ABSTRACT

Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO 2 based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO 2 . There was substantial inter-and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO 2 ) rather than severe (defined as approximately 0.1% pO 2 ). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.

A newly developed water-soluble phosphor suitable for measuring oxygen pressure in the blood (Green 2W) was used for noninvasive, in vivo imaging of oxygen distribution in the vascular systems of mice. Oxygen quenches the phosphorescence of Green 2W, measured in the presence of 2% albumin, according to the Stern-volmer relationship. This oxygen-dependent quenching of phosphorescence has been used to obtain digital maps of the oxygen distribution in the tissue vasculature. EMT-6 mammary carcinoma tumors were grown by injecting 1 x 10(6) cells in 0.1-ml carrier into the subcutaneous space over the muscle on the hindquarter. When the tumors were approximately 8 mm in diameter, 300 micrograms of phosphorescence probe (Green 2W; absorption maximum 636 nm) was injected into the tail vein. The mice were immobilized with intraperotoneal Ketamine (133 mg/kg) and Xylazine (10 mg/kg) and illuminated with flashes (< 4-microseconds t1/2) of light of 630 +/- 12 nm. The emitted phosphorescence (790-nm maximum) was imaged an intensified CCD camera. Images were collected beginning at 30, 50, 80, 120, 180, 240, 420, and 2500 microseconds after the flash and used to calculate digital maps of the phosphorescence lifetimes and oxygen pressure. Both the illumination light and the phosphorescence were in the near-infrared region of the spectrum, where tissue has greatly decreased absorbance. The light therefore readily passed through the skin and centimeter thicknesses of tissue. The oxygen maps could be obtained by illuminating from the side of the mouse opposite the camera (and tumor). The tumors were readily observed as regions with oxygen pressures substantially below those of the surrounding tissue. Thus, phosphorescence measurements can noninvasively detect volumes of tissue with below-normal oxygen pressure in the presence of much larger volumes of tissue with normal oxygen pressures. In addition, tissue oxygen pressures can be monitored in real time, even through centimeter thicknesses of tissue.

The standard of care for glioblastoma (GB) is surgery followed by concurrent radiation therapy (RT) and temozolomide (TMZ) and then adjuvant TMZ. This regime is associated with increased survival but also increased occurrence of equivocal imaging findings, e.g., tumor progression (TP) versus treatment effect (TE), which is also referred to as pseudoprogression (PsP). Equivocal findings make decisions regarding further treatment difficult and often delayed. Because none of the current imaging assays have proven sensitive and specific for differentiation of TP versus TE/PsP, we investigated whether blood-derived microvesicles (MVs) would be a relevant assay. METHODS: 2.8 ml of citrated blood was collected from patients with GB at the time of their RT simulation, at the end of chemoradiation therapy (CRT), and multiple times following treatment. MVs were collected following multiple centrifugations (300g, 2500g, and 15,000g). The pellet from the final spin was analyzed using flow cytometry. A diameter of approximately 300 nm or greater and Pacific Blue–labeled Annexin V positivity were used to identify the MVs reported herein. RESULTS: We analyzed 19 blood samples from 11 patients with GB. MV counts in the patients with stable disease or TE/PsP were significantly lower than patients who developed TP (P = .014). CONCLUSION: These preliminary data suggest that blood analysis for MVs from GB patients receiving CRT may be useful to distinguish TE/PsP from TP. MVs may add clarity to standard imaging for decision making in patients with equivocal imaging findings.

No abstract

Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-lH-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)

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.

Increased Na+-K+ATPase activity during epinephrine treatment or hemorrhage contributes to muscle lactate production. Hypoxia is not necessarily the sole cause of hyperlactatemia during and after hemorrhagic shock.