Julia A. O'Hara

Carboplatin as a potentiator of radiation therapy

A rationale for coordinating the administration of carboplatin with radiation to achieve enhancement of cancer therapy is developed. This approach is based upon a review of the reports of effects in a variety of systems, effects attributed to interactions between cisplatin or other platinum analogs and radiation. Two major effects include radiosensitization (RS) of hypoxic cells with platinum present during irradiation and potentiation of cell kill with platinum complexes administered after irradiation. Both these effects are expected to result in an improved therapeutic ratio. The latter effect may include inhibition of recovery from radiation-induced potentially lethal damage (PLD) and sublethal damage (SLD). Evidence for RS by carboplatin with an enhancement ratio (ER) of 1.8 is presented in Chinese hamster lung cells (V79) irradiated in culture under hypoxic conditions. Potentiation of radiation therapy in mice bearing a transplanted mouse mammary tumor (MTG-B) is reported as a supra-additive tumor growth delay when 60 mg/kg carboplatin is administered either 30 minutes before or immediately after 20 Gy of X-irradiation. Improved efficacy resulting from ongoing clinical trials coordinating cisplatin with radiation should support the role for carboplatin as a potentiator of radiation therapy since this second generation complex of platinum also interacts with radiation and larger concentrations of platinum should be attainable in tumors using the new drug.

Changes in Oxygenation of Intracranial Tumors With Carbogen: a BOLD MRI and EPR Oximetry Study

To examine, using blood oxygen level dependent (BOLD) MRI and EPR oximetry, the changes in oxygenation of intracranial tumors induced by carbogen breathing.

Effect on regrowth delay in a murine tumor of scheduling split-dose irradiation based on direct pO2 measurements by electron paramagnetic resonance oximetry

Tumor reoxygenation after irradiation may contribute to a tumors response to subsequent doses of radiation. The timing of reoxygenation in RIF-1 murine tumors was determined using electron paramagnetic resonance (EPR) oximetry with intratumoral implantation of an oxygen-sensitive paramagnetic material (India ink) to monitor the pO2 in individual murine tumors before, during and after three different irradiation schemes. Radiation was given as a single 20-Gy dose or was split into two 10-Gy doses where the second dose of radiation was delivered at the minimum postirradiation tumor pO2 (24-h interval, hypoxic group) or where the second dose of radiation was delivered after reoxygenation had occurred (72-h interval, oxygenated group). The end point for tumor response was time taken to reach double the volume at the time of treatment. There were significantly longer tumor doubling times in the oxygenated compared to the hypoxic group, indicating that the measured changes in pO2 reflected changes in tumor radiosensitivity. A 24-h interval between doses resulted in a delay of reoxygenation in the tumors, while a 72-h interval resulted in a second cycle of hypoxia/reoxygenation. Our results suggest that repeated direct measurements of pO2 in tumors by EPR oximetry could be useful in timing radiation doses to achieve improved local control of tumors.

Production of DNA double-strand breaks by interactions between carboplatin and radiation: a potential mechanism for radiopotentiation.

Hemopoiesis is the product of two components: the hemopoietic tissue and the regulatory stromal microenvironment in which it resides. Plutonium-239, incorporated during fetal development, is known to cause deficient hemopoiesis. A predetermined equivalent gamma-ray dose has now been used in combination with cross-transplantation experiments to separate these two components and define where the damage arises. It was confirmed that 1.8 Gy gamma irradiation at midterm gestation caused a 40% reduction in the hemopoietic stem (spleen colony-forming) cell population of their offspring which persisted to at least 24 weeks of age. Spleen colony formation after sublethal doses of gamma rays reflected this reduced complement of endogenous stem cells. The regulatory hemopoietic microenvironment, measured as fibroblastoid colony-forming cells, was similarly depleted. Normal growth of the CFU-S population after transplantation into standard recipients showed that the quality of the stem cell population in the offspring of irradiated mothers was not affected. By contrast, when used as recipients of a bone marrow transplant from either normal or irradiated offspring, the offspring of irradiated mothers were unable to support normal growth: there was a twofold difference in the number of CFU-S per femur for at least 100 days after transplantation. There were 70% fewer CFU-F in the femur 1 month after bone marrow transplantation when the offspring of irradiated mothers were used as transplant recipients compared to when normal offspring were used. This not only confirmed their reduced capacity to host normal stem cells but also indicated that CFU-F in the transplant were unable to compensate for the poor microenvironment in the irradiated offspring hosts. It is concluded that irradiation at midterm gestation damages the developing regulatory microenvironment but not the hemopoietic stem cell population that it hosts.

Enhancement of radiation-induced cell kill by platinum complexes (carboplatin and iproplatin) in V79 cells

Two second generation platinum complexes currently undergoing clinical chemotherapeutic trials, carboplatin (CBDCA) and iproplatin (CHIP), were evaluated for their ability to alter the survival of cultured Chinese hamster V79 cells following irradiation. Two protocols were employed. In the first, the drug was added to preplated cells, some of which were subsequently made hypoxic with nitrogen gas. These hypoxic cells were irradiated following 1 hour exposure to drug and survival was assessed by standard colony forming unit (CFU) methods. Enhancement ratios (ER) of approximately 1.4 were obtained for irradiation under hypoxic conditions, if the cells were exposed to equitoxic doses of CBDCA (500 microM) CHIP (50 microM). In the second series of experiments, cells were treated with 10 Gy in air and then incubated for various times prior to trypsinization and serial dilution of single cell suspensions. Six hours after irradiation, cells treated with X rays alone had recovered to produce a surviving fraction twice that of cells trypsinized immediately after irradiation (not held). Post-irradiation administration of CBDCA (50 microM) or CHIP (20 microM), at a time when free radical-mediated radiosensitization would not be possible, operationally inhibited this recovery from radiation-induced potentially lethal damage (PLD). Inhibition, expressed as recovery inhibition factor (RIF) after 6 hr with drug, was 2.0 for CBDCA and 1.2 for CHIP. These results suggest that the rationale for designing clinical trials to exploit interactions between cisplatin and radiation might also extend to include combined modality therapy using radiation with either of these two platinum complexes.

In Vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy

Abstract In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)–induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 ± 25 nm emission band relative to the fluorescence intensity integrated over the entire 400–600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.

The pO2 in a Murine Tumor after Irradiation: An In Vivo Electron Paramagnetic Resonance Oximetry Study

Using electron paramagnetic resonance (EPR) oximetry with the oxygen-sensitive paramagnetic material, fusinite, we have measured the partial pressure of oxygen (pO2) in the mouse mammary adenocarcinoma MTG-B. The average pO2 in untreated tumors was low (about 5 mm Hg) and decreased with tumor growth. Magnetic resonance imaging and histological examination were used to localize the position of the fusinite with respect to tumor margins and vascularization. The pO2 was generally higher in the periphery than in the center of the tumors, but there was considerable variation among tumors both during normal growth and after radiation treatment. After a single 20-Gy dose, a characteristic pattern of change in tumor pO2 was observed. In irradiated tumors, there was an initial reduction in pO2 (minimum occurred 6 h postirradiation) which was followed by a transient increase in pO2 to levels higher than the preirradiation pO2 (maximum occurred 48 h postirradiation). This work demonstrates postirradiation changes in pO2 of potential radiobiological significance. Compared to other oxygen assessment techniques, EPR oximetry is very useful because it can assess pO2 in the same region of the tumor over the course of tumor growth and during response to treatment. Thus EPR could be used to identify potentially radioresistant tumors as well as to identify tumors with slow reoxygenation.

IMAGING TUMOR VARIATION IN RESPONSE TO PHOTODYNAMIC THERAPY IN PANCREATIC CANCER XENOGRAFT MODELS

PURPOSE A treatment monitoring study investigated the differential effects of orthotopic pancreatic cancer models in response to interstitial photodynamic therapy (PDT), and the validity of using magnetic resonance imaging as a surrogate measure of response was assessed. METHODS AND MATERIALS Different orthotopic pancreatic cancer xenograft models (AsPC-1 and Panc-1) were used to represent the range of pathophysiology observed in human beings. Identical dose escalation studies (10, 20, and 40J/cm) using interstitial verteporfin PDT were performed, and magnetic resonance imaging with T2-weighted and T1-weighted contrast were used to monitor the total tumor volume and the vascular perfusion volume, respectively. RESULTS There was a significant amount of necrosis in the slower-growing Panc-1 tumor using high light dose, although complete necrosis was not observed. Lower doses were required for the same level of tumor kill in the faster-growing AsPC-1 cell line. CONCLUSIONS The tumor growth rate and vascular pattern of the tumor affect the optimal PDT treatment regimen, with faster-growing tumors being relatively easier to treat. This highlights the fact that therapy in human beings shows a heterogeneous range of outcomes, and suggests a need for careful individualized treatment outcomes assessment in clinical work.

MRI-coupled Fluorescence Tomography Quantifies EGFR Activity in Brain Tumors

RATIONALE AND OBJECTIVES This report demonstrates the diagnostic potential of magnetic resonance imaging (MRI)-coupled fluorescence molecular tomography (FMT) to determine epidermal growth factor receptor (EGFR) status in brain cancer. MATERIALS AND METHODS Two orthotopic glioma xenograft models were used in this study: one represented high EGFR expression and the other low expression. Nude mice were inoculated with cells from either one of the tumor lines or were used in a sham surgery control group. Animals were imaged using a unique MRI-FMT scanner 48 hours after intravenous injection of a near-infrared fluorophore bound to epidermal growth factor (EGF) ligand. Coronal images of fluorescence activity of the injected dye in the mouse brain were recovered using the MRI images as anatomical templates. RESULTS In vivo images of fluorescence activity showed significant differences between animal populations, an observation confirmed by receiver operating characteristic analysis that revealed 100% sensitivity and specificity between animal groups implanted with EGFR((+)) and EGFR((-)) tumor lines. Similar performance was observed between EGFR((+)) and sham surgery control animals. CONCLUSIONS This preclinical study suggests that MRI-FMT with fluorescent EGF provides excellent discrimination between tumors based on EGFR status. Reliable quantification of receptor status using minimally invasive techniques would be an important innovation for investigating new and existing cancer treatments that target these cellular mechanisms in research animals, and may be applied to identify receptor amplification in human brain cancer patients. This study represents the first systematic multianimal validation of receptor-specific imaging using MRI-guided fluorescence tomography.

Simultaneous measurement of rat brain cortex PtO2 using EPR oximetry and a fluorescence fiber-optic sensor during normoxia and hyperoxia

Electron paramagnetic resonance (EPR) oximetry is a promising, relatively non-invasive method of monitoring tissue partial pressure of oxygen (PtO(2)) that has proven useful in following changes in PtO(2) under various physiologic and pathophysiologic conditions. Optimal utilization of the method will be facilitated by systematic comparisons with other available methods. Here, we report on the absolute values and changes of rat brain PtO(2) using EPR oximetry and the OxyLite, an oxygen monitor based on fluorescence quenching, at adjacent locations in the same brain. EPR oximetry utilizes an implanted oxygen-sensitive material and reports tissue PtO(2) at the surface of the material. OxyLite measures PtO(2) using the fluorescence lifetime of a chromophore fixed to the tip of an optical fiber that is inserted into tissue. Measurements were made at a depth of 2-3 mm into the cortex during normoxia and during breathing of carbogen (95% O(2):5% CO(2)) followed by a return to normoxia. We conclude that in this study (1) PtO(2) values reported by the two methods are similar but not exactly the same, (2) both methods can record a baseline and rapid changes in PtO(2), (3) changes in PtO(2) induced by increasing FiO(2) from 0.26 to 0.95 (carbogen) were similar by the two methods and (4) in some rats breathing carbogen, absolute values of PtO(2) were above the sensitive range of the OxyLite method.