Phospho-AXL is widely expressed in glioblastoma and associated with significant shorter overall survival

Abstract

Receptor tyrosine kinase AXL (RTK-AXL) is regarded as a suitable target in glioblastoma (GBM) therapy. Since AXL kinase inhibitors are about to get approval for clinical use, patients with a potential benefit from therapy targeting AXL need to be identified. We therefore assessed the expression pattern of Phospho-AXL (P-AXL), the biologically active form of AXL, in 90 patients with newly diagnosed GBM, which was found to be detectable in 67 patients (corresponding to 74%). We identified three main P-AXL expression patterns: i) exclusively in the tumor vasculature (13%), ii) in areas of hypercellularity (35%), or iii) both, in the tumor vasculature and in hypercellular areas of the tumor tissue (52%). Pattern iii) is associated with significant decrease in overall survival (Hazard ratio 2.349, 95% confidence interval 1.069 to 5.162, *p=0.03). Our data suggest that P-AXL may serve as a therapeutic target in the majority of GBM patients.

Keywords: Pathology Section; glioblastoma multiforme (GBM); glomeroid tufts; overall survival; phospho-Axl (P-AXL); receptor tyrosine kinase AXL (AXL).

Figure 1

Immunohistochemical evaluation of serial sections of GBM tissue samples revealed the expression of both intracellular AXL phospho-sites - pTyr691 (A., C.) and pTyr779 (B., D.) - which were phosphorylated and expressed similarly in the tumor tissue. Immunofluorescent labeling of AXL (F., J.) and AXL phospho-site pTyr779 (G., K.) demonstrated colocalization (H., L.) of both antigens. Counterstaining was performed with 4’, 6-diamidino-2-phenylindoleI (DAPI; E., I.). (Scale bar: 100 μm A.-D., 20 μm A.-D.; inserts), 50 μm E.-L.)

Figure 2. Immunohistochemical double-staining of GBM tissue samples with antibodies against P-AXL (brown) and fibronectin (green) revealed three main P-AXL expression patterns

In samples classified as “tissue type”, P-AXL expression was mainly seen in areas of hypercellularity (A., C., E.). Tumor vessels which were highlighted by fibronectin staining were negative for P-AXL (arrows). Samples classified as “vessel type” showed exclusive P-AXL staining within the tumor vasculature (B., D., F.; arrowheads). The third pattern was characterized by P-AXL expression in both, the tumor tissue and vasculature (G., H.). P-AXL expression was detected in tubular (B., D., G.; arrowheads) and glomeruloid (F., H.; arrowheads) vascular proliferation. (Scale bar: 50 μm A.-D., F.-H.; 20 μm E.).

Figure 3

The expression pattern of P-AXL in hypercellular areas was further classified as focal (10-50%; A., C.) or diffuse (≥ 50%; B., D.). Focal expression was seen e.g. in pseudopalisades (A.; dashed line) or scattered clusters of tumor cells (C.; arrows). Other GBM samples demonstrated broad/global P-AXL expression (B., D.) which was pronounced in areas which often showed a typical herringbone-like pattern (D.; arrowheads). (Scale bar: 50 μm).

Figure 4

Immunohistochemical and immunofluorescent double-staining of GBM tissue samples revealed strong P-AXL expression in CD31 positive endothelial cells (A., B., D.; arrowheads), no colocalization with aSMA (B., C., D.), and partial colabeling with PDGFR-ß positive pericytes (E.; arrowheads) in microvascular proliferation. Glioma cells adjacent to microvascular proliferation (dashed lines) showed strong immunopositivity for PDGFR-ß (E.; arrows). P-AXL was further expressed by neoplastic glioma cells as noted by colabeling with GFAP (F.-H.; arrowheads), MAP2 (I.-K.; arrowheads), Nestin (L.-N.; arrowheads), and ZEB1 O.-Q.; arrowheads). (Scale bar: 20 μm A., 50 μm B.-Q.).

Figure 5. Kaplan Meier curve showing overall survival in days in GBM patients with P-AXL expression i) exclusively in the tumor vasculature, ii) in the hypercellular tumor tissue, or both iii) in tumor vasculature and hypercellular tumor tissue

The overall survival of patients with P-AXL expression in vital tumor tissue and tumor vessels (iii) was significantly reduced compared to patients with P-AXL expression in tumor tissue without vascular expression (ii). Log-rank (Mantel-Cox) Test *p = 0.0335, HR 2.349, 95% CI1.069 to 5.162.