Radiation Oncology - Latest Articles

Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells

Geeta Sharungbam — 2012-05-17T00:00:00Z

Background: Quantitative analysis of transcriptional regulation of genes is a prerequisite for a betterunderstanding of the molecular mechanisms of action of different radiation qualities such asphoton, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR(qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpretingthese results it is critical to normalize the expression levels of the target genes by that ofappropriately selected endogenous control genes (ECGs) or housekeeping genes. We soughtto systematically investigate common ECG candidates for their stability after differentradiation modalities in different human cell lines by qRT-PCR. We aimed to identify themost robust set of ECGs or housekeeping genes for transcriptional analysis in irradiationstudies. Methods: We tested the expression stability of 32 ECGs in three human cancer cell lines. Theepidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and thepancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbonions. Expression Heat maps, clustering and statistic algorithms were employed using SUMOsoftware package. The expression stability was evaluated by computing: mean, standarddeviation, ANOVA, coefficient of variation and the stability measure (M) given by thegeNorm algorithm. Results: Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs(p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function ofdifferent radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, oneof the most frequently used ECG, was differentially regulated as the function of differentradiation qualities (p [less than or equal to] 0.01). A comprehensive search for the most stable ECGs using thegeNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization.In contrast, 6 ECGs were required to properly normalize expression data in the more variableA549 cells. Considering both variables tested, i.e. cell type and radiation qualities, 5 genes--RPLP0, UBC, PPIA, TBP and PSMC4-- were identified as the consensus set of stable ECGs. Conclusions: Caution is warranted when selecting the internal control gene for the qRT-PCR geneexpression studies. Here, we provide a template of stable ECGs for investigation of radiationinduced gene expression.

Functional dosimetric metrics for predicting radiation-induced lung injury in non-small cell lung cancer patients treated with chemoradiotherapy

Dongqing Wang — 2012-05-17T00:00:00Z

Background: Radiation-induced lung injury (RILI) is an important dose-limiting toxicity during thoracicradiotherapy. The purpose of this study is to investigate single photon emission computedtomography (SPECT) perfusion-weighted functional dose-volume histogram (FDVH) forpredicting RILI in non-small cell lung cancer (NSCLC) patients treated with definitivechemoradiotherapy. Methods: Fifty-seven locally advanced NSCLC patients receiving chemoradiotherapy were enrolledprospectively. Patients had treatment scans and dose calculations to provide a standard dosevolumehistogram (DVH). Fusion of SPECT and computed tomography scans providedperfusion-weighted FDVH and associated functional dosimetric parameters (relative volumesof functional lung receiving more than a threshold dose of 5 - 60 Gy at increments of 5 Gy[FV5 - FV60]). The predictive abilities of FDVH and DVH were calculated and comparedbased on the area under receiver operating characteristic (ROC) curve (AUC). Results: The accumulative incidence of [greater than or equal to] 2 grade RILI was 19.3% with a median follow-up of 12months. Univariate analysis showed that the functional (FV5 - FV60) and standard (V5 -V40) parameters were associated with RILI (all value of p < 0.05). Close correlationsbetween a variety of functional and standard parameters were found. By ROC curve analysis,functional metrics (AUCs were 0.784 - 0.869) provided similarly (p value 0.233 - 1.000)predictive outcome to standard metrics (AUCs were 0.695 - 0.902) in lower - median doselevel parameters (FV5 - FV40). However, FDVH seemed to add some predictive value inhigher dose level, the best statistical significance for comparing FV60 with V60 was 0.693vs. 0.511 (p = 0.055). Conclusions: Functional metrics are identified as reliable predictors for RILI, however, this observationstill needs to be further verified using a larger sample size.

An athymic rat model of cutaneous radiation injury designed to study human tissue-based wound therapy

Lucas Rifkin — 2012-05-08T00:00:00Z

PurposeTo describe a pilot study for a novel preclinical model used to test human tissue-basedtherapies in the setting of cutaneous radiation injury. Methods: A protocol was designed to irradiate the skin of athymic rats while sparing the body andinternal organs by utilizing a non-occlusive skin clamp along with an x-ray image guidedstereotactic irradiator. Each rat was irradiated both on the right and the left flank with acircular field at a 20 cm source-to-surface distance (SSD). Single fractions of 30.4 Gy, 41.5Gy, 52.6 Gy, 65.5 Gy, and 76.5 Gy were applied in a dose-finding trial. Eight additionalwounds were created using the 41.5 Gy dose level. Each wound was photographed and thepercentage of the irradiated area ulcerated at given time points was analyzed using ImageJsoftware. Results: No systemic or lethal sequelae occurred in any animals, and all irradiated skin areas in themulti-dose trial underwent ulceration. Greater than 60% of skin within each irradiated zoneunderwent ulceration within ten days, with peak ulceration ranging from 62.1% to 79.8%.Peak ulceration showed a weak correlation with radiation dose (r = 0.664). Mean ulcerationrate over the study period is more closely correlated to dose (r = 0.753). With the highest doseexcluded due to contraction-related distortions, correlation between dose and averageulceration showed a stronger relationship (r = 0.895). Eight additional wounds created using41.5 Gy all reached peak ulceration above 50%, with all healing significantly butincompletely by the 65-day endpoint. Conclusions: We developed a functional preclinical model which is currently used to evaluate humantissue-based therapies in the setting of cutaneous radiation injury. Similar models may bewidely applicable and useful the development of novel therapies which may improveradiotherapy management over a broad clinical spectrum.

Clinical outcomes and toxicity using Stereotactic Body Radiotherapy (SBRT) for advanced cholangiocarcinoma

Brandon Barney — 2012-05-03T00:00:00Z

Background: To report single-institutional clinical outcomes and toxicity with SBRT forcholangiocarcinoma. Methods: From March 2009 to July 2011, 10 patients with 12 unresectable primary (n = 6) or recurrent(n = 6) cholangiocarcinoma lesions underwent abdominal SBRT. Sites treated included liver(n = 10), abdominal lymph nodes (n = 1), and adrenal gland (n = 1). SBRT was delivered inthree (n = 2) or five (n = 10) consecutive daily fractions over one week. The medianprescription dose was 55 Gy (range, 45-60). Treatment response was graded by RECISTv.1.1, and toxicities were scored by CTCAE v.4.0. Data was analyzed using the Kaplan-Meier method to determine rates of local control (LC), freedom from distant progression(FFDM) and overall survival (OS). Results: The median follow-up was 14 months (range, 2-26 months). LC, defined as freedom fromprogression within the SBRT field, was 100%, but four patients treated to intrahepatic sitesexperienced progression elsewhere in the liver. Estimates for FFDM at 6 and 12 months were73% and 31%, respectively. Sites of disease relapse included liver (n = 3), liver and lymphnodes (n = 1), liver and lungs (n = 1), lymph nodes (n = 1), and mesentery (n = 1). OSestimates for the cohort at 6 and 12 months were 83% and 73%, respectively. The mostcommon Grade [greater than or equal to]2 early toxicities were Grade 2 nausea and vomiting (n = 5) andgastrointestinal pain (n = 2). Late [greater than or equal to]2 toxicities included Grade 2 gastrointestinal pain (n = 3),Grade 3 biliary stenosis (n = 1), and Grade 5 liver failure (n = 1). Conclusions: SBRT shows promise as an effective local therapy for properly-selected patients withcholangiocarcinoma. Further follow-up is needed to better quantify the risk of latecomplications associated with SBRT.

The prognostic value of expression of HIF1alpha, EGFR and VEGF-A, in localized prostate cancer for intermediate- and high-risk patients treated with radiation therapy with or without androgen deprivation therapy

Damien Weber — 2012-04-30T00:00:00Z

PurposeAndrogens stimulate the production of hypoxia-inducible factor (HIF1alpha) and ultimately vascular endothelial growth factor (VEGF-A). Additionally, epithelial growth factor (EGF) mediates HIF1alpha production. Carbonic anhydrase IX (CAIX) expression is associated with tumor cell hypoxia in a variety of malignancies. This study assesses the prognostic relation between HIF1alpha, VEGF-A, EGF Receptor and CAIX expression by immunochemistry in diagnostic samples of patients with intermediate- and high-risk localized prostate cancer treated with radiation therapy, with or without androgen deprivation therapy (ADT).Materials and methodsBetween 1994 and 2004, 103 prostate cancer patients (mean age, 68.7 +/- 6.2), with prostate cancer (mean PSA, 13.3 +/- 3.7), were treated with radiation therapy (RT, median dose, 74 Gy). Fifty seven (55.3%) patients received ADT (median duration, 6 months; range, 0 - 24). Median follow-up was 97.6 months (range, 5.9 - 206.8) Results: Higher EGFR expression was significantly (p = 0.04) correlated with higher Gleason scores. On univariate analysis, HIF1alpha nuclear expression was a significant (p = 0.02) prognostic factor for biological progression-free survival (bPFS). A trend towards significance (p = 0.05) was observed with EGFR expression and bPFS. On multivariate analysis, low HIF1alpha nuclear (p = 0.01) and high EGFR (p = 0.04) expression remained significant adverse prognostic factors. Conclusions: Our study suggests that high nuclear expression of HIF1alpha and low EGFR expression in diagnostic biopsies of prostate cancer patients treated with RT +/- ADT is associated with a good prognosis.

Association of single nucleotide polymorphisms in the genes ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with risk of severe erythema after breast conserving radiotherapy

Annette Raabe — 2012-04-26T00:00:00Z

PurposeTo examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position 509), XPD (codon 751), and XRCC1 (codon 399) with the risk of severe erythema after breast conserving radiotherapy.Methods and materialsRetrospective analysis of 83 breast cancer patients treated with breast conserving radiotherapy. A total dose of 50.4 Gy was administered, applying 1.8 Gy/fraction within 42 days. Erythema was evaluated according to the Radiation Therapy Oncology Group (RTOG) score. DNA was extracted from blood samples and polymorphisms were determined using either the Polymerase Chain Reaction based Restriction-Fragment-Length-Polymorphism (PCR-RFL) technique or Matrix-Assisted-Laser-Desorption/Ionization -Time-Of-Flight-Mass-Spectrometry (MALDI-TOF). Relative excess heterozygosity (REH) was investigated to check compatibility of genotype frequencies with Hardy-Weinberg equilibrium (HWE). In addition, p-values from the standard exact HWE lack of fit test were calculated using 100,000 permutations. HWE analyses were performed using R. Results: Fifty-six percent (46/83) of all patients developed erythema of grade 2 or 3, with this risk being higher for patients with large breast volume (odds ratio, OR = 2.55, 95% confidence interval, CI: 1.03-6.31, p = 0.041). No significant association between SNPs and risk of erythema was found when all patients were considered. However, in patients with small breast volume the TGFB1 SNP was associated with erythema (p = 0.028), whereas the SNP in XPD showed an association in patients with large breast volume (p = 0.046). A risk score based on all risk alleles was neither significant in all patients nor in patients with small or large breast volume. Risk alleles of most SNPs were different compared to a previously identified risk profile for fibrosis. Conclusions: The genetic risk profile for erythema appears to be different for patients with small and larger breast volume. This risk profile seems to be specific for erythema as compared to a risk profile for fibrosis.

Determination of cytokine protein levels in oral secretions in patients undergoing radiotherapy for head and neck malignancies

Deborah Citrin — 2012-04-26T00:00:00Z

Background: Cytokines may be elevated in tumor and normal tissues following irradiation. Cytokine expression in these tissues may predict for toxicity or tumor control. The purpose of this pilot study was to determine the feasibility of measuring local salivary cytokine levels using buccal sponges in patients receiving chemo-radiation for head and neck malignancies.Patients and Methods11 patients with epithelial malignancies of the head and neck were recruiting to this study. All patients received radiotherapy to the head and neck region with doses ranging between 60 - 67.5 Gy. Chemotherapy was delivered concurrently with radiation in all patients. Salivary samples were obtained from high dose and low dose regions prior to treatment and at three intervals during treatment for assessment of cytokine levels (IL-4, IL-6, IL-8, IL-10, EGF, MCP-1, TNF-alpha, and VEGF). Results: Cytokine levels were detectable in the salivary samples. Salivary cytokine levels of IL-4, IL-6, IL-8, EGF, MCP-1, TNF- alpha , and VEGF were higher in the high dose region compared to the low dose region at all time points (p < 0.05). A trend toward an increase in cytokine levels as radiation dose increased was observed for IL-6, IL-8, MCP-1, and TNF-alpha. Conclusion: Assessment of salivary cytokine levels may provide a novel method to follow local cytokine levels during radiotherapy and may provide a mechanism to study cytokine levels in a regional manner.

Dosimetric consequences of translational and rotational errors in frame-less image-guided radiosurgery

Matthias Guckenberger — 2012-04-24T00:00:00Z

Background: To investigate geometric and dosimetric accuracy of frame-less image-guided radiosurgery(IG-RS) for brain metastases.Methods and materialsSingle fraction IG-RS was practiced in 72 patients with 98 brain metastases. Patientpositioning and immobilization used either double- (n = 71) or single-layer (n = 27)thermoplastic masks. Pre-treatment set-up errors (n = 98) were evaluated with cone-beam CT(CBCT) based image-guidance (IG) and were corrected in six degrees of freedom without anaction level. CBCT imaging after treatment measured intra-fractional errors (n = 64). Pre- andpost-treatment errors were simulated in the treatment planning system and target coverageand conformity were evaluated. Three scenarios of 0 mm, 1 mm and 2 mm GTV-to-PTV(gross tumor volume) safety margins (SM) were simulated. Results: Errors prior to IG were 3.9 mm +/- 1.7 mm (3D vector) and the maximum rotational error was1.7degrees +/- 0.8degrees on average. The post-treatment 3D error was 0.9 mm +/- 0.6 mm. No differencesbetween double- and single-layer masks were observed. Simulation of RS without imageguidancereduced target coverage and conformity to 75% +/- 19% and 60% +/- 25% of plannedvalues. Each 3D set-up error of 1 mm decreased target coverage and dose conformity by 6%and 10% on average, respectively, with a large inter-patient variability. Pre-treatmentcorrection of translations only but not rotations did not affect target coverage and conformity.Post-treatment errors reduced target coverage by >5% in 14% of the patients. A 1 mm safetymargin fully compensated intra-fractional patient motion. Conclusions: IG-RS with online correction of translational errors achieves high geometric and dosimetricaccuracy. Intra-fractional errors decrease target coverage and conformity unless compensatedwith appropriate safety margins.

The combination of olaparib and camptothecin for effective radiosensitization

Katsutoshi Miura — 2012-04-23T00:00:00Z

Background: Poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme involved in the repair of radiation-induced single-strand DNA breaks. PARP inhibitors such as olaparib (KU-0059436, AZD-2281) enhance tumor sensitivity to radiation and to topoisomerase I inhibitors like camptothecin (CPT). Olaparib is an orally bioavailable inhibitor of PARP-1 and PARP-2 that has been tested in multiple clinical trials. The purpose of this study was to investigate the characteristics of the sensitizing effect of olaparib for radiation and CPT in order to support clinical application of this agent. Methods: DLD-1 cells (a human colorectal cancer cell line) and H1299 cells (a non-small cell lung cancer cell line) with differences of p53 gene status were used. The survival of these cells was determined by clonogenic assay after treatment with drugs and X-ray irradiation. The gammaH2AX focus formation assay was performed to examine the influence of olaparib on induction and repair of double-stranded DNA breaks after exposure to radiation or CPT. Results: A radiosensitizing effect of olaparib was seen even at 0.01 muM. Its radiosensitizing effect after exposure for 2 h was similar to that after 24 h. H1299 cells with depletion or mutation of p53 were more radioresistant than H1299 cells with wild-type p53. However, similar enhancement of radiosensitization by olaparib was observed with all of the tested cell lines regardless of the p53 status. Olaparib also sensitized cells to CPT. This sensitizing effect was seen at low concentrations of olaparib such as 0.01 muM, and its sensitizing effect was the same at both 0.01 muM and 1 muM. The combination of olaparib and CPT had a stronger radiosensitizing effect. The results of the gammaH2AX focus assay corresponded with the clonogenic assay findings. Conclusion: Olaparib enhanced sensitivity to radiation and CPT at low concentrations and after relatively short exposure times such as 2 h. The radiosensitizing effect of olaprib was not dependent on the p53 status of tumor cells. These characteristics could be advantageous for clinical radiotherapy since tumor cells may be exposed to low concentrations of olaparib and/or may have different levels of p53 mutation. The combination of olaparib and CPT had a stronger radiosensitizing effect, indicating that combining a PARP inihibitor with a topoiomerase I inhibitor could be promising for clinical radiosensitization.

Factors influencing heterogeneity of radiation induced DNA-damage measured by the alkaline comet assay

Clemens Seidel — 2012-04-20T00:00:00Z

Background: To investigate whether different conditions of DNA structure and radiation treatment couldmodify heterogeneity of response. Additionally to study variance as a potential parameter ofheterogeneity for radiosensitivity testing. Methods: Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intactchromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histonedepletedDNA with 90 mM DMSO as antioxidant. Response to single (I-III) and twice (IV)irradiation with 4 Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity ofDNA damage was determined by calculation of variance of DNA-damage (V) and meanvariance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA). Results: Heterogeneity of initial DNA-damage (I, 0 min repair) increased without histones (II).Absence of histones was balanced by addition of antioxidants (III). Repair reducedheterogeneity of all samples (with and without irradiation). However double irradiation plusrepair led to a higher level of heterogeneity distinguishable from single irradiation and repairin intact cells. Increase of mean DNA damage was associated with a similarly elevatedvariance of DNA damage (r = +0.88). Conclusions: Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration,repair and radiation dose and was positively correlated with DNA damage. Experimentalconditions might be optimized by reducing scatter of comet assay data by repair andantioxidants, potentially allowing better discrimination of small differences. Amount ofheterogeneity measured by variance might be an additional useful parameter to characterizeradiosensitivity.