Nuclear factor IA is expressed in astrocytomas and is associated with improved survival

Abstract

Nuclear factor IA (NFIA) is a transcription factor that specifies glial cell identity and promotes astrocyte differentiation during embryonic development. Its expression and function in gliomas are not known. Here, we examined NFIA protein expression in gliomas and its association with clinical outcome in pediatric malignant astrocytomas. We analyzed expression of NFIA by immunohistochemistry in 88 existing glioma specimens from Childrens Hospital Los Angeles and the University of Southern California. Association between NFIA expression and progression-free survival (PFS) was examined in high-grade astrocytomas for which clinical data were available (n = 23, all children). NFIA was highly expressed in astrocytomas of all grades, but only in a minority of cells in oligodendroglial tumors. NFIA was expressed on a higher percentage of tumor cells in low-grade astrocytomas (91 +/- 5% and 77 +/- 14% in World Health Organization [WHO] I and II, respectively) compared with high-grade astrocytomas (48 +/- 18% and 37 +/- 16% in WHO III and IV, respectively; P < .001, low- vs high-grade astrocytomas). There was a significant association between NFIA expression and PFS in children with astrocytoma WHO grade III or IV (Cox regression P = .019; logrank trend test for NFIA tertiles P = .0040 and NFIA quartiles P = .014). The association was not consistently significant in this small series of patients after adjustment was made for WHO grade III or IV. This is the first study to demonstrate expression of NFIA protein in astrocytomas and its association with grades of astrocytoma and PFS, suggesting that NFIA may play a role in astrocytoma biology.

Fig. 1.

NFIA expression is restricted to astrocyte-lineage cells in normal human cerebral cortex. Imunohistochemical analysis of NFIA (brown) in paraffin sections of human gliomas, counterstained with hematoxylin (blue) as described in the Materials and Methods section. (A) Cerebral cortex, (B) SVZ, (C) white matter, and (D) double staining with NFIA (brown) and GFAP (pink). Arrows show astrocytic cells, which express both NFIA and GFAP. Arrowheads indicate neurons, which express neither marker.

Fig. 2.

NFIA is highly expressed in human astrocytomas. (A) H&E and immunohistochemistry for NFIA of 4 representative pediatric astrocytomas WHO grades I–IV. Left column is H&E stain (×400), middle column is anti-NFIA stain (×400), and right column is a 2-fold magnification of the inset from the middle column. Endothelial cells, which lack NFIA immunoreactivity, were used as an internal negative control (arrowheads). (B) Quantification of NFIA expression in pediatric and adult gliomas and normal brains. Bars represent mean ± SD for pilocytic astrocytoma (I, n = 21), astrocytoma (II, n = 21), anaplastic astrocytoma (III, n = 20), and GBM (IV, n = 12). Comparison is with nontumor containing cerebrum (NL, n = 10), oligodendroglioma (II, n = 7), and anaplastic oligodendroglioma (III, n = 5). *P < .0001 in the continuum of the 4 WHO grades of astrocytoma (one-way ANOVA) and P < .001 between the low-grade (WHO I and II) and the high-grade (WHO III and IV) astrocytomas (Student's t-test). P value is also <.0001 between astrocytomas and oligodendrogliomas (t-test). See Table 2 for details.

Fig. 3.

NFIA is expressed highly in perivascular tumor cells in glioblastomas. Immunohistochemical stain for NFIA in GBM: (A and B) perivascular infiltrating tumor cells at the tumor border (top row; magnification: left panel ×100 and right panel ×400). (C) Nonperivascular area of tumor showing lower expression of NFIA in the tumor cells (same patient; magnification ×400). (D) Percent NFIA positive cells compared with total tumor cells in perivascular compared with nonperivascular areas in 2 cases of GBM. Shown is the analysis of 3 representative high-power fields from 2 invasive adult GBMs that were quantified for perivascular NFIA expressing tumor cells compared with nonperivascular ones. NFIA-positive cells are brown, nuclear stain is blue.

Fig. 4.

NFIA is only minimally expressed in oligodendroglial tumors. Immunohistochemical analysis of NFIA in oligoastrocytomas (top row), oligodendrogliomas (middle row), and anaplastic oligodendrogliomas (lower row) was performed as in Fig. 2. Shown are representative fields. Quantification of the percent NFIA-positive tumor cells is shown in Fig. 2B. In oligoastrocytomas, cells with astrocytic features (increased pleomorphism and atypia; arrows) expressed NFIA, but cells with oligodendroglial features (round nuclei and perinuclear halos; arrowheads) were negative. In the pure oligodendrogliomas (middle and lower panels), only rare cells expressed NFIA.

Fig. 5.

Upregulation of NFIA mRNA (microarrays) is associated with longer OS in adult GBM patients. Kaplan–Meier plots (OS) analyzed and derived from the publicly available REMBRANDT expression microarray glioma data set for adult GBM patients grouped according to NFIA mRNA expression level (4 of 4 NFIA probesets available).¹⁷ Red curves (high NFIA expression): ≥2-fold increase in NFIA expression compared with nontumor brain samples, yellow curves (intermediate): intermediate between >2-fold increase and >2-fold decrease of NFIA mRNA (ie, >0.5- and <2.0-fold NFIA expression level compared with nontumor brain sample), or green (low): ≥2-fold decrease in NFIA expression compared with nontumor tissue. Blue line depicts all GBM samples combined. The table under the 4 panels shows sample number and P values calculated by the REMBRANDT website.

Fig. 6.

Higher NFIA expression is associated with longer PFS in pediatric high-grade astrocytoma patients (WHO III and IV). Kaplan–Meier plots of PFS in pediatric patients with high-grade astrocytoma (WHO III and IV). (A) Patients grouped into 3 equal groups according to % tumor cells expressing NFIA: high NFIA (blue dashed), intermediate NFIA (black, solid), and low NFIA (red, solid). P = .0040 was by logrank test for trend. Tick marks indicate length of follow-up in 4 patients without disease progression. (B) Patients grouped by WHO grade: grade IV, GBM (red line), and grade III, anaplastic astrocytoma (dashed blue line). P = .0112 by logrank test (Mantel–Cox). Numbers of patients are shown in the panels. Median survival is shown in Table 3.