The Relationship among Hypoxia, Proliferation, and Outcome in Patients with De Novo Glioblastoma: A Pilot Study

Abstract

The hypoxia and proliferation index increase with grade in human glial tumors, but there is no agreement whether either has prognostic importance in glioblastomas. We evaluated these end points individually and together in 16 de novo human glioblastomas using antibodies against the 2-nitroimidazole hypoxia detection agent EF5 and the proliferation detection agent Ki-67. Frozen tumor tissue sections were fluorescence-stained for nuclei (Hoechst 33342), hypoxia (anti-EF5 antibodies), and proliferation (anti-Ki-67 antibodies). EF5 binding adjacent to Ki-67+ cells, overall EF5 binding, the ratio of these values, and the proliferation index were evaluated. Patients were classified using recursive partitioning analysis and followed up until recurrence and/or death. Recursive partitioning analysis was statistically significant for survival (P = .0026). Overall EF5 binding, EF5 binding near Ki-67+ cells, and proliferation index did not predict recurrence. Two additional survival analyses based on ratios of the overall EF5 binding to EF5 binding near Ki-67+ cells were performed. High and low ratio values were determined by two cutoff points: (a) the 50% value for the ratio [EF5/Ki-67(Binding)]/[Tumor(binding)] = Ratio(EF5 50%) and (b) the median EF5 value (75.6%) of the ratio [EF5/Ki-67(Binding)]/[Tumor(binding)] = Ratio(patients median). On the basis of the Ratio(EF5 50%), recurrence (P = .0074) and survival (P = .0196) could be predicted. Using the Ratio(patients median), only survival could be predicted (P = .0291). In summary, patients had a worse prognosis if the [EF5/Ki-67(Binding)]/[Tumor(binding)] ratio was high. A hypothesis for the mechanisms and translational significance of these findings is discussed.

Figure 1

Examples of EF5, Ki-67 (proliferation), and Hoechst 33342 (viable cells) binding in GBs. Color codes: hypoxia (EF5 binding, red), viable cells (Hoechst 33342, blue) and proliferation (Ki-67, green). Left panel: patient 39; right panel: patient 55. Both patients have regions of moderate hypoxia, but in patient 39, all proliferating cells are in oxic regions compared with patient 55, where proliferating hypoxic cells are seen. It should be noted that yellow cells in this image are moderately hypoxic and proliferating, but many proliferating cells close to the moderately hypoxic regions are mildly hypoxic.

Figure 2

Analysis of EF5 value surrounding Ki-67+ cells: The upper left image depicts nuclei stained with Hoechst 33342 (the same process is done using Ki-67-stained cells, not shown). The upper central image is the point map that results from the counting algorithm described in text. A similar map is generated from the Ki-67 image (not shown). The upper right image shows the correlation between the counted points and the original Hoechst 33342 image. The mask shown in the lower left is generated from the upper left and middle images using the identified nuclei as reference points for cells and the base Hoechst image to threshold against. In combination, this yields an accurate map of viable tissue regions. The lower middle image shows the EF5 image after calibration and a Gaussian blur to eliminate hard edges and maintain the overall binding levels. The lower right image shows the combination of all images: the tissue mask has been applied to eliminate off tissue areas (black), calibrated EF5 binding levels are shown in varying intensities of red, green dots are Ki-67+ cells, and the blue rings are the 50-µm regions around these cells in which the PO₂ levels are calculated.

Figure 3

Analysis of time to tumor recurrence in 16 newly diagnosed GB patients based on their [EF5/Ki-67_binding]/[Tumor_binding] ratio. Patients with a Ratio_{EF5 50%} less than 50% (n = 4; dotted line) have a significantly longer time to recurrence than those with a Ratio_{EF5 50%} more than 50% (n = 12; solid line); P = .0074. Using the cutoff point of Ratio_{patients median}, this factor was not significant for recurrence (data not shown).

Figure 4

Analysis of time to patient survival in 16 newly diagnosed GB patients based on their [EF5/Ki-67_binding]/[Tumor_binding] ratio. (A) Patients with a Ratio_{EF5 50%} less than 50% (n = 4; dotted line) have a significantly longer time to recurrence than those with a RatioEF5 50% more than 50% (n = 12; solid line); P = .0196. (B) Comparing patients below and above of the Ratio_{patients median} value, patients had a significantly longer time to recurrence when this value was greater than the median (75.6%; n = 6); P = .0291 (dotted line).

Figure 5

Multivariate analysis of 16 newly diagnosed GB patients based on their [EF5/Ki-67_binding]/[Tumor_binding] ratio and their RPA classification. When considered along with RPA classification for overall survival, Ratio_{EF5 50%} was found to be an independent prognostic factor (P = .038). When considered along with RPA classification for overall survival, Ratio_{patients median} was found to be of borderline significance prognostic factor (P = .09). Dotted line = Ratio_{EF5 50%} < 50%, solid line = Ratio_{EF5 50%} < 50%.