Neoplastic cells are a rare component in human glioblastoma microvasculature

Abstract

Microvascular proliferation is a key biological and diagnostic hallmark of human glioblastoma, one of the most aggressive forms of human cancer. It has recently been suggested that stem-like glioblastoma cells have the capacity to differentiate into functional endothelial cells, and that a significant proportion of the vascular lining in tumors has a neoplastic origin. In principle, this finding could significantly impact the efficacy and development of antiangiogenic therapies targeting the vasculature. While the potential of stem-like cancer cells to form endothelium in culture seems clear, in our clinical experience using a variety of molecular markers, neoplastic cells do not contribute significantly to the endothelial-lined vasculature of primary human glioblastoma. We sought to confirm this impression by analyzing vessels in glioblastoma previously examined using chromogenic in situ hybridization (CISH) for EGFR and immunohistochemistry for mutant IDH1. Vessels containing cells expressing these definitive neoplastic markers were identified in a small fraction of tumors, but only 10% of vessel profiles examined contained such cells and when identified these cells comprised less than 10% of the vascular cellularity in the cross section. Interestingly, these rare intravascular cells showing EGFR amplification by CISH or mutant IDH1 protein by immunohistochemistry were located in the middle or outer portions of vessel walls, but not amongst the morphologic boundaries of the endothelial lining. To more directly address the capacity of glioblastoma cells to contribute to the vascular endothelium, we performed double labeling (Immunofluorescence/FISH) for the endothelial marker CD34 and EGFR gene locus. This analysis did not identify EGFR amplified CD34+ endothelial cells within vascular linings, and further supports our observation that incorporation of glioblastoma cells into the tumor vessels is, at best, extremely rare of questionable clinical or therapeutic significance.

Figure 1. Glioma specific molecular alterations are not a common feature of endothelial tumor vasculature in clinical samples

(a) PDGFRA amplification in tumor cells (arrow) but not in endothelial cells (arrowheads) in adjacent vessel (V). (b and c) Molecular alterations in neoplastic cells by immunohistochemistry, but not in vasculature (V), including EGFR overexpression (b) and PTEN loss (c). Quantification of vessels in tumors stained for IDH1 R132H mutant protein by immunohistochemistry (d-f) or EGFR amplification by CISH (g-i) revealed that most tumor cells showed positive staining, whereas the vessels (V) were devoid of signal (d and g). Tumor cells were found to crowd the perivascular region (e and h), but only rare vessels contained positive cells within the vascular wall (f and i).

Figure 2. Lack of significant EGFR alterations in CD34 positive endothelial cells

EGFR amplifications in glioblastoma neoplastic cells (arrowheads), but lacking in associated CD34+ endothelial cells (arrows)(a). Orange autofluorescence frequently identified intraluminal red blood cells (asterisks). Only a minority of CD34+ cells demonstrated extra EGFR copies, and at a similar frequency in glioblastomas with and without chromosome 7/EGFR abnormalities (b).