mass and the associated parenchymal changes that they cause. Histologic features are dominated by pseudorosettes and rosettes, although other features such as ependymal canals, apical cilia, and blepharoplasts can be seen more rarely.^3,8,13 Subtypes of ependymoma are recognized in the World Health Organization (WHO) classification system of tumors of the human CNS, including classic, papillary, clear-cell, tanycytic, and anaplastic; all of these subtypes have been reported in cats.^7,11,13 To date, no data exist on the relationship between the histologic subtype and prognosis of feline ependymoma; however, detailed outcome data are available for human ependymomas based on the WHO classification scheme. ⁷

Immunolabeling for glial acidic fibrillary protein (GFAP) has long been used to bolster the histologic diagnosis of ependymoma. Labeling is noted most consistently in the pseudorosettes and more sporadically in other areas of the tumor.^7,13 Negative labeling for GFAP has been recorded in feline ependymoma and should not be used to disfavor the diagnosis. More sporadic immunolabeling of feline ependymomas has been noted with pancytokeratin, similar to the scenario encountered in human ependymomas. ¹³ Oligodendrocyte transcription factor (OLIG2) was first identified as a critical mediator of oligodendrocyte differentiation during development and has been widely recognized to be strongly expressed in a variety of gliomas, including oligodendroglioma and astrocytoma.^2,10 OLIG2 immunolabeling is scant in both human and canine ependymoma, with <1% of neoplastic cell immunolabeling in a canine ependymoma study.^7,8 There are no reports of the pattern of OLIG2 immunolabeling in feline ependymoma. Here we describe the clinicopathologic findings and the GFAP and OLIG2 immunolabeling features of 19 cases of feline ependymoma.

Cases of feline ependymomas were identified retrospectively from the archives of the Athens Veterinary Diagnostic Laboratory (College of Veterinary Medicine, University of Georgia, Athens, GA, USA), the New York State Animal Health Diagnostic Center (College of Veterinary Medicine, Cornell University, Ithaca, NY, USA), the Texas A&M Veterinary Medical Diagnostic Laboratory (College Station, TX, USA), and the personal archives of one author (A.D. Miller) between January 2005 and December 2021. All cases were re-reviewed (by A.D. Miller) and assigned a histologic subtype based on the WHO classification scheme and previously published reports in the cat. ¹³

Formalin-fixed, paraffin-embedded tissues were immunolabeled for GFAP (Z0334, rabbit polyclonal; Agilent) and OLIG2 (ab109186, rabbit monoclonal; Abcam) via an automated immunohistochemistry (IHC) system (Bond Mac; Leica).^11,13 In brief, protocols for both antibodies are as follows: slides were dewaxed with Bond dewax solution (Leica). Bond epitope retrieval solution 2 (Leica) was applied for 30 min (OLIG2). Peroxide block was applied for 5 min (GFAP and OLIG2). For GFAP, antibody was diluted at 1:3,000 and applied for 15 min. For OLIG2, antibody was diluted at 1:2,000 and applied for 60 min. Following primary antibody incubation, Bond polymer reagent was applied to both for 30 min (Leica). This was followed by Bond polymer refine detection (Leica) for 10 min. Lastly, hematoxylin was applied for 5 min. Positive controls for both antibodies consisted of feline brain tissue and internal positive control. Negative controls consisted of isotype-matched antibodies. GFAP was performed for additional support of the diagnosis of ependymoma. For GFAP, the labeling was consistently cytoplasmic, and intensity (negative, weak, moderate, strong) and distribution (none, <25%, 25–50%, 51–75%, >75%) was evaluated. The pattern of OLIG2 immunolabeling in the neoplastic cell population was recorded according to location (intranuclear), intensity (negative, weak, moderate, strong), and distribution (none, <25%, 25–50%, 51–75%, >75%).

We included 19 cases in our study (Suppl. Table 1). The average age of cats was 9 y, and breeds included domestic shorthaired (11 cases), domestic longhaired (5 cases), Siamese (2 cases), and Ragdoll (1 case). No sex predilection was noted, with an approximately even breakdown between male (9 cases) and female (10 cases) cats. When available for review, clinical signs included head pressing, behavioral changes, abnormal gait, circling, and altered mentation (Suppl. Table 1). Sixteen cases were autopsies, and 3 cases were biopsies. For all of the autopsy cases, masses were roughly spherical, smooth and white-to-tan on section, and often bulged into the adjacent neuroparenchyma. Neuroanatomic locations included the lateral ventricle (11 cases), third ventricle (3 cases), mesencephalic aqueduct (2 cases), fourth ventricle (1 case), and spinal cord (1 case). One biopsy case did not have the location recorded, and location could not be determined from the sample submitted.

Histologically, ependymomas were classified as classic (18 cases) and clear-cell (1 case; Table 1). Cases 6, 13, and 14 have been published in a case series of feline ependymoma; case 10 was published in a retrospective study of feline glioma.^11,12 Briefly, the classic ependymomas were characterized by pseudorosettes and rosettes of tall columnar ependymal cells that occasionally formed papillary projections (Fig. 1). The clear-cell ependymoma had a similar growth pattern, but was formed by cells with abundant, clear cytoplasm. Cytoplasmic GFAP immunolabeling was negative in 5 cases, weak in 5 cases, and strong in 9 cases (Fig. 2). The distribution of GFAP immunolabeling was absent (5 cases), <25% (3 cases, all corresponding to a weak intensity pattern), 25–50% (4 cases), 51–75% (4 cases), and >75% (3 cases). OLIG2 immunolabeling was always intranuclear. OLIG2 immunolabeling intensity was negative in 2 cases (Fig. 3), weak in 4 cases (Fig. 4), and strong in 13 cases (Figs. 5, 6). The distribution of OLIG2 immunolabeling was absent (2 cases), <25% (6 cases), 25–50% (6 cases; Fig. 4), 51–75% (2 cases; Fig. 5), and >75% (3 cases; Fig. 6).

Table 1.

Histologic diagnosis and immunolabeling pattern, distribution, and intensity for GFAP and OLIG2 in 19 cases of feline ependymoma.

Case	Ependymoma type	GFAP immunolabeling intensity	GFAP labeling distribution, %	OLIG2 immunolabeling intensity	OLIG2 labeling distribution, %
1	Classic	Weak	<25	Strong	25–50
2	Classic	Strong	>75	Strong	25–50
3	Classic	Negative	NA	Strong	>75
4	Classic	Strong	51–75	Strong	25–50
5	Classic	Weak	<25	Strong	>75
6	Classic	Weak	51–75	Strong	>75
7	Classic	Strong	>75	Strong	25–50
8	Classic	Strong	25–50	Strong	25–50
9	Classic	Strong	25–50	Weak	<25
10	Classic	Strong	25–50	Negative	NA
11	Classic	Negative	NA	Strong	25–50
12	Classic	Weak	<25	Weak	<25
13	Clear-cell	Negative	NA	Strong	<25
14	Classic	Negative	NA	Negative	NA
15	Classic	Strong	51–75	Strong	51–75
16	Classic	Negative	NA	Weak	<25
17	Classic	Weak	25–50	Strong	51–75
18	Classic	Strong	>75	Weak	<25
19	Classic	Strong	51–75	Strong	<25

GFAP = glial fibrillary acidic protein; NA = not applicable; OLIG2 = oligodendrocyte transcription factor 2.

Figures 1-6.

Feline ependymoma. Figure 1. Classic feline ependymoma with prominent pseudorosettes and cords of neoplastic ependymal cells; case 3. H&E. Figure 2. Abundant cytoplasmic GFAP immunolabeling of neoplastic ependymal cells forming the pseudorosettes; case 19. Figure 3. OLIG2 immunolabeling is absent in the neoplastic cells; case 14. Figure 4. Weak intranuclear OLIG2 immunolabeling in <25% of the neoplastic cells; case 16. Figure 5. Strong intranuclear OLIG2 immunolabeling in 25–50% of the neoplastic cells; case 7. Figure 6. Strong intranuclear OLIG2 immunolabeling in >75% of the neoplastic cells; case 3.

OLIG2 is a diagnostic IHC marker that labels oligodendrocyte precursor cells in the developing brain, oligodendrocytes in the adult brain, and broadly labels glial neoplasms including oligodendrogliomas, astrocytomas, and undefined gliomas in which, based on the type of neoplasm, it can also label cells that are dually positive for GFAP, among other markers. ⁴ Given the labeling pattern for OLIG2 in gliomas, it has been used increasingly as a diagnostic immunomarker in humans and veterinary species.^4,5 The diagnostic utility of OLIG2 is strengthened by limited or absent immunolabeling in other primary tumors of the CNS in humans, including ependymomas and embryonal tumors.^6,9 This utility was confirmed in a case series of canine ependymomas in which sparse OLIG2 immunolabeling was recorded in ependymal neoplasms compared to oligodendroglioma, which had robust immunolabeling. ⁸ This immunolabeling specificity is in direct contrast to our study, in which we found a wide and diverse OLIG2 immunolabeling pattern among feline ependymomas. This immunolabeling pattern appears to be unique among species in which neoplastic ependymal OLIG2 immunolabeling has been studied.^8,9

Although the embryologic origin of ependymal cells is not fully defined, the consensus is that ependymal cells are terminally differentiated cells that are either differentiated from radial glial cells or are derived from clonal proliferation of multiprogenitor stem cells of the subventricular zone. ¹ The significance of OLIG2 labeling in feline ependymoma is unclear, but it may represent retained stemness in the neoplastic ependymal cells in cats or lineage differences in the differentiation of neoplastic glial cells in the feline CNS compared to other species. Strong immunolabeling for OLIG2 has been reported in feline oligodendroglioma, with labeling absent in astrocytomas. ¹¹ Importantly, and unexpectedly, widespread OLIG2 immunolabeling occurred in the feline ependymomas that we studied, and OLIG2 was less specific to oligodendroglioma in cats compared to other species. Therefore, OLIG2 should not be relied upon as a specific diagnostic immunomarker for feline oligodendroglioma.