Stephen M. Hahn

Hypoxia is important in the biology and aggression of human glial brain tumors.

We investigated whether increasing levels of tissue hypoxia, measured by the binding of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] or by Eppendorf needle electrodes, were associated with tumor aggressiveness in patients with previously untreated glial brain tumors. We hypothesized that more extensive and severe hypoxia would be present in tumor cells from patients bearing more clinically aggressive tumors. Hypoxia was measured with the 2-nitroimidazole imaging agent EF5 in 18 patients with supratentorial glial neoplasms. In 12 patients, needle electrode measurements were made intraoperatively. Time to recurrence was used as an indicator of tumor aggression and was analyzed as a function of EF5 binding, electrode values and recursive partitioning analysis (RPA) classification. On the basis of EF5 binding, WHO grade 2 tumors were characterized by modest cellular hypoxia (pO2s ≈ 10%) and grade 3 tumors by modest-to-moderate hypoxia (pO2s ≈ 10%- 2.5%). Severe hypoxia (≈0.1% oxygen) was present in 5 of 12 grade 4 tumors. A correlation between more rapid tumor recurrence and hypoxia was demonstrated with EF5 binding, but this relationship was not predicted by Eppendorf measurements.

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, Tempol

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.

Comparative Measurements of Hypoxia in Human Brain Tumors Using Needle Electrodes and EF5 Binding

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 pO2 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 pO2. 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% pO2) rather than severe (defined as approximately 0.1% pO2). 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.

Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage.

The protective effect of stable nitroxide radicals against oxidative damage was studied using cardiomyocyte cultures obtained from newborn rats. Monolayered cardiomyocytes were exposed to H2O2 and the effect on spontaneous beating and leakage of LDH was determined. Hydrogen peroxide irreversibly blocked rhythmic beating and resulted in a significant membrane injury as shown by release of LDH. The injury was prevented by catalase which removes H2O2 and by cell-permeable, metal-chelating agents such as desferrioxamine or bipyridine. In contrast, reagents which are excluded from the cell such as superoxide dismutase or DTPA did not protect the cells against H2O2. Five- and six-membered ring, stable nitroxide radicals which have previously been shown to chemically act as low-molecular weight, membrane-permeable, SOD-mimetic compounds provided full protection. The nitroxides prevented leakage of LDH and preserved normal cardiomyocyte contractility, presumably by intercepting intracellular O2-radicals. Alternatively, protection may result through nitroxides reacting with reduced transition metal ions or by detoxifying secondary organic radicals.

Factors Affecting the Risk of Brain Metastases After Definitive Chemoradiation for Locally Advanced Non–Small-Cell Lung Carcinoma

PURPOSE As therapy for locally advanced non-small-cell lung carcinoma (NSCLC) improves, brain metastases (BM) may become a greater problem. We analyzed our chemoradiation experience for patients at highest risk for the brain as the first failure site. METHODS Records for 150 consecutive patients with stage II/III NSCLC treated definitively with chemoradiation from June 1992 to June 1998 at the University of Pennsylvania were reviewed. Most patients (89%) received cisplatin, paclitaxel, or both. All had negative brain imaging before treatment. Posttreatment brain imaging was performed for suspicious symptoms. Incidence of BM was examined as a function of age, sex, histology, stage, performance status, weight loss, tumor location, surgery, radiation dose, initial radiation field, chemotherapy regimen, and chemotherapy timing. RESULTS Crude and 2-year actuarial rates of BM were 19% and 30%, respectively. Among pretreatment parameters, stage IIIB was associated with a higher risk of BM (P <.04) versus stage II/IIIA. Histology alone was not significant (P <.12), although patients with IIIB nonsquamous tumors had an exceptionally high 2-year BM rate of 42% (P <.01 v all others). Examining treatment-related parameters, crude and 2-year actuarial risk of BM were 27% and 39%, respectively, in patients receiving chemotherapy before radiotherapy and 15% and 20%, respectively, when radiotherapy was not delayed (P <.05). On multivariate analysis, timing of chemotherapy (P <.01) and stage IIIA versus IIIB (P <.01) remained significant. CONCLUSION Patients with later stage, nonsquamous NSCLC, particularly those receiving induction chemotherapy, have sufficiently common BM rates to justify future trials including prophylactic cranial irradiation.

The RAS signal transduction pathway and its role in radiation sensitivity.

RAS has been shown to increase radiation resistance. Upstream and downstream pathways from RAS could thus be targets for manipulation of radiosensitivity. EGFR expression and AKT phosphorylation are also associated with the response to radiation. A retrospective study evaluating EGFR and AKT in patients treated with multimodality therapy found a significant association between P-AKT and treatment failure. Moreover, these data are strengthened by in vitro studies showing that inhibition of EGFR, RAS, PI3K, and AKT radiosensitized cancer cell lines. We have previously shown that PI3K is a mediator of RAS-induced radiation resistance. We now suggest that EGFR, which is upstream of PI3K, may also mediate resistance through a common pathway. In addition to EGFR and RAS, PTEN can also regulate the PI3K pathway. Identifying a common signal for EGFR, RAS, or PTEN that results in radiation resistance may uncover targets for developing molecular-based radiosensitization protocols for tumors resistant to radiation and thus improve local control.

Nelfinavir Down-regulates Hypoxia-Inducible Factor 1α and VEGF Expression and Increases Tumor Oxygenation: Implications for Radiotherapy

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1alpha, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1alpha/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.

A phase I study of topical Tempol for the prevention of alopecia induced by whole brain radiotherapy.

Purpose: Complete alopecia is a universal complication of whole brain radiation therapy which contributes to patient anxiety over treatment. Tempol, a nitroxide radioprotector, has been shown to protect against radiation-induced alopecia in an animal model. This phase Ib study was designed to evaluate the safety and side effect profile of topical Tempol in patients with brain metastases being treated with whole brain radiotherapy. Experimental Design: Twelve patients with metastatic cancer to the brain were enrolled in the study between October 2000 and February 2003. Tempol (70 mg/ml concentration solution) was applied topically to the scalp 15 minutes before and washed off immediately after the completion of each of 10 fractions of whole brain radiation. Pharmacokinetic studies to evaluate the systemic absorption of Tempol were performed. Patients were assessed for toxicity before, during, and after Tempol administration. A secondary end point of the study, hair retention, was also scored. Results: Eleven patients were treated with topical Tempol. Adverse events that were considered possibly, probably, or definitely related to Tempol, included asymptomatic grade 2 (two patients) and grade 1 (one patient) hypoglycemia, grade 1 forehead skin redness (one patient), grade 1 dry scalp (one patient), and grade 1 tingling sensation on the scalp (one patient). Tempol was not detected in blood samples from more than 50% of the patients. Mean maximum Tempol levels for individual patients at any time point varied from 0.4 to 3.1 μmol/L. Hair retention was localized to the base of the scalp where the Tempol solution pooled after application in the first four patients on the study. Subsequently, full scalp hair retention was seen in three of final five evaluable patients after gauze had been wrapped around the head to hold the solution against the scalp. Conclusions: This study demonstrates that topical application of Tempol to the scalp before whole brain radiation is safe and well tolerated. Evidence of protection against radiation-induced alopecia was observed. A phase II study that uses a gel formulation to increase the exposure of scalp to Tempol has been initiated.

Epidermal growth factor receptor inhibition modulates the microenvironment by vascular normalization to improve chemotherapy and radiotherapy efficacy.

Background Epidermal growth factor receptor (EGFR) inhibitors have shown only modest clinical activity when used as single agents to treat cancers. They decrease tumor cell expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF). Hypothesizing that this might normalize tumor vasculature, we examined the effects of the EGFR inhibitor erlotinib on tumor vascular function, tumor microenvironment (TME) and chemotherapy and radiotherapy sensitivity. Methodology/Principal Findings Erlotinib treatment of human tumor cells in vitro and mice bearing xenografts in vivo led to decreased HIF-1α and VEGF expression. Treatment altered xenograft vessel morphology assessed by confocal microscopy (following tomato lectin injection) and decreased vessel permeability (measured by Evans blue extravasation), suggesting vascular normalization. Erlotinib increased tumor blood flow measured by Power Doppler ultrasound and decreased hypoxia measured by EF5 immunohistochemistry and tumor O2 saturation measured by optical spectroscopy. Predicting that these changes would improve drug delivery and increase response to chemotherapy and radiation, we performed tumor regrowth studies in nude mice with xenografts treated with erlotinib and either radiotherapy or the chemotherapeutic agent cisplatin. Erlotinib therapy followed by cisplatin led to synergistic inhibition of tumor growth compared with either treatment by itself (p<0.001). Treatment with erlotinib before cisplatin led to greater tumor growth inhibition than did treatment with cisplatin before erlotinib (p = 0.006). Erlotinib followed by radiation inhibited tumor regrowth to a greater degree than did radiation alone, although the interaction between erlotinib and radiation was not synergistic. Conclusions/Significance EGFR inhibitors have shown clinical benefit when used in combination with conventional cytotoxic therapy. Our studies show that targeting tumor cells with EGFR inhibitors may modulate the TME via vascular normalization to increase response to chemotherapy and radiotherapy. These studies suggest ways to assess the response of tumors to EGFR inhibition using non-invasive imaging of the TME.

Short-Term and Long-Term Health Risks of Nuclear-Power-Plant Accidents

Recent natural disasters in Japan led to a partial meltdown at the Fukushima nuclear power plant. This article reviews the history of such accidents, along with the short-term and long-term health risks associated with environmental exposure to nuclear fission products.