Three-dimensional nuclear telomere architecture is associated with differential time to progression and overall survival in glioblastoma patients

Abstract

The absence of biological markers allowing for the assessment of the evolution and prognosis of glioblastoma (GBM) is a major impediment to the clinical management of GBM patients. The observed variability in patients' treatment responses and in outcomes implies biological heterogeneity and the existence of unidentified patient categories. Here, we define for the first time three GBM patient categories with distinct and clinically predictive three-dimensional nuclear-telomeric architecture defined by telomere number, size, and frequency of telomeric aggregates. GBM patient samples were examined by three-dimensional fluorescent in situ hybridization of telomeres using two independent three-dimensional telomere-measurement tools (TeloView program [P(1)] and SpotScan system [P(2)]). These measurements identified three patients categories (categories 1-3), displaying significant differences in telomere numbers/nucleus (P(1) = .0275; P(2) <or= .0001), telomere length (P(1) and P(2) = .0275), and number of telomeric aggregates (P(1) = .0464; P(2) <or= .0001). These categories corresponded to patients with long-term, intermediate, and short-term survival, respectively (P = .0393). The time to progression analyses showed significant differences between the three categories (P = .0167). There was a correlation between time to progression, median survival, and nuclear telomere architecture. Our study suggests a link between patient outcome and three-dimensional nuclear-telomere organization and highlights the potential clinical power of telomere signatures as a new prognostic, predictive, and potentially pharmacodynamic biomarker in GBM. Furthermore, novel automated three-dimensional high-throughput scanning as developed here permits to obtain data from 300 nuclei in 20 minutes. This method is applicable to any cell type and scanning application.

Figure 1

Two-dimensional (A, C, E) and three-dimensional images (B, D, F) of human GBM nuclei (blue) and their telomeres (red) imaged and visualized with AxioVision 4.6. Nuclear telomere distribution patterns define TAs indicated with an arrowin the three categories of patient: (A, B) from P7 in category 1, (C, D) from P9 in category 2, and (E, F) from P1 in category 3. The arrows point to the telomere aggregates.

Figure 2

Number of telomeres versus relative intensities of fluorescent signal analyzed by TeloView. Lines and arrows define the three categories of cells. (A) Category 1, P7. (B) Category 2, P9. (C) Category 3, P1. P = .0275.

Figure 3

Screenshots of human GBM nuclei (blue) and telomeres (red) with ScanView 6.0. Upper panel: Screenshot of a selected field of the gallery of 20 scanned cells. Bottompanel: The corresponding histogram showing the distribution of classified defined cells based on the number of telomeres/cell. (A) Category 1, P7. (B) Category 2, P9. (C) Category 3, P1. For patient details, see Table 1.

Figure 4

Kaplan-Meier curves depicting the TTP and overall survival of the three categories of population defined by telomere organization.

Figure 5

(A) Human GBM nuclei presenting breakage bridge fusion (BBF), with their telomere distribution. Arrows point to nuclear bridges indicative of breakage bridge fusion cycles. (B) Human GBM nuclei presenting nuclear fusion. These data are observed in all three patient categories. A qualitative evaluation showed that these characteristic figures of GBM are most prominent in category 3, followed by category 2 and category 1.