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1.  Genome-wide association study identifies a region on chromosome 11q14.3 associated with late rectal bleeding following radiation therapy for prostate cancer* 
Background and Purpose
Rectal bleeding can occur following radiotherapy for prostate cancer and negatively impacts quality of life for cancer survivors. Treatment and clinical factors do not fully predict for rectal bleeding, and genetic factors may be important.
Materials and Methods
A genome-wide association study (GWAS) was performed to identify SNPs associated with development of late rectal bleeding following radiotherapy for prostate cancer. Logistic regression was used to test association between 614,453 SNPs and rectal bleeding in a discovery cohort (79 cases, 289 controls), and top-ranking SNPs were tested in a replication cohort (108 cases, 673 controls) from four independent sites.
Results
rs7120482 and rs17630638, which tag a single locus on chromosome 11q14.3, reached genome-wide significance for association with rectal bleeding (combined p-values 5.4×10−8 and 6.9×10−7 respectively). Several other SNPs had p-values trending towards genome-wide significance, and a polygenic risk score including these SNPs shows a strong rank-correlation with rectal bleeding (Sommers’ d = 5.0×10−12 in the replication cohort).
Conclusions
This GWAS identified novel genetic markers of rectal bleeding following prostate radiotherapy. These findings could lead to development of a predictive assay to identify patients at risk for this adverse treatment outcome so that dose or treatment modality could be modified.
doi:10.1016/j.radonc.2013.05.001
PMCID: PMC3787843  PMID: 23719583
radiogenomics; prostate cancer; rectal toxicity; genome-wide association study
2.  Radiation induces aerobic glycolysis through reactive oxygen species 
Background and purpose
Although radiation induced reoxygenation has been thought to increase radiosensitivity, we have shown that its associated oxidative stress can have radioprotective effects, including stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is known to regulate many of the glycolytic enzymes, thereby promoting aerobic glycolysis, which is known to promote treatment resistance. Thus, we hypothesized that reoxygenation after radiation would increase glycolysis. We previously showed that blockade of oxidative stress using a superoxide dismutase (SOD) mimic during reoxygenation can downregulate HIF-1 activity. Here we tested whether concurrent use of this drug with radiotherapy would reduce the switch to a glycolytic phenotype.
Materials and methods
40 mice with skin fold window chambers implanted with 4T1 mammary carcinomas were randomized into (1) no treatment, (2) radiation alone, (3) SOD mimic alone, and (4) SOD mimic with concurrent radiation. All mice were imaged on the ninth day following tumor implantation (30 h following radiation treatment) following injection of a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Hemoglobin saturation was measured by using hyperspectral imaging to quantify oxygenation state.
Results
Mice treated with radiation showed significantly higher 2-NBDG fluorescence compared to controls (p = 0.007). Hemoglobin saturation analysis demonstrated reoxygenation following radiation, coinciding with the observed increase in glycolysis. The concurrent use of the SOD mimic with radiation demonstrated a significant reduction in 2-NBDG fluorescence compared to effects seen after radiation alone, while having no effect on reoxygenation.
Conclusions
Radiation induces an increase in tumor glucose demand approximately 30 h following therapy during reoxygenation. The use of an SOD mimic can prevent the increase in aerobic glycolysis when used concurrently with radiation, without preventing reoxygenation.
doi:10.1016/j.radonc.2013.02.013
PMCID: PMC3770265  PMID: 23541363
Warburg effect; Radiation; Reoxygenation; Aerobic glycolysis; Reactive oxygen species
3.  Predictive value of hypoxia, proliferation and tyrosine kinase receptors for EGFR-inhibition and radiotherapy sensitivity in head and neck cancer models 
Background and Purpose
EGFR-inhibitor Cetuximab (C225) improves the efficacy of radiotherapy in only a subgroup of HNSCC patients. Identification of predictive tumor characteristics is essential to improve patient selection.
Material and Methods
Response to C225 and/or radiotherapy was assessed with tumor growth delay assays in 4 HNSCC xenograft models with varying EGFR-expression levels. Hypoxia and proliferation was quantified with immunohistochemistry and expression of proteins involved in C225-resistance with western blot.
Results
EGFR-expression did not predict response to C225 and/or radiotherapy. Reduction of hypoxia by C225 was only observed in SCCNij202, which was highly sensitive to C225. Proliferation changes correlated with response to C225 and C225 combined with radiotherapy, as proliferation decreased after C225 treatment in C225-sensitive SCCNij202 and after combined treatment in SCCNij185, which showed a synergistic effect to combined C225-radiotherapy. Furthermore, C225-resistant SCCNij153 tumors expressed high levels of (activated) HER3 and MET.
Conclusions
EGFR-expression is needed for C225-response, but is not sufficient to predict response to C225 with or without radiotherapy. However, basal expression of additional growth factor receptors and effects on proliferation, but not hypoxia, correlated with response to combined C225-radiotherapy treatment and are potential clinically relevant predictive biomarkers.
doi:10.1016/j.radonc.2013.02.001
PMCID: PMC3627829  PMID: 23453541
head and neck cancer; EGFR-inhibition; tumor microenvironment; radiotherapy; tyrosine kinase receptors
4.  Aortic Dose Constraints when Reirradiating Thoracic Tumors 
Background and Purpose
Improved radiation delivery and planning has allowed, in some instances, for the retreatment of thoracic tumors. We investigated the dose limits of the aorta wherein grade 5 aortic toxicity was observed after reirradiation of lung tumors.
Material and Methods
In a retrospective analysis, 35 patients were identified, between 1993 and 2008, who received two rounds of external beam irradiation that included the aorta in the radiation fields of both the initial and retreatment plans. We determined the maximum cumulative dose to 1 cm3 of the aorta (the composite dose) for each patient, normalized these doses to 1.8 Gy/fraction, and corrected them for long-term tissue recovery between treatments (NIDR).
Results
The median time interval between treatments was 30 months (range, 1–185 months). The median follow-up of patients alive at analysis was 42 months (range, 14–70 months). Two of the 35 patients (6%) were identified as having grade 5 aortic toxicities. There was a 25% rate of grade 5 aortic toxicity for patients receiving composite doses ≥120.0 Gy (vs. 0% for patients receiving <120.0 Gy) (P=0.047).
Conclusions
Grade 5 aortic toxicities were observed with composite doses ≥120.0 Gy (NIDR ≥90.0 Gy) to 1 cm3 of the aorta.
doi:10.1016/j.radonc.2013.02.002
PMCID: PMC3921976  PMID: 23453540
reirradiation; aortic toxicity; radiation toxicity; lung cancer
5.  Developing a class solution for Prostate Stereotactic Ablative Body Radiotherapy (SABR) using Volumetric Modulated Arc Therapy (VMAT) 
Radiotherapy and Oncology  2014;110(2):298-302.
Background and purpose
To develop a class solution for prostate Stereotactic Ablative Radiotherapy (SABR) using Volumetric Modulated Arc Therapy (VMAT).
Materials and methods
Seven datasets were used to compare plans using one 360° arc (1FA), one 210° arc (1PA), two full arcs and two partial arcs. Subsequently using 1PA, fifteen datasets were compared using (i) 6 mm CTV–PTV margins, (ii) 8 mm CTV–PTV margins and (iii) including the proximal SV within the CTV. Monaco™ 3.2 (Elekta™) was used for planning with the Agility™ MLC system (Elekta™).
Results
Highly conformal plans were produced using all four arc arrangements. Compared to 1FA, 1PA resulted in significantly reduced rectal doses, and monitor units and estimated delivery times were reduced in six of seven cases. Using 6 mm CTV–PTV margins, planning constraints were met for all fifteen datasets. Using 8 mm margins required relaxation of the uppermost bladder constraint in three cases to achieve adequate coverage, and, compared to 6 mm margins, rectal and bladder doses significantly increased. Including the proximal SV required relaxation of the uppermost bladder and rectal constraints in two cases, and rectal and bladder doses significantly increased.
Conclusions
Prostate SABR VMAT is optimal using 1PA. 6 mm CTV–PTV margins, compatible with daily fiducial-based IGRT, are consistently feasible in terms of target objectives and OAR constraints.
doi:10.1016/j.radonc.2013.10.036
PMCID: PMC3969574  PMID: 24332021
Image Guided RadioTherapy (IGRT); Prostate cancer; Stereotactic Ablative Body Radiotherapy (SABR); Volumetric Modulated Arc Therapy (VMAT)
6.  Second primary cancers after radiation for prostate cancer: A systematic review of the clinical data and impact of treatment technique 
Radiotherapy and Oncology  2014;110(2):213-228.
The development of a radiation induced second primary cancer (SPC) is one the most serious long term consequences of successful cancer treatment. This review aims to evaluate SPC in prostate cancer (PCa) patients treated with radiotherapy, and assess whether radiation technique influences SPC. A systematic review of the literature was performed to identify studies examining SPC in irradiated PCa patients. This identified 19 registry publications, 21 institutional series and 7 other studies. There is marked heterogeneity in published studies. An increased risk of radiation-induced SPC has been identified in several studies, particularly those with longer durations of follow-up. The risk of radiation-induced SPC appears small, in the range of 1 in 220 to 1 in 290 over all durations of follow-up, and may increase to 1 in 70 for patients followed up for more than 10 years, based on studies which include patients treated with older radiation techniques (i.e. non-conformal, large field). To date there are insufficient clinical data to draw firm conclusions about the impact of more modern techniques such as IMRT and brachytherapy on SPC risk, although limited evidence is encouraging. In conclusion, despite heterogeneity between studies, an increased risk of SPC following radiation for PCa has been identified in several studies, and this risk appears to increase over time. This must be borne in mind when considering which patients to irradiate and which techniques to employ.
doi:10.1016/j.radonc.2013.12.012
PMCID: PMC3988985  PMID: 24485765
Prostate cancer; Second primary cancer; Systematic review
7.  Spinal cord tolerance to single-session uniform irradiation in pigs: implications for a dose-volume effect 
Background and Purpose
This study was performed to test the hypothesis that spinal cord radiosensitivity is significantly modified by uniform versus laterally non-uniform dose distributions.
Materials and Methods
A uniform dose distribution was delivered to a 4.5–7.0cm length of cervical spinal cord in 22 mature Yucatan minipigs for comparison with a companion study in which a laterally non-uniform dose was given[1]. Pigs were allocated into four dose groups with mean maximum spinal cord doses of 17.5±0.1 Gy(n=7), 19.5±0.2 Gy(n=6), 22.0±0.1 Gy(n=5), and 24.1±0.2 Gy(n=4). The study endpoint was motor neurologic deficit determined by a change in gait within one year. Spinal cord sections were stained with a Luxol fast blue/periodic acid Schiff combination.
Results
Dose-response curves for uniform versus non-uniform spinal cord irradiation were nearly identical with ED50's (95% confidence interval) of 20.2 Gy(19.1–25.8) and 20.0 Gy(18.3–21.7), respectively. No neurologic change was observed for either dose distribution when the maximum spinal cord dose was ≤17.8 Gy while all animals experienced deficits at doses ≥21.8Gy.
Conclusion
No dose-volume effect was observed in pigs for the dose distributions studied and the endpoint of motor neurologic deficit; however, partial spinal cord irradiation resulted in less debilitating neurologic morbidity and histopathology.
doi:10.1016/j.radonc.2012.08.007
PMCID: PMC3526692  PMID: 22985780
dose-volume effect; spinal cord tolerance; normal tissue tolerance; stereotactic spinal radiosurgery; swine
8.  Proton therapy radiation pneumonitis local dose–response in esophagus cancer patients 
Purpose
This study quantifies pulmonary radiation toxicity in patients who received proton therapy for esophagus cancer.
Materials/methods
We retrospectively studied 100 esophagus cancer patients treated with proton therapy. The linearity of the enhanced FDG uptake vs. proton dose was evaluated using the Akaike Information Criterion (AIC). Pneumonitis symptoms (RP) were assessed using the Common Toxicity Criteria for Adverse Events version 4.0 (CTCAEv4). The interaction of the imaging response with dosimetric parameters and symptoms was evaluated.
Results
The RP scores were: 0 grade 4/5, 7 grade 3, 20 grade 2, 37 grade 1, and 36 grade 0. Each dosimetric parameter was significantly higher for the symptomatic group. The AIC winning models were 30 linear, 52 linear quadratic, and 18 linear logarithmic. There was no significant difference in the linear coefficient between models. The slope of the FDG vs. proton dose response was 0.022 for the symptomatic and 0.012 for the asymptomatic (p = 0.014). Combining dosimetric parameters with the slope did not improve the sensitivity or accuracy in identifying symptomatic cases.
Conclusions
The proton radiation dose response on FDG PET/CT imaging exhibited a predominantly linear dose response on modeling. Symptomatic patients had a higher dose response slope.
doi:10.1016/j.radonc.2012.09.003
PMCID: PMC3696882  PMID: 23127772
Radiation pneumonitis; Proton therapy; Positron emission tomography
9.  Review of clinical brachytherapy uncertainties: Analysis guidelines of GEC-ESTRO and the AAPM☆ 
Radiotherapy and Oncology  2014;110(1):199-212.
Background and purpose
A substantial reduction of uncertainties in clinical brachytherapy should result in improved outcome in terms of increased local control and reduced side effects. Types of uncertainties have to be identified, grouped, and quantified.
Methods
A detailed literature review was performed to identify uncertainty components and their relative importance to the combined overall uncertainty.
Results
Very few components (e.g., source strength and afterloader timer) are independent of clinical disease site and location of administered dose. While the influence of medium on dose calculation can be substantial for low energy sources or non-deeply seated implants, the influence of medium is of minor importance for high-energy sources in the pelvic region. The level of uncertainties due to target, organ, applicator, and/or source movement in relation to the geometry assumed for treatment planning is highly dependent on fractionation and the level of image guided adaptive treatment. Most studies to date report the results in a manner that allows no direct reproduction and further comparison with other studies. Often, no distinction is made between variations, uncertainties, and errors or mistakes. The literature review facilitated the drafting of recommendations for uniform uncertainty reporting in clinical BT, which are also provided. The recommended comprehensive uncertainty investigations are key to obtain a general impression of uncertainties, and may help to identify elements of the brachytherapy treatment process that need improvement in terms of diminishing their dosimetric uncertainties. It is recommended to present data on the analyzed parameters (distance shifts, volume changes, source or applicator position, etc.), and also their influence on absorbed dose for clinically-relevant dose parameters (e.g., target parameters such as D90 or OAR doses). Publications on brachytherapy should include a statement of total dose uncertainty for the entire treatment course, taking into account the fractionation schedule and level of image guidance for adaptation.
Conclusions
This report on brachytherapy clinical uncertainties represents a working project developed by the Brachytherapy Physics Quality Assurances System (BRAPHYQS) subcommittee to the Physics Committee within GEC-ESTRO. Further, this report has been reviewed and approved by the American Association of Physicists in Medicine.
doi:10.1016/j.radonc.2013.11.002
PMCID: PMC3969715  PMID: 24299968
Brachytherapy; Dosimetry; Uncertainties; Treatment planning
10.  Coxsackie- and adenovirus receptor as a novel marker of stem cells in treatment-resistant non-small cell lung cancer 
Background
Treatment resistance resulting from the presence of cancer stem cells (CSCs) remains a challenge in cancer treatment. Little is known about possible markers of CSCs in treatment-resistant non-small cell lung cancer (NSCLC). We explored the coxsackie- and adenovirus receptor (CAR) as one such marker of CSCs in models of treatment-resistant NSCLC.
Materials and methods
Resistant H460 and A549 cell lines were established by repeated exposure to paclitaxel or fractionated radiation. CSC markers were measured by western blotting and flow cytometry. We also established stable CAR-overexpressing and stable shRNA-CAR-knockdown cell lines and assessed their survival, invasiveness, and tumorigenic capabilities with clonogenic, telomerase, Matrigel, and tumor formation assays.
Results
CAR expression was associated with CSC phenotype both in vitro and in vivo. CAR-overexpressing cells were more treatment-resistant, self-renewing, and tumorigenic than were parental cells, and shRNA-mediated knockdown of CAR expression was sufficient to inhibit these functions. CAR expression also correlated with the epithelial-mesenchymal transition.
Conclusions
We showed for the first time that CAR is a marker of CSCs and may affect the activities of CSCs in treatment-resistant NSCLC. CAR may prove to be a target for CSC treatment and a predictor of treatment response in patients with NSCLC.
doi:10.1016/j.radonc.2012.09.002
PMCID: PMC3845342  PMID: 23022172
Coxsackie-adenovirus receptor; treatment resistance; NSCLC; cancer stem cells; molecular markers
11.  Effects of Lipopolysaccharide on the response of C57BL/6J mice to whole thorax irradiation 
Background and Purpose
Inflammatory and fibrogenic processes play a crucial role in the radiation-induced injury in the lung. The aim of the present study was to examine whether additive LPS exposure in the lung (to simulate respiratory infection) would affect pneumonitis or fibrosis associated with lung irradiation.
Material and Methods
Wildtype C57Bl/6J (WT-C57) and TNFα, TNFR1 and TNFR2 knockout (−/−) mice, in C57Bl/6J background, were given whole thorax irradiation (10Gy) with or without post-irradiation intratracheal administration of LPS (50μg/mice). Functional deficit was examined by measuring breathing rate at various times after treatment. Real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry were used to analyse the protein expression and m-RNA of Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumour Necrosis Factor alpha (TNFα) and Transforming Growth Factor beta (TGFβ) in the lung at various times after treatment. Inflammatory cells were detected by Mac-3 (macrophages) and Toluidine Blue (mast cells) staining. Collagen content was estimated by hydroxyproline (total collagen) and Sircol assay (soluble collagen). Levels of oxidative damage were assessed by 8-hydroxy-2-deoxyguanosine (8-OHdG) staining.
Results
LPS exposure significantly attenuated the breathing rate increases following irradiation of WT-C57, TNFR1−/− and TNFR2−/− mice and to a lesser extent in TNFα−/− mice. Collagen content was significantly reduced after LPS treatment in WT-C57, TNFR1−/− and TNFα−/− mice and there was a trend in TNFR2−/− mice. Similarly there were lower levels of inflammatory cells and cytokines in the LPS treated mice.
Conclusions
This study reveals a mitigating effect of early exposure to LPS on injury caused by irradiation on lungs of C57Bl mice. The results suggest that immediate infection post irradiation may not impact lung response negatively in radiation-accident victims, however, further studies are required in different animal models, and with specific infectious agents, to confirm and extend our findings.
doi:10.1016/j.radonc.2012.08.003
PMCID: PMC3632095  PMID: 22985778
TNF alpha −/−; TNFR1−/− and TNFR2−/− mice; lung; radiation; lipopolysaccharide (LPS); pneumonitis; fibrosis
12.  A semiautomatic CT-based ensemble segmentation of lung tumors: comparison with oncologists’ delineations and with the surgical specimen 
Purpose
To assess the clinical relevance of a semiautomatic CT-based ensemble segmentation method, by comparing it to pathology and to CT/PET manual delineations by five independent radiation oncologists in non-small cell lung cancer (NSCLC).
Materials and Methods
For twenty NSCLC patients (stage Ib – IIIb) the primary tumor was delineated manually on CT/PET scans by five independent radiation oncologists and segmented using a CT based semi-automatic tool. Tumor volume and overlap fractions between manual and semiautomatic-segmented volumes were compared. All measurements were correlated with the maximal diameter on macroscopic examination of the surgical specimen. Imaging data is available on www.cancerdata.org.
Results
High overlap fractions were observed between the semi-automatically segmented volumes and the intersection (92.5 ± 9.0, mean ± SD) and union (94.2 ± 6.8) of the manual delineations. No statistically significant differences in tumor volume were observed between the semiautomatic segmentation (71.4 ± 83.2 cm3, mean ± SD) and manual delineations (81.9 ± 94.1 cm3; p = 0.57). The maximal tumor diameter of the semiautomatic-segmented tumor correlated strongly with the macroscopic diameter of the primary tumor (r = 0.96).
Conclusion
Semiautomatic segmentation of the primary tumor on CT demonstrated high agreement with CT/PET manual delineations and strongly correlated with the macroscopic diameter considered the “gold standard”. This method may be used routinely in clinical practice and could be employed as a starting point for treatment planning, target definition in multi-center clinical trials or for high throughput data mining research. This method is particularly suitable for peripherally located tumors.
doi:10.1016/j.radonc.2012.09.023
PMCID: PMC3749821  PMID: 23157978
CT; Auto-segmentation; Inter-observer variability; NSCLC delineation
13.  Correlation of PET Images of Metabolism, Proliferation and Hypoxia to Characterize Tumor Phenotype in Patients with Cancer of the Oropharynx 
Spatial organization of tumor phenotype is of great interest to radiotherapy target definition and outcome prediction. We characterized tumor phenotype in patients with cancers of the oropharynx through voxel-based correlation of PET images of metabolism, proliferation, and hypoxia.
Methods
Patients with oropharyngeal cancer received 18F-fluorodeoxyglucose (FDG) PET/CT, 18F-fluorothymidine (FLT) PET/CT, and 61Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) PET/CT. Images were co-registered and standardized uptake values (SUV) were calculated for all modalities. Voxel-based correlation was evaluated with Pearson’s correlation coefficient in tumor regions. Additionally, sensitivity studies were performed to quantify the effects of image segmentation, registration, noise, and segmentation on R.
Results
On average, FDG PET and FLT PET images were most highly correlated (RFDG:FLT=0.76, range 0.53–0.85), while Cu-ATSM PET showed greater heterogeneity in correlation to other tracers (RFDG:Cu-ATSM=0.64, range 0.51–0.79; RFLT:Cu-ATSM=0.61, range 0.21–0.80). Of the tested parameters, correlation was most sensitive to image registration. Misregistration of one voxel lead to ΔRFDG=0.25, ΔRFLT=0.39, and ΔRCu-ATSM=0.27. Image noise and reconstruction also had quantitative effects on correlation. No significant quantitative differences were found between GTV, expanded GTV, or CTV regions.
Conclusions
Voxel-based correlation represents a first step into understanding spatial organization of tumor phenotype. These results have implications for radiotherapy target definition and provide a framework to test outcome prediction based on pretherapy distribution of phenotype.
doi:10.1016/j.radonc.2012.09.012
PMCID: PMC3494460  PMID: 23068711
Correlation; FDG; FLT; Cu-ATSM; PET
14.  Tumour microenvironment heterogeneity affects the perceived spatial concordance between the intratumoural patterns of cell proliferation and 18F-fluorothymidine uptake 
Background and purpose
PET imaging with 18F-fluorothymidine (18F-FLT) can potentially be used to identify tumour subvolumes for selective dose escalation in radiation therapy. The purpose of this study is to analyse the co-localization of intratumoural patterns of cell proliferation with 18F-FLT tracer uptake.
Materials and methods
Mice bearing FaDu or SQ20B xenograft tumours were injected with 18F-FLT, and bromodeoxyuridine (proliferation marker). Ex vivo images of the spatial pattern of intratumoural 18F-FLT uptake and that of bromodeoxyuridine DNA incorporation were obtained from thin tumour tissue sections. These images were segmented by thresholding and relative operating characteristic (ROC) curves and Dice similarity indices were evaluated.
Results
The thresholds at which maximum overlap occurred between FLT-segmented areas and areas of active cell proliferation were significantly different for the two xenograft tumour models, whereas the median Dice values were not. However, ROC analysis indicated that segmented FLT images were more specific at detecting the proliferation pattern in FaDu tumours than in SQ20B tumours.
Conclusion
Highly dispersed patterns of cell proliferation observed in certain tumours can affect the perceived spatial concordance between the spatial pattern of 18F-FLT uptake and that of cell proliferation even when a high-resolution ex vivo autoradiography imaging is used for 18F-FLT imaging.
doi:10.1016/j.radonc.2012.02.011
PMCID: PMC3387509  PMID: 22444241
FLT; tumour cell proliferation; mouse tumour; autoradiography; image-guided radiotherapy
15.  Spatially resolved regression analysis of pre-treatment FDG, FLT and Cu-ATSM PET from post-treatment FDG PET: an exploratory study 
Purpose
To quantify associations between pre-radiotherapy and post-radiotherapy PET parameters via spatially resolved regression.
Materials and methods
Ten canine sinonasal cancer patients underwent PET/CT scans of [18F]FDG (FDGpre), [18F]FLT (FLTpre), and [61Cu]Cu-ATSM (Cu-ATSMpre). Following radiotherapy regimens of 50 Gy in 10 fractions, veterinary patients underwent FDG PET/CT scans at three months (FDGpost). Regression of standardized uptake values in baseline FDGpre, FLTpre and Cu-ATSMpre tumour voxels to those in FDGpost images was performed for linear, log-linear, generalized-linear and mixed-fit linear models. Goodness-of-fit in regression coefficients was assessed by R2. Hypothesis testing of coefficients over the patient population was performed.
Results
Multivariate linear model fits of FDGpre to FDGpost were significantly positive over the population (FDGpost~0.17 FDGpre, p=0.03), and classified slopes of RECIST non-responders and responders to be different (0.37 vs. 0.07, p=0.01). Generalized-linear model fits related FDGpre to FDGpost by a linear power law (FDGpost~FDGpre0.93, p<0.001). Univariate mixture model fits of FDGpre improved R2 from 0.17 to 0.52. Neither baseline FLT PET nor Cu-ATSM PET uptake contributed statistically significant multivariate regression coefficients.
Conclusions
Spatially resolved regression analysis indicates that pre-treatment FDG PET uptake is most strongly associated with three-month post-treatment FDG PET uptake in this patient population, though associations are histopathology-dependent.
doi:10.1016/j.radonc.2012.05.002
PMCID: PMC3489967  PMID: 22682748
PET; FDG; FLT; Cu-ATSM; dose painting; regression analysis; sinonasal cancer
16.  Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo 
Purpose
To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy.
Material and Methods
Stable transfectant 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 varified 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 locally radiation. Tumor volume were 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 combined with local irradiation resulted in significant tumor regression, as 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 improves the tumor control in combination with radiotherapy.
doi:10.1016/j.radonc.2012.07.011
PMCID: PMC3747309  PMID: 22938726
hypoxia; radiosensitization; cytosine deaminase; uracil phosphoribosyltransferase; 5-flurocytosine
17.  Radiosensitisation of bladder cancer cells by panobinostat is modulated by Ku80 expression☆ 
Radiotherapy and Oncology  2013;108(3):429-433.
Background and purpose
In muscle-invasive bladder cancer there is an urgent need to identify relatively non-toxic radiosensitising agents for use in elderly patients. Histone deacetylase inhibitors radiosensitise tumour cells but not normal cells in vitro and variously downregulate DNA damage signalling, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair proteins. We investigated panobinostat (PAN) as a potential radiosensitiser in bladder cancer cells.
Materials and methods
Clonogenic assays were performed in RT112 bladder cancer cells, and RT112 cells stably knocked down for RAD51 or Ku80 by shRNAi. Resolution of γH2AX foci was determined by immunofluorescence confocal microscopy, cell cycle progression by FACS analysis and protein expression by western blotting.
Results
PAN had a greater radiosensitising effect in Ku80KD than RT112 or RAD51KD cells; enhancement ratios 1.35 for Ku80KD at 10 nM (IC20 for Ku80KD) and 1.31 for RT112 and RAD51KD at 25 nM (IC40 for both). PAN downregulated MRE11, NBS1 and RAD51, but not Ku70 and Ku80, increased γH2AX foci formation in a dose-dependent manner and delayed γH2AX foci repair after ionising radiation.
Conclusions
PAN acts as a radiosensitiser in bladder cancer cell lines, and appears to target HR rather than NHEJ. As muscle-invasive bladder tumours have reduced Ku-DNA binding, PAN could be particularly useful as a radiosensitiser in bladder cancer.
doi:10.1016/j.radonc.2013.06.021
PMCID: PMC3824066  PMID: 23932191
Bladder cancer; Histone deacetylase inhibitor; Ku80; Panobinostat; Radiosensitisation
18.  G2 Checkpoint Abrogator Abates the Antagonistic Interaction between Antimicrotubule Drugs and Radiation Therapy 
Background and Purpose
We previously demonstrated that radiation may arrest tumor cells at G2 phase, which in turn prevents the cytotoxicity of antimicrotubule drugs and results in antagonistic interaction between these two modalities. Herein we tested whether G2 abrogators would attenuate the above antagonistic interaction and improve the therapeutic efficacy of combination therapy between radiation and antimicrotubule drugs.
Materials and Methods
Breast cancer BCap37 and epidermoid carcinoma KB cell lines were administered with radiation, UCN-01 (a model drug of G2 abrogator), paclitaxel or vincristine, alone or in combinations. The antitumor activities of single and combined treatments were analyzed by a series of cytotoxic, apoptosis, cell cycle, morphological and biochemical assays.
Results
UCN-01 significantly enhanced the cytotoxicity of radiation, antimitotic drugs, and their combined treatments in vitro. Further investigations demonstrated that UCN-01 attenuated radiation-induced G2 arrest, and subsequently repressed the inhibitory effect of radiation on drug-induced mitotic arrest and apoptosis.
Conclusions
This is the first report demonstrating that G2 checkpoint abrogation represses the inhibitory effect of radiation on antimicrotubule drugs, which may be implicated in cancer combination therapy. Considering that G2 abrogators are under extensive evaluation for cancer treatment, our findings provide valuable information for this class of promising compounds.
doi:10.1016/j.radonc.2012.04.021
PMCID: PMC3423477  PMID: 22682751
radiation therapy; antimicrotubule drugs; UCN-01; breast cancer; apoptosis; cell cycle arrest
19.  Comparison of Dose Decrement from Intrafraction Motion for Prone and Supine Prostate Radiotherapy 
Background and Purpose
Dose effects of intrafraction motion during prone prostate radiotherapy are unknown. We compared prone and supine treatment using real-time tracking data to model dose coverage.
Material and Methods
Electromagnetic tracking data was analyzed for 10 patients treated prone, and 15 treated supine, with IMRT for localized prostate cancer. Plans were generated using 0, 3, and 5 mm PTV expansions. Manual beam-hold interventions were applied to reposition the patient when translations exceeded a predetermined threshold. A custom software application (SWIFTER) used intrafraction tracking data acquired during beam-on to model delivered prostate dose, by applying rigid body transformations to the prostate structure contoured at simulation within the planned dose cloud. The delivered minimum prostate dose as a percentage of planned dose (Dmin%), and prostate volume covered by the prescription dose as a percentage of the planned volume (VRx%) were compared for prone and supine treatment.
Results
Dmin% was reduced for prone treatment for 0 (p=0.02) and 3 mm (p=0.03) PTV margins. VRx% was reduced for prone treatment only for 0 mm margins (p=0.002). No significant differences were found using 5 mm margins.
Conclusions
Intrafraction motion has a greater impact on target coverage for prone compared to supine prostate radiotherapy. PTV margins of 3 mm or less correlate with a significant decrease in delivered dose for prone treatment.
doi:10.1016/j.radonc.2012.06.008
PMCID: PMC3423556  PMID: 22809590
Prostate; Radiotherapy; Prone; Tracking; Calypso
20.  Adjuvant chemoradiation versus surgery alone for adenocarcinoma of the ampulla of Vater ☆ 
Background and purpose
To examine the role of adjuvant chemoradiation (CRT) in patients with resected ampullary adenocarcinoma.
Materials and methods
The records of patients who underwent curative surgery for ampullary adenocarcinoma at a single institution between 1992 and 2007 were reviewed. Final analysis included 111 patients, 45% of which also received adjuvant CRT.
Results
Median overall survival (OS) was 36.2 months for all patients. Adverse prognostic factors for OS included T stage (T3/4 vs. T1/T2, p = 0.046), node status (positive vs. negative, p < 0.001), and histological grade (grade 3 vs. 1/2, p = 0.09). Patients receiving CRT were more likely to have advanced T-stage (p = 0.001), node positivity (p < 0.001), and poor histologic grade (p = 0.015). Patients who received CRT were also significantly younger (p = 0.001). On univariate analysis, adjuvant CRT failed to result in a significant difference in survival when compared to surgery alone (median OS: 33.4 vs. 36.2 months, p = 0.969). Patients with node-positive resections who underwent CRT had a non-significant improvement in survival (median OS: 21.6 vs. 13.0 months, p = 0.092). Thirty-three percent of patients developed distant metastasis. Common sites of distant metastasis included liver (23%) and peritoneum (7%).
Conclusions
Adjuvant chemoradiation following curative resection for ampullary adenocarcinoma did not lead to a statistically significant benefit in overall survival. A significant proportion of patients still developed distant metastatic disease suggesting a need for more effective systemic adjuvant therapy.
doi:10.1016/j.radonc.2009.05.006
PMCID: PMC3700350  PMID: 19541379
Ampulla of Vater; Ampullary cancer; Radiotherapy; Adjuvant therapy; Chemoradiation
21.  Symptomatic cardiac toxicity is predicted by dosimetric and patient factors rather than changes in 18F-FDG PET determination of myocardial activity after chemoradiotherapy for esophageal cancer 
Radiotherapy and Oncology  2012;104(1):72-77.
Purpose
To determine factors associated with symptomatic cardiac toxicity in patients with esophageal cancer treated with chemoradiotherapy.
Material and Methods
We retrospectively evaluated 102 patients treated with chemoradiotherapy for locally advanced esophageal cancer. Our primary endpoint was symptomatic cardiac toxicity. Radiation dosimetry, patient demographic factors, and myocardial changes seen on 18F-FDG PET were correlated with subsequent cardiac toxicity. Cardiac toxicity measured by RTOG and CTCAE v3.0 criteria was identified by chart review.
Results
During the follow up period, 12 patients were identified with treatment related cardiac toxicity, 6 of which were symptomatic. The mean heart V20 (79.7% vs. 67.2%, p=0.05), V30 (75.8% vs. 61.9%, p=0.04), and V40 (69.2% vs. 53.8%, p=0.03) were significantly higher in patients with symptomatic cardiac toxicity than those without. We found the threshold for symptomatic cardiac toxicity to be a V20, V30 and V40 above 70%, 65% and 60%, respectively. There was no correlation between change myocardial SUV on PET and cardiac toxicity, however, a greater proportion of women suffered symptomatic cardiac toxicity compared to men (p=0.005).
Conclusions
A correlation did not exist between percent change in myocardial SUV and cardiac toxicity. Patients with symptomatic cardiac toxicity received significantly greater mean V20, 30 and 40 values to the heart compared to asymptomatic patients. These data need validation in a larger independent data set.
doi:10.1016/j.radonc.2012.04.016
PMCID: PMC3389132  PMID: 22682539
Chemoradiotherapy; Esophageal cancer; Cardiac Toxicity
22.  USE OF FRACTIONAL DOSE-VOLUME HISTOGRAMS TO MODEL RISK OF ACUTE RECTAL TOXICITY AMONG PATIENTS TREATED ON RTOG 94-06 
Background and Purpose
For toxicities occurring during the course of radiotherapy, it is conceptually inaccurate to perform normal-tissue complication probability analyses using the complete dose-volume histogram. The goal of this study was to analyze acute rectal toxicity using a novel approach in which the fit of the Lyman-Kutcher-Burman (LKB) model is based on the fractional rectal dose-volume histogram (DVH).
Materials and Methods
Grade ≥2 acute rectal toxicity was analyzed in 509 patients treated on Radiation Therapy Oncology Group (RTOG) protocol 94-06. These patients had no field reductions or treatment-plan revisions during therapy, allowing the fractional rectal DVH to be estimated from the complete rectal DVH based on the total number of dose fractions delivered.
Results
The majority of patients experiencing Grade ≥2 acute rectal toxicity did so before completion of radiotherapy (70/80=88%). Acute rectal toxicity depends on fractional mean rectal dose, with no significant improvement in the LKB model fit when the volume parameter differs from n=1. The incidence of toxicity was significantly lower for patients who received hormone therapy (P=0.024).
Conclusions
Variations in fractional mean dose explain the differences in incidence of acute rectal toxicity, with no detectable effect seen here for differences in numbers of dose fractions delivered.
doi:10.1016/j.radonc.2012.04.023
PMCID: PMC3413409  PMID: 22673726
prostate cancer; acute rectal toxicity; dose-volume histogram; normal-tissue complication probability; Lyman model
23.  [18F]-FDG UPTAKE DOSE RESPONSE CORRELATES WITH RADIATION PNEUMONITIS IN LUNG CANCER PATIENTS 
Purpose
To quantify the post-radiotherapy 2-[18F]-fluoro-2-deoxyglucose (FDG) pulmonary uptake dose-response in lung cancer patients and determine its relationship with radiation pneumonitis symptoms.
Methods and Materials
The data from 24 patients treated for lung cancer with thoracic radiotherapy who received restaging PET/CT imaging between 4 and 12 weeks after radiotherapy completion were evaluated. Their radiation dose distribution was registered with the post-treatment restaging PET/CT. Using histogram analysis, the voxel average FDG-PET uptake versus radiation dose was obtained for each case and linear regression was performed. The resulting slope, the pulmonary metabolic radiation response (PMRR), was used to characterize the dose-response. The Common Toxicity Criteria version 3 was used to score clinical pulmonary toxicity symptoms. Receiver operating characteristic (ROC) curves were used to determine the level of FDG uptake v. dose, MLD, V5, V10, V20, and V30 that can best predict symptomatic and asymptomatic patients.
Results
The median time between radiotherapy completion and FDG-PET imaging was 59 days (range, 26–70 days). The median of the mean SUV from lung that received 0–5 Gy was 1.00 (range, 0.37–1.48), 5–10 Gy was 1.01 (range, 0.37–1.77), 10 –20 Gy was 1.04 (0.42–1.53), and > 20 Gy was 1.29 (range, 0.41–8.01). Using the dose range of 0 Gy to the maximum dose minus 10 Gy, hierarchical linear regression model of the radiation dose and normalized FDG uptake per case found an adequate fit with the linear model. Pneumonitis scores were: Grade 0 for 13, Grade 1 for 5, Grade 2 for 6, and Grade 3, 4 or 5 for none. Using a PMRR threshold of 0.017 yields an associated true positive rate of 0.67 and false positive rate of 0.15 with average error of 30%. A V5 threshold of 57.6 gives an associated true positive rate of 0.67 and false positive rate of 0.05 with a 20% average error.
Conclusion
The metabolic radiation pneumonitis dose response was evaluated from post-treatment FDG-PET/CT imaging. Statistical modeling found a linear relationship. The FDG uptake dose response and V5 correlated with symptomatic radiation pneumonitis.
doi:10.1016/j.radonc.2012.04.003
PMCID: PMC3626502  PMID: 22578806
Radiation pneumonitis; Pulmonary injury; Computed tomography; Positron emission tomography
24.  Necrosis predicts benefit from hypoxia-modifying therapy in patients with high risk bladder cancer enrolled in a phase III randomised trial☆ 
Radiotherapy and Oncology  2013;108(1):40-47.
Background and purpose
Addition of carbogen and nicotinamide (hypoxia-modifying agents) to radiotherapy improves the survival of patients with high risk bladder cancer. The study investigated whether histopathological tumour features and putative hypoxia markers predicted benefit from hypoxia modification.
Materials and methods
Samples were available from 231 patients with high grade and invasive bladder carcinoma from the BCON phase III trial of radiotherapy (RT) alone or with carbogen and nicotinamide (RT + CON). Histopathological tumour features examined were: necrosis, growth pattern, growing margin, and tumour/stroma ratio. Hypoxia markers carbonic anhydrase-IX and glucose transporter-1 were examined using tissue microarrays.
Results
Necrosis was the only independent prognostic indicator (P = 0.04). Necrosis also predicted benefit from hypoxia modification. Five-year overall survival was 48% (RT) versus 39% (RT + CON) (P = 0.32) in patients without necrosis and 34% (RT) versus 56% (RT + CON) (P = 0.004) in patients with necrosis. There was a significant treatment by necrosis strata interaction (P = 0.001 adjusted). Necrosis was an independent predictor of benefit from RT + CON versus RT (hazard ratio [HR]: 0.43, 95% CI 0.25–0.73, P = 0.002). This trend was not observed when there was no necrosis (HR: 1.64, 95% CI 0.95–2.85, P = 0.08).
Conclusions
Necrosis predicts benefit from hypoxia modification in patients with high risk bladder cancer and should be used to select patients; it is simple to identify and easy to incorporate into routine histopathological examination.
doi:10.1016/j.radonc.2013.05.017
PMCID: PMC3885794  PMID: 23773411
Bladder cancer; Hypoxic modification; Necrosis; Biomarker; Radiotherapy; Carbogen and nicotinamide
25.  Heterogeneity in head and neck IMRT target design and clinical practice 
Purpose
To assess patterns of H&N IMRT practice with particular emphasis on elective target delineation.
Materials and methods
Twenty institutions with established H&N IMRT expertise were solicited to design clinical target volumes for the identical H&N cancer case. To limit contouring variability, a primary tonsil GTV and ipsilateral level II node were pre-contoured. Participants were asked to accept this GTV, and contour their recommended CTV and PTV. Dose prescriptions, contouring time, and recommendations regarding chemotherapy were solicited.
Results
All 20 institutions responded. Remarkable heterogeneity in H&N IMRT design and practice was identified. Seventeen of 20 centers recommended treatment of bilateral necks whereas 3/20 recommended treatment of the ipsilateral neck only. The average CTV volume was 250 cm3 (range 37–676 cm3). Although there was high concordance in coverage of ipsilateral neck levels II and III, substantial variation was identified for levels I, V, and the contralateral neck. Average CTV expansion was 4.1 mm (range 0–15 mm). Eight of 20 centers recommended chemotherapy (cisplatin), whereas 12/20 recommended radiation alone. Responders prescribed on average 69 and 68 Gy to the tumor and metastatic node GTV, respectively. Average H&N target volume contouring time was 102.5 min (range 60–210 min).
Conclusion
This study identifies substantial heterogeneity in H&N IMRT target definition, prescription, neck treatment, and use of chemotherapy among practitioners with established H&N IMRT expertise. These data suggest that continued efforts to standardize and simplify the H&N IMRT process are desirable for the safe and effective global advancement of H&N IMRT practice.
doi:10.1016/j.radonc.2012.02.010
PMCID: PMC3694728  PMID: 22405806
IMRT; Head and neck; Target design; CTV definition; Contouring

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