WT1 expression distinguishes astrocytic tumor cells from normal and reactive astrocytes

Abstract

Particularly in small brain biopsies, it might be difficult to distinguish reactive astrogliosis from low-grade or infiltration zones of high-grade astrocytomas. So far no immunohistochemical marker allows a reliable distinction. Recently, the over-expression of Wilms' tumor gene product WT1 was reported in astrocytic tumor cells. However, no sufficient data on WT1 expression in normal or reactive astrocytes are available. Therefore, we investigated WT1 expression in paraffin-embedded brain sections from 28 controls, 48 cases with astrogliosis of various etiology and 219 astrocytomas [World Health Organization (WHO) grades I-IV] by immunohistochemistry. In normal brains and in astrogliosis, expression of WT1 was restricted to endothelial cells. In astrocytomas, WT1-positive tumor cells were found in pilocytic astrocytomas (66.7% of cases), diffuse astrocytomas (52.7%) WHO grade II (52.7%), anaplastic astrocytomas (83.4%) and glioblastomas (98.1%). Overall, the majority of all astrocytic neoplasms (84.5%) expressed WT1. Establishing a cut-off value of 0% immunoreactive tumor cells served to recognize neoplastic astrocytes with 100% specificity and 68% sensitivity and was associated with positive and negative predictive values of 1 and 0.68, respectively. Therefore, WT1 expression in astrocytes indicates a neoplastic origin and might represent an important diagnostic tool to differentiate reactive from neoplastic astrocytes by immunohistochemistry.

Figure 1

WT1 immunoreactivity in normal brain and Wilms' tumor. In normal brain specimens expression of WT1 is restricted to endothelial cells, both in gray (A) and in white matter (B). No WT1 expression is found in glial cells (A, B). In Wilms' tumor serving as positive control the vast majority of tumor cells and endothelial cells showed strong nuclear expression of WT1 (C) (scale bar: 100 µm).

Figure 2

WT1 immunoreactivity in reactive astrogliosis. Thin pathological vessels of a cavernoma [A, hematoxylin and eosin (H&E)] are surrounded by reactive astrocytes with eosinophilic cytoplasm (inset). Reactive astrocytes are strongly positive for glial fibrillary acidic protein (GFAP, B), whereas WT1 expression remains restricted to endothelial cells of capillaries and cavernomatous vessels (C). Residual infarction aged 4 months shows glial scar formation with reactive astrocytes (D, H&E), which express GFAP (E). In the same region WT1 immunoreactivity also remains restricted to endothelial cells (F). Astrogliosis because of a brain metastasis of an adenocarcinoma (G, H&E). Reactive astrocytes express GFAP (H) but not WT1 (I). Note WT1 expression in tumor vessels and in brain capillaries (scale bars: A–I, 200 µm; insets, 50 µm).

Figure 3

Illustration of WT1 immunoreactivity scores. Representative photomicrographs illustrating WT1 immunoreactivity scores in glioblastomas. A. WT1 expression is limited to endothelial cells of capillaries (score 0). B. A few WT1‐positive cell processes and single (<1%) WT1+ tumor cells (arrows) are present (score 1). Glioblastomas showing WT1 expression in 1–19% (C, score 2), in 20 to 50% (D, score 3) or in more than 50% of tumor cells (E, score 4). Isotype control demonstrating specificity of the primary WT1 antibody (F) (scale bar: 100 µm).

Figure 4

Distribution of different WT1 scores in neoplastic glial cells. Overall illustration of the frequency of different WT1 scores in astrocytomas grades I–IV WHO. Note absence of WT1 expression in normal brain and astrogliosis (all cases score 0, not shown). In astrocytomas of each WHO grade, all WT1 scores are present in variable frequency, respectively.

Figure 5

WT1 immunoreactivity in astrocytomas. Immunostaining for WT1 in astrocytic neoplasms. A. Glioblastoma (grade IV WHO) with areas of pseudopalisading necrosis (score 4); B. Glioblastoma with WT1 enhancement around pathological tumor vessels (score 3); C. Another glioblastoma with WT1 expression predominating in pleomorphic tumor cells (score 3); D. Anaplastic astrocytoma, grade III WHO (score 3); E. Gemistocytic astrocytoma grade II WHO (score 2); and F. Pilocytic astrocytoma grade I WHO (score 3) show cytoplasmic WT1 in varying number of positive cells (scale bars: A–F, 100 µm).

Figure 6

Diagnostic value of WT1 in daily routine. A–C. A 69‐year‐old patient with a left paraventricular mass lesion considered foremost clinically to be a glioblastoma. Stereotactic biopsy showed mild to moderate increase in cellularity and moderate nuclear pleomorphism but no mitotic activity, necrosis or microvascular proliferation (A, H&E stain). Therefore, the differential diagnosis between reactive astrogliosis and the infiltration zone of an astrocytoma was difficult. Assumption of an infiltration zone being more likely was strengthened by WT1 expression in astrocytic cells (B). The surgical specimen proved the neoplastic nature of the lesion and was finally diagnosed as a glioblastoma (C, H&E stain). D–F. A 59‐year‐old patient with a clinically unclear parahippocampal lesion, with glioblastoma or a lymphoma consided foremost. No steroids were applied prior to the biopsy. Because of complications during the stereotactic procedure only a very small tissue specimen was obtained intraoperative diagnosis. The frozen section revealed a moderate increase in cell density with some gemistocytic astrocytes (D, H&E stain). WT1 expression on frozen‐tissue was limited to endothelial cells (E), and there were no Ki67‐positive cells (not shown). Therefore, a reactive astrogliosis was considered to be more likely than the infiltration zone of a glioma. During the following weeks, rapid spread of the lesion further favored glioblastoma. Two months after stereotaxic biopsy an open surgical biopsy was performed yielding the diagnosis of a primary cerebral B‐cell lymphoma (F).