A framework for defining FLASH dose rate for pencil beam scanning.

PURPOSE: To develop a method of (a) calculating the dose rate of voxels within a proton field delivered using pencil beam scanning (PBS), and (b) reporting a representative dose rate for the PBS treatment field that enables correspondence between multiple treatment modalities. This method takes into account the unique spatiotemporal delivery patterns of PBS FLASH radiotherapy. METHODS: The dose rate at each voxel of a PBS radiation field is approximately the quotient of the voxel's dose and "effective" irradiation time. Each voxel's "effective" irradiation time starts when the cumulative dose rises above a chosen threshold value, and stops when its cumulative dose reaches its total dose minus the same threshold value. The above calculation yields a distribution of dose rates for the voxels within a PBS treatment field. To report a representative dose rate for the PBS field, we propose a user-selectable parameter of pth percentile of the dose rate distribution, such that (100 - p) % of the field is above the corresponding dose rate. To demonstrate the method described above, we design FLASH transmission fields using 250 MeV protons and calculate the PBS dose rate distributions in both two-dimensional (2D) and three-dimensional (3D) models. To further evaluate the formalism, we provide an example of a clinical PBS treatment field. RESULTS: With the 2D PBS transmission field, it is demonstrated that the time to accumulate the total dose at a voxel is limited to a fraction of the delivery time of the entire field. In addition, the spatial distributions of dose and dose rate are quite different within the field. For the 10 × 10 cm2 PBS field irradiating a 3D water phantom, the prescribed dose of 10 Gy at 10 cm depth is delivered in 1.0 s. The dose rate decreases in the irradiated volume with increasing depth (until the Bragg peak) due to increase of beam spot size by Coulomb scattering. For example, 95% of the irradiated volume between 0 and 10 cm depth receive >40 Gy/s, whereas between 0-20 cm and 0-30 cm depth, 95% of the irradiated volume received >36 Gy/s and >24 Gy/s, respectively. For the clinical PBS treatment field, the scanning pattern conforms to the PTV. PBS dose rate data are presented for the PTV and adjacent normal organs. CONCLUSION: We have developed a method of calculating the dose rate distribution of a PBS proton field and have recommended nomenclature for reporting PBS treatment dose rate. We believe that standardizing the method for calculating and reporting PBS treatment dose rates, in a manner that corresponds with other treatment modalities, will advance the research and potential application of PBS FLASH radiotherapy.

Visualizing the antivascular effect of bortezomib on the hypoxic tumor microenvironment.

Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1α in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.

Hypoxia-targeted triple suicide gene therapy radiosensitizes human colorectal cancer cells.

The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissue, may provide an opportunity for cancer-specific gene therapy. The purpose of the present study was to investigate whether hypoxia-driven triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to ganciclovir (GCV) and 5-fluorocytosine (5-FC), and sensitizes human colorectal cancer to radiation in vitro and in vivo. Stable transfectant of human colorectal HCT8 cells was established which expressed hypoxia-inducible vectors (HRE-TK/eGFP and HRE-CD/UPRT/mDsRed). Hypoxia-induced expression/function of TK, CD and UPRT was verified by western blot analysis, flow cytometry, fluorescent microscopy and cytotoxicity assay of GCV and 5-FC. Significant radiosensitization effects were detected after 5-FC and GCV treatments under hypoxic conditions. In the tumor xenografts, the distribution of TK/eGFP and CD/UPRT/mDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC and GCV in mice in combination with local irradiation resulted in tumor regression, as compared with prodrug or radiation treatments alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple suicide gene therapy may have the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

Respiration-phase-matched digital tomosynthesis imaging for moving target verification: a feasibility study.

PURPOSE: To develop a respiration-phase-matched digital tomosynthesis (DTS) technique to monitor moving targets, and to evaluate its accuracy for various imaging parameters and anatomical characteristics. METHODS: Previously developed 3D-DTS techniques, registering onboard DTS (OB-DTS, reconstructed from onboard projections) to reference DTS (R-DTS, reconstructed from DRRs of 3D reference CT), are inadequate to monitor moving targets. The authors' proposed respiration-phase-matched DTS technique registers OB-DTS to R-DTS reconstructed from DRRs generated by the same phase images of 4D reference CT as the corresponding onboard projections. To evaluate the improved accuracy of the author's technique, the authors performed thoracic phantom studies using (1) simulation with the 4D digital extended-cardiac-torso (XCAT) phantom, and (2) experiments with an anthropomorphic motion phantom. The studies were performed for various: respiratory cycle (RC), scan angle, and fraction of RC contained therein. Also, the authors assessed the accuracy of their technique relative to target size/location, and respiration inconsistencies from the R-DTS to OB-DTS. RESULTS: In both simulation and experimental studies, the respiration-phase-matched DTS technique is significantly more accurate in determining moving target positions. For 324 different scenarios simulated by XCAT, the respiration-phase-matched DTS technique localizes the 3D target position to errors of 1.07 ± 0.57 mm (mean ± S.D.), as compared to (a) 2.58 ± 1.37 and (b) 7.37 ± 4.18 mm, for 3D-DTS using 3D reference CT of (a) average intensity projection and (b) free-breathing CT. For 60 scenarios evaluated through experimental study, the uncertainties corresponding to those above are 1.24 ± 0.87, 2.42 ± 1.80, and 5.77 ± 6.45 mm, respectively. For a given scan angle, the accuracy of respiration-phase-matched DTS technique is less dependent on RC and the fraction of RC included in the scan. Increasing scan angle improves its accuracy. For different target locations, the targets near the chest wall or in the middle of lung provide higher registration accuracy compared to those near the mediastinum and diaphragm. Larger targets provide higher registration accuracy than small targets. Different respiratory cycle inconsistencies from R-DTS to OB-DTS minimally affect the registration accuracy. Increasing the respiratory amplitude inconsistencies will decrease the accuracy. CONCLUSIONS: The respiration-phase-matched DTS is more accurate and robust in determining moving target positions than 3D-DTS. It has potential application in pretreatment setup, post-treatment analysis, and intrafractional target verification.

Pilot study of PET imaging of 124I-iodoazomycin galactopyranoside (IAZGP), a putative hypoxia imaging agent, in patients with colorectal cancer and head and neck cancer.

BACKGROUND: Hypoxia within solid tumors confers radiation resistance and a poorer prognosis. 124I-iodoazomycin galactopyranoside (124I-IAZGP) has shown promise as a hypoxia radiotracer in animal models. We performed a clinical study to evaluate the safety, biodistribution, and imaging characteristics of 124I-IAZGP in patients with advanced colorectal cancer and head and neck cancer using serial positron emission tomography (PET) imaging. METHODS: Ten patients underwent serial whole-torso (head/neck to pelvis) PET imaging together with multiple whole-body counts and blood sampling. These data were used to generate absorbed dose estimates to normal tissues for 124I-IAZGP. Tumors were scored as either positive or negative for 124I-IAZGP uptake. RESULTS: There were no clinical toxicities or adverse effects associated with 124I-IAZGP administration. Clearance from the whole body and blood was rapid, primarily via the urinary tract, with no focal uptake in any parenchymal organ. The tissues receiving the highest absorbed doses were the mucosal walls of the urinary bladder and the intestinal tract, in particular the lower large intestine. All 124I-IAZGP PET scans were interpreted as negative for tumor uptake. CONCLUSIONS: It is safe to administer 124I-IAZGP to human subjects. However, there was insufficient tumor uptake to support a clinical role for 124I-IAZGP PET in colorectal cancer and head and neck cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00588276.

The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: evidence of hypoxia-induced potentially lethal damage.

PURPOSE: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill. MATERIALS AND METHODS: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N(2) gas containing 0, 0.1 or 0.5% O(2). Cell survival was determined by colony formation assay immediately after 0-96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2-24 h before survival assay. RESULTS: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O(2) concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair. CONCLUSION: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.

Technologies of image guidance and the development of advanced linear accelerator systems for radiotherapy.

As advanced radiotherapy approaches for targeting the tumor and sparing the normal tissues have been developed, the image guidance of therapy has become essential to directing and confirming treatment accuracy. To approach these goals, image guidance devices now include kV on-board imagers, kV/MV cone-beam CT systems, CT-on-rails, and mobile and in-room radiographic/fluoroscopic systems. Nonionizing sources, such as ultrasound and optical systems, and electromagnetic devices have been introduced to monitor or track the patient and/or tumor positions during treatment. In addition, devices have been designed specifically for monitoring and/or controlling respiratory motion. Optimally, image-guided radiation therapy systems should possess 3 essential elements: (1) 3D imaging of soft tissues and tumors, (2) efficient acquisition and comparison of the 3D images, and (3) an efficacious process for clinically meaningful intervention. Understanding and using these tools effectively is central to current radiotherapy practice. The implementation and integration of these devices continue to carry practical challenges, which emphasize the need for further development of the technologies and their clinical applications.

18F-fluromisonidazole PET imaging as a biomarker for the response to 5,6-dimethylxanthenone-4-acetic acid in colorectal xenograft tumors.

UNLABELLED: The aim of this study was to evaluate (18)F-fluromisonidazole ((18)F-FMISO) PET for monitoring the tumor response to the antivascular compound 5,6-dimethylxanthenone-4-acetic acid (DMXAA; vadimezan). METHODS: (18)F-FMISO PET was performed 3 h before and 24 h after treatment with DMXAA (20 mg/kg) in mice bearing HT29 xenograft tumors. Pimonidazole was coadministered with the first (18)F-FMISO injection, and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) was coadministered with the second one. Hoechst 33342 was administered 5 min before sacrifice. Digital autoradiograms of tumor sections were acquired; this acquisition was followed by immunofluorescence microscopic visualization of pimonidazole, EF5, the Hoechst 33342, CD31, and α-smooth muscle actin. RESULTS: DMXAA treatment resulted in a marked reduction in the (18)F-FMISO mean standardized uptake value (SUV(mean)) in approximately half of the treated tumors. The reduction in SUV(mean) correlated with a decrease in the fraction of tumor area staining positive for both EF5 and pimonidazole. Compared with untreated controls, tumors with decreasing SUV(mean) had significantly fewer perfused microvessels. CONCLUSION: (18)F-FMISO PET could distinguish between different tumor responses to DMXAA treatment. However, a reduction in (18)F-FMISO SUV(mean) after DMXAA treatment was indicative of reduced perfusion and therefore delivery of (18)F-FMISO, rather than a reduction in tumor hypoxia.

Response to multiple radiation doses of human colorectal carcinoma cells infected with recombinant adenovirus containing dominant-negative Ku70 fragment.

PURPOSE: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. METHODS AND MATERIALS: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. RESULTS: Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was approximately 1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2-1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. CONCLUSION: Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

Dose-rate effects in external beam radiotherapy redux.

Recent developments in external beam radiotherapy, both in technical advances and in clinical approaches, have prompted renewed discussions on the potential influence of dose-rate on radio-response in certain treatment scenarios. We consider the multiple factors that influence the dose-rate effect, e.g. radical recombination, the kinetics of sublethal damage repair for tumors and normal tissues, the difference in alpha/beta ratio for early and late reacting tissues, and perform a comprehensive literature review. Based on radiobiological considerations and the linear-quadratic (LQ) model we estimate the influence of overall treatment time on radio-response for specific clinical situations. As the influence of dose-rate applies to both the tumor and normal tissues, in oligo-fractionated treatment using large doses per fraction, the influence of delivery prolongation is likely important, with late reacting normal tissues being generally more sensitive to the dose-rate effect than tumors and early reacting tissues. In conventional fractionated treatment using 1.8-2Gy per fraction and treatment times of 2-1 min, the influence of dose-rate is relatively small. Lastly, the dose-rate effect in external beam radiotherapy is governed by the overall beam-on-time, not by the average linac dose-rate, nor by the instantaneous dose-rate within individual linac pulses which could be as high as 3 x 10(6)MU/min.

The effect of mild temperature hyperthermia on tumour hypoxia and blood perfusion: relevance for radiotherapy, vascular targeting and imaging.

Clinically achievable mild temperature local hyperthermia (<43 degrees C) has been demonstrated to be an effective adjuvant to radiotherapy in pre-clinical and clinical studies. In this article, we briefly review the recent progress in the following areas: (1) the effect of mild temperature hyperthermia (MTH) on tumour hypoxia and blood perfusion as assessed by dual marker immunohistochemistry (IHC); (2) the kinetics of MTH induced changes in tumour hypoxia; (3) the potential role of heat-induced tumour reoxygenation on radio- and chemo-sensitisation; (4) the potential role of MTH in combination with vascular targeting agents (VTAs) on tumour response; and (5) non-invasive detection of changes in tumour oxygenation and blood perfusion. It is shown that MTH, by itself or in combination with VTAs, leads to changes in tumour perfusion and oxygenation with potential for radio- and chemo-sensitisation.

High 18F-FDG uptake in microscopic peritoneal tumors requires physiologic hypoxia.

UNLABELLED: The objective of this study was to examine (18)F-FDG uptake in microscopic tumors grown intraperitoneally in nude mice and to relate this to physiologic hypoxia and glucose transporter-1 (GLUT-1) expression. METHODS: Human colon cancer HT29 and HCT-8 cells were injected intraperitoneally into nude mice to generate disseminated tumors of varying sizes. After overnight fasting, animals, breathing either air or carbogen (95% O(2) + 5% CO(2)), were intravenously administered (18)F-FDG together with the hypoxia marker pimonidazole and cellular proliferation marker bromodeoxyuridine 1 h before sacrifice. Hoechst 33342, a perfusion marker, was administered 1 min before sacrifice. After sacrifice, the intratumoral distribution of (18)F-FDG was assessed by digital autoradiography of frozen tissue sections. Intratumoral distribution was compared with the distributions of pimonidazole, GLUT-1 expression, bromodeoxyuridine, and Hoechst 33342 as visualized by immunofluorescent microscopy. RESULTS: Small tumors (diameter, <1 mm) had high (18)F-FDG accumulation and were severely hypoxic, with high GLUT-1 expression. Larger tumors (diameter, 1-4 mm) generally had low (18)F-FDG accumulation and were not significantly hypoxic, with low GLUT-1 expression. Carbogen breathing significantly decreased (18)F-FDG accumulation and tumor hypoxia in microscopic tumors but had little effect on GLUT-1 expression. CONCLUSION: There was high (18)F-FDG uptake in microscopic tumors that was spatially associated with physiologic hypoxia and high GLUT-1 expression. This enhanced uptake was abrogated by carbogen breathing, indicating that in the absence of physiologic hypoxia, high GLUT-1 expression, by itself, was insufficient to ensure high (18)F-FDG uptake.

Detection of hypoxia in microscopic tumors using 131I-labeled iodo-azomycin galactopyranoside (131I-IAZGP) digital autoradiography.

PURPOSE: Previous studies have shown that tumors less than 1 mm diameter derived from HT29 colorectal cancer cells are extremely hypoxic when grown intraperitoneally or intradermally in nude mice, whereas those of greater size (approximately 1-4 mm diameter) are not significantly hypoxic. The object of this study was to determine if digital autoradiography using the radiolabeled hypoxia imaging tracer iodo-azomycin galactopyranoside ((131)I-IAZGP) could detect hypoxia in this model. METHODS: Microscopic HT29 tumors were grown as disseminated peritoneal disease and intradermally in nude mice. Tumors ranged in size from a few hundred microns to several millimeters in diameter. Animals were intravenously administered (131)I-IAZGP and pimonidazole 2 h before sacrifice. Following sacrifice, the intratumoral distribution of (131)I-IAZGP was assessed by digital autoradiography and compared with immunofluorescence microscopic images of pimonidazole binding and carbonic anhydrase IX (CAIX) expression. RESULTS: The distributions of (131)I-IAZGP, pimonidazole, and CAIX expression were similar. Tumors less than 1 mm diameter displayed high (131)I-IAZGP uptake; these tumors also stained strongly for pimonidazole and CAIX. Larger tumors (approximately 1-4 mm diameter) were not significantly hypoxic and had low (131)I-IAZGP accumulation. CONCLUSION: (131)I-IAZGP can detect hypoxia in microscopic tumors. Microscopic tumors are useful models for the validation of hypoxia radiotracers, and digital autoradiography is an appropriate technique for studying the distribution of hypoxia radiotracers in microscopic tumors.

A robotic system for 18F-FMISO PET-guided intratumoral pO2 measurements.

An image-guided robotic system was used to measure the oxygen tension (pO2) in rodent tumor xenografts using interstitial probes guided by tumor hypoxia PET images. Rats with approximately 1 cm diameter tumors were anesthetized and immobilized in a custom-fabricated whole-body mold. Imaging was performed using a dedicated small-animal PET scanner (R4 or Focus 120 microPET) approximately 2 h after the injection of the hypoxia tracer 18F-fluoromisonidazole (18F-FMISO). The coordinate systems of the robot and PET were registered based on fiducial markers in the rodent bed visible on the PET images. Guided by the 3D microPET image set, measurements were performed at various locations in the tumor and compared to the corresponding 18F-FMISO image intensity at the respective measurement points. Experiments were performed on four tumor-bearing rats with 4 (86), 3 (80), 7 (162), and 8 (235) measurement tracks (points) for each experiment. The 18F-FMISO image intensities were inversely correlated with the measured pO2, with a Pearson coefficient ranging from -0.14 to -0.97 for the 22 measurement tracks. The cumulative scatterplots of pO2 versus image intensity yielded a hyperbolic relationship, with correlation coefficients of 0.52, 0.48, 0.64, and 0.73, respectively, for the four tumors. In conclusion, PET image-guided pO2 measurement is feasible with this robot system and, more generally, this system will permit point-by-point comparison of physiological probe measurements and image voxel values as a means of validating molecularly targeted radiotracers. Although the overall data fitting suggested that 18F-FMISO may be an effective hypoxia marker, the use of static 18F-FMISO PET postinjection scans to guide radiotherapy might be problematic due to the observed high variation in some individual data pairs from the fitted curve, indicating potential temporal fluctuation of oxygen tension in individual voxels or possible suboptimal imaging time postadministration of hypoxia-related trapping of 18F-FMISO.

Modeling acute and chronic hypoxia using serial images of 18F-FMISO PET.

PURPOSE: Two types of tumor hypoxia most likely exist in human cancers: Chronic hypoxia due to the paucity of blood capillaries and acute hypoxia due to temporary shutdoWn of microvasculatures or fluctuation in the red cell flux. In a recent hypoxia imaging study using 18F-FMISO PET, the authors observed variation in tracer uptake in two sequential images and hypothesized that variation in acute hypoxia may be the cause. In this study, they develop an iterative optimization method to delineate chronic and acute hypoxia based on the 18F-FMISO PET serial images. METHODS: They assume that (1) chronic hypoxia is the same in the two scans and can be represented by a Gaussian distribution, while (2) acute hypoxia varies in the two scans and can be represented by Poisson distributions. For validation, they used Monte Carlo simulations to generate pairs of 18F-FMISO PET images with known proportion of chronic and acute hypoxia and then applied the optimization method to the simulated serial images, yielding excellent fit between the input and the fitted results. They then applied this method to the serial 18F-FMISO PET images of 14 patients with head and neck cancers. RESULTS: The results show good fit of the chronic hypoxia to Gaussian distributions for 13 out of 14 patients (with R2>0.7). Similarly, acute hypoxia appears to be well described by the Poisson distribution (R2>0.7) with three exceptions. The model calculation yielded the amount of acute hypoxia, which differed among the patients, ranging from approximately 13% to 52%, with an average of approximately 34%. CONCLUSIONS: This is the first effort to separate acute and chronic hypoxia from serial PET images of cancer patients.

Considering marker visibility during leaf sequencing for segmental intensity-modulated radiation therapy.

PURPOSE: Segmental intensity-modulated radiation therapy (IMRT) delivers a sequence of segments to obtain a desired intensity distribution. Many leaf sequencing algorithms for segmental IMRT have been developed with the aim of reducing delivered monitor units (MUs) and (or) number of segments and, consequently, to reduce the total treatment delivery time. With the development of real-time detection technology, it is useful to develop leaf sequencing algorithms that consider the detecting probability of markers implanted into or near the target volume. METHODS: In this study, the authors defined the concept of marker visibility to denote the marker's detecting probability and proposed a new leaf sequencing algorithm based on the Kamath algorithm. The new algorithm first uses the Kamath algorithm to generate an initial leaf sequence and then performs a series of column transformations to obtain a new leaf sequence that is optimal in terms of MU efficiency and marker visibility. The authors evaluated the performance of the new algorithm with six artificial fields that had randomly generated intensity matrices and 15 clinical fields that had intensity matrices from the IMRT plans for three prostate cancer patients. RESULTS: Compared to the Kamath algorithm, the new algorithm does not increase the total delivered intensity but increases the marker visibility. For the artificial fields, the marker visibility increased from 66.67% to 91.67% for small (5 x 5) radiation fields, from 39.29% to 42.86% for medium size (10 x 10) fields, and from 31.48% to 37.04% for large (20 x 20) fields. For the clinical fields, the marker visibility increased 9%-20% for four fields, 20%-30% for three fields, 30%-40% for two fields, and more than 40% for one field. However, the marker visibility did not change for 4 out of 15 fields. CONCLUSIONS: The authors developed a new leaf sequencing algorithm for optimal MU efficiency and marker visibility and also rigorously proved its optimality.

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

PURPOSE: To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). METHODS: Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. RESULTS: Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. CONCLUSIONS: We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the evaluation of radiolabeled hypoxia tracers.

Atrasentan (ABT-627) enhances perfusion and reduces hypoxia in a human tumor xenograft model.

The endothelin-1 antagonist, Atrasentan (ABT-627) was used to modify perfusion in the human tumor xenograft model, HT29, growing in nude mice. Atrasentan produced a significant increase in perfusion, as measured in vivo by Gd-DTPA DCE-MRI. Changes in tumor hypoxia were assessed by comparing the binding of two hypoxia tracers, pimonidazole and EF5 given before and after Atrasentan administration. In vehicle-treated controls, the distribution of EF5 and pimonidazole was very similar. However, Atrasentan treatment was associated with decreased uptake of the second hypoxia tracer (EF5), relative to the first (pimonidazole). Although Atrasentan had no independent effect on the growth of HT29 tumors, Atrasentan combined with 20 Gy radiation led to a modest but significant increase in tumor growth delay compared to radiation alone.