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PURPOSE: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. MATERIALS AND METHODS: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. RESULTS: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. CONCLUSIONS: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

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INTRODUCTION: Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function. MATERIALS AND METHODS: Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy ((19)F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts. RESULTS: Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor (19)F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes. CONCLUSION: This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings.

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The recent wave of enthusiasm for image guidance in radiation therapy is largely due to the advent of on-line imaging devices. The current narrow definition of image-guided radiotherapy (IGRT), in fact, essentially connotes the use of near real-time imaging during treatment delivery to reduce uncertainties in target position and should therefore be termed IGRT-D. However, a broader (and more appropriate) context of image-guidance should include: (1) detection and diagnosis, (2) delineation of target and organs at risk, (3) determining biological attributes, (4) dose distribution design, (5) dose delivery assurance and (6) deciphering treatment response through imaging i.e. the 6 D's of IGRT. Strategies to advance these areas will be discussed.

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BACKGROUND AND PURPOSE: To study the effect of breathing motion on gross tumor volume (GTV) coverage for lung tumors using dose-volume histograms and relevant dosimetric indices. PATIENTS AND METHODS: Treatment plans were chosen for 12 patients treated at our institution for lung carcinoma. GTV volumes of these patients ranged from 1.2 to 97.3 cm(3). A margin of 1-2 cm was used to generate the planning target volume (PTV). Additional margins of 0.6-1.0 cm were added to the PTV when designing treatment portals. For the purposes of TCP calculation, the prescription dose was assumed to be 70 Gy to remove the effects of prescription differences. Setup error was incorporated into the evaluation of treatment plans with a systematic component of sigma(RL) = 0.2 cm, sigma(AP) = 0.2 cm, and sigma(SI) = 0.3 cm and a random component of sigma(RL) = 0.3 cm, sigma(AP) = 0.3 cm, and sigma(SI) = 0.3 cm. Breathing motion was incorporated into these plans based on an independent analysis of fluoroscopic movies of the diaphragm for 7 patients. The systematic component of breathing motion (sigma(RL) = 0.3 cm, sigma(AP) = 0.2 cm, and sigma(SI) = 0.6 cm) was incorporated into the treatment plans on a slice by slice basis. The intrafractional component of breathing motion (sigma(RL) = 0.3 cm, sigma(AP) = 0.2 cm, and sigma(SI) = 0.6 cm) was incorporated by averaging the dose calculation over all displacements of the breathing cycle. Each patient was simulated 500 times to discern the range of possible outcomes. The simulations were repeated for a worst case scenario which used only breathing data with a large diaphragmatic excursion, both with and without intrafractional breathing motion. RESULTS: Dose to 95% of the GTV (D95), volume of the GTV receiving 95% of the prescription dose (V95) and TCP changed an average of -1.4+/-4.2, -1.0+/-3.3, and -1.4+/-3.8%, respectively, with the incorporation of normal breathing effects. In the worst case scenario (heavy breathers), D95 and V95 changed an average of -9.8+/-10.1 and -8.3+/-11.3%, respectively, and TCP changed by -8.1+/-9.1%. GTVs with volumes greater than 60 cm(3) showed stronger sensitivity to breathing especially if the shape was non-ellipsoidal. In the normal breathing case, the probability of a decrease in D95, V95, or TCP of a magnitude greater than 10% is less than 4%, and in the worse case scenario this probability is approximately 30-40% with intrafractional breathing motion included, and less than 10% with intrafractional breathing motion not included. CONCLUSIONS: With the PTV margins routinely used at our center, the effects of normal breathing on coverage are small on the average, with a less than 4% chance of a 10% or greater decrease in D95, V95, or TCP. However, in patients with large respiration-induced motion, the effect can be significant and efforts to identify such patients are important.

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