External Beam Irradiation Preferentially Inhibits the Endochondral Pathway of Fracture Healing: A Rat Model.

BACKGROUND: External beam irradiation is an accepted treatment for skeletal malignancies. Radiation acts on both cancerous and normal cells and, depending on the balance of these effects, may promote or impair bone healing after pathologic fracture. Previous studies suggest an adverse effect of radiation on endochondral ossification, but the existence of differential effects of radiation on the two distinct bone healing pathways is unknown. QUESTIONS/PURPOSES: The purpose of this study was to investigate the differential effects of external beam irradiation on endochondral compared with intramembranous ossification with intramedullary nail and plate fixation of fractures inducing the two respective osseous healing pathways through assessment of (1) bone biology by histomorphometric analysis of cartilage area and micro-CT volumetric assessment of the calcified callus; and (2) mechanical properties of the healing fracture by four-point bending failure analysis of bending stiffness and strength. METHODS: Thirty-six male Sprague-Dawley rats underwent bilateral iatrogenic femur fracture: one side was repaired with an intramedullary nail and the other with compression plating. Three days postoperatively, half (n = 18) received 8-Gray external beam irradiation to each fracture. Rodents were euthanized at 1, 2, and 4 weeks postoperatively (n = 3/group) for quantitative histomorphometry of cartilage area and micro-CT assessment of callus volume. The remaining rodents were euthanized at 3 months (n = 9/group) and subjected to four-point bending tests to assess stiffness and maximum strength. RESULTS: Nailed femurs that were irradiated exhibited a reduction in cartilage area at both 2 weeks (1.08 ± 1.13 mm versus 37.32 ± 19.88 mm; 95% confidence interval [CI] of the difference, 4.32-68.16 mm; p = 0.034) and 4 weeks (4.60 ± 3.97 mm versus 39.10 ± 16.28 mm; 95% CI of the difference, 7.64-61.36 mm; p = 0.023) compared with nonirradiated fractures. There was also a decrease in the volume ratio of calcified callus at 4 weeks (0.35 ± 0.08 versus 0.51 ± 0.05; 95% CI of the difference, 0.01-0.31; p = 0.042) compared with nonirradiated fractures. By contrast, there was no difference in cartilage area or calcified callus between irradiated and nonirradiated plated femurs. The stiffness (128.84 ± 76.60 N/mm versus 26.99 ± 26.07 N/mm; 95% CI of the difference, 44.67-159.03 N/mm; p = 0.012) and maximum strength (41.44 ± 22.06 N versus 23.75 ± 11.00 N; 95% CI of the difference, 0.27-35.11 N; p = 0.047) of irradiated plated femurs was greater than the irradiated nailed femurs. However, for nonirradiated femurs, the maximum strength of nailed fractures (36.05 ± 17.34 N versus 15.63 ± 5.19 N; 95% CI of the difference, 3.96-36.88 N; p = 0.022) was greater than plated fractures, and there was no difference in stiffness between the nailed and plated fractures. CONCLUSIONS: In this model, external beam irradiation was found to preferentially inhibit endochondral over intramembranous ossification with the greatest impairment in healing of radiated fractures repaired with intramedullary nails compared with those fixed with plates. Future work with larger sample sizes might focus on further elucidating the observed differences in mechanical properties. CLINICAL RELEVANCE: This work suggests that there may be a rationale for compression plating rather than intramedullary nailing of long bone fractures in select circumstances where bony union is desirable, adjunctive radiation treatment is required, and bone stock is sufficient for plate and screw fixation.

Validation of a modern second-check dosimetry system using a novel verification phantom.

PURPOSE: To evaluate the Mobius second-check dosimetry system by comparing it to ionization-chamber dose measurements collected in the recently released Mobius Verification Phantom™ (MVP). For reference, a comparison of these measurements to dose calculated in the primary treatment planning system (TPS), Varian Eclipse with the AcurosXB dose algorithm, is also provided. Finally, patient dose calculated in Mobius is compared directly to Eclipse to demonstrate typical expected results during clinical use of the Mobius system. METHODS: Seventeen anonymized intensity-modulated clinical treatment plans were selected for analysis. Dose was recalculated on the MVP in both Eclipse and Mobius. These calculated doses were compared to doses measured using an A1SL ionization-chamber in the MVP. Dose was measured and analyzed at two different chamber positions for each treatment plan. Mobius calculated dose was then compared directly to Eclipse using the following metrics; target mean dose, target D95%, global 3D gamma pass rate, and target gamma pass rate. Finally, these same metrics were used to analyze the first 36 intensity modulated cases, following clinical implementation of the Mobius system. RESULTS: The average difference between Mobius and measurement was 0.3 ± 1.3%. Differences ranged from -3.3 to + 2.2%. The average difference between Eclipse and measurement was -1.2 ± 0.7%. Eclipse vs. measurement differences ranged from -3.0 to -0.1%. For the 17 anonymized pre-clinical cases, the average target mean dose difference between Mobius and Eclipse was 1.0 ± 1.1%. Average target D95% difference was -0.9 ± 2.0%. Average global gamma pass rate, using a criteria of 3%, 2 mm, was 94.4 ± 3.3%, and average gamma pass rate for the target volume only was 80.2 ± 12.3%. Results of the first 36 intensity-modulated cases, post-clinical implementation of Mobius, were similar to those seen for the 17 pre-clinical test cases. CONCLUSION: Mobius correctly calculated dose for each tested intensity modulated treatment plan, agreeing with measurement to within 3.5% for all cases analyzed. The dose calculation accuracy and independence of the Mobius system is sufficient to provide a rigorous second-check of a modern TPS.

RIP1 and RIP3 complex regulates radiation-induced programmed necrosis in glioblastoma.

Radiation-induced necrosis (RN) is a relatively common side effect of radiation therapy for glioblastoma. However, the molecular mechanisms involved and the ways RN mechanisms differ from regulated cell death (apoptosis) are not well understood. Here, we compare the molecular mechanism of cell death (apoptosis or necrosis) of C6 glioma cells in both in vitro and in vivo (C6 othotopically allograft) models in response to low and high doses of X-ray radiation. Lower radiation doses were used to induce apoptosis, while high-dose levels were chosen to induce radiation necrosis. Our results demonstrate that active caspase-8 in this complex I induces apoptosis in response to low-dose radiation and inhibits necrosis by cleaving RIP1 and RI. When activation of caspase-8 was reduced at high doses of X-ray radiation, the RIP1/RIP3 necrosome complex II is formed. These complexes induce necrosis through the caspase-3-independent pathway mediated by calpain, cathepsin B/D, and apoptosis-inducing factor (AIF). AIF has a dual role in apoptosis and necrosis. At high doses, AIF promotes chromatinolysis and necrosis by interacting with histone H2AX. In addition, NF-κB, STAT-3, and HIF-1 play a crucial role in radiation-induced inflammatory responses embedded in a complex inflammatory network. Analysis of inflammatory markers in matched plasma and cerebrospinal fluid (CSF) isolated from in vivo specimens demonstrated the upregulation of chemokines and cytokines during the necrosis phase. Using RIP1/RIP3 kinase specific inhibitors (Nec-1, GSK'872), we also establish that the RIP1-RIP3 complex regulates programmed necrosis after either high-dose radiation or TNF-α-induced necrosis requires RIP1 and RIP3 kinases. Overall, our data shed new light on the relationship between RIP1/RIP3-mediated programmed necrosis and AIF-mediated caspase-independent programmed necrosis in glioblastoma.

Assessment of a three-dimensional (3D) water scanning system for beam commissioning and measurements on a helical tomotherapy unit.

Beam scanning data collected on the tomotherapy linear accelerator using the TomoScanner water scanning system is primarily used to verify the golden beam profiles included in all Helical TomoTherapy treatment planning systems (TOMO TPSs). The user is not allowed to modify the beam profiles/parameters for beam modeling within the TOMO TPSs. The authors report the first feasibility study using the Blue Phantom Helix (BPH) as an alternative to the TomoScanner (TS) system. This work establishes a benchmark dataset using BPH for target commissioning and quality assurance (QA), and quantifies systematic uncertainties between TS and BPH. Reproducibility of scanning with BPH was tested by three experienced physicists taking five sets of measurements over a six-month period. BPH provides several enhancements over TS, including a 3D scanning arm, which is able to acquire necessary beam-data with one tank setup, a universal chamber mount, and the OmniPro software, which allows online data collection and analysis. Discrepancies between BPH and TS were estimated by acquiring datasets with each tank. In addition, data measured with BPH and TS was compared to the golden TOMO TPS beam data. The total systematic uncertainty, defined as the combination of scanning system and beam modeling uncertainties, was determined through numerical analysis and tabulated. OmniPro was used for all analysis to eliminate uncertainty due to different data processing algorithms. The setup reproducibility of BPH remained within 0.5 mm/0.5%. Comparing BPH, TS, and Golden TPS for PDDs beyond maximum depth, the total systematic uncertainties were within 1.4mm/2.1%. Between BPH and TPS golden data, maximum differences in the field width and penumbra of in-plane profiles were within 0.8 and 1.1 mm, respectively. Furthermore, in cross-plane profiles, the field width differences increased at depth greater than 10 cm up to 2.5 mm, and maximum penumbra uncertainties were 5.6mm and 4.6 mm from TS scanning system and TPS modeling, respectively. Use of BPH reduced measurement time by 1-2 hrs per session. The BPH has been assessed as an efficient, reproducible, and accurate scanning system capable of providing a reliable benchmark beam data. With this data, a physicist can utilize the BPH in a clinical setting with an understanding of the scan discrepancy that may be encountered while validating the TPS or during routine machine QA. Without the flexibility of modifying the TPS and without a golden beam dataset from the vendor or a TPS model generated from data collected with the BPH, this represents the best solution for current clinical use of the BPH.

Feasibility study of performing IGRT system daily QA using a commercial QA device.

The purpose of this study was to investigate the feasibility of using a single QA device for comprehensive, efficient daily QA of a linear accelerator (Linac) and three image-guided stereotactic positioning systems (IGSPSs). The Sun Nuclear Daily QA 3 (DQA3) device was used to perform daily dosimetry and mechanical accuracy tests for an Elekta Linac, as well as daily image geometric and isocenter coincidence accuracy tests for three IGSPSs: the AlignRT surface imaging system; the frameless SonArray optical tracking System (FSA) and the Elekta kV CBCT. The DQA3 can also be used for couch positioning, repositioning, and rotational tests during the monthly QA. Based on phantom imaging, the Linac coordinate system determined using AlignRT was within 0.7 mm/0.6° of that of the CBCT system. The difference is attributable to the different calibration methods that are utilized for these two systems. The laser alignment was within 0.5 mm of the isocenter location determined with the three IGSPSs. The ODI constancy was ± 0.5 mm. For gantry and table angles of 0°, the mean isocenter displacement vectors determined using the three systems were within 0.7 mm and 0.6° of one another. Isocenter rotational offsets measured with the systems were all ≤ 0.5°. For photon and electron beams tested over a period of eight months, the output was verified to remain within 2%, energy variations were within 2%, and the symmetry and flatness were within 1%. The field size and light-radiation coincidence were within 1mm ± 1 mm. For dosimetry reproducibility, the standard deviation was within 0.2% for all tests and all energies, except for photon energy variation which was 0.6%. The total measurement time for all tasks took less than 15 minutes per QA session compared to 40 minutes with our previous procedure, which utilized an individual QA device for each IGSPS. The DQA3 can be used for accurate and efficient Linac and IGSPS daily QA. It shortens QA device setup time, eliminates errors introduced by changing phantoms to perform different tests, and streamlines the task of performing dosimetric checks.

MiR-34a modulates ionizing radiation-induced senescence in lung cancer cells.

MicroRNAs (miRNAs) are a new class of gene expression regulators that have been implicated in tumorigenesis and modulation of the responses to cancer treatment including that of human non-small cell lung cancer (NSCLC). However, the role of miR-34a in ionizing radiation (IR)-induced senescence in NSCLC cells remains poorly understood. Here we report that IR-induced premature senescence correlates with upregulation of miR-34a expression in NSCLC cells. Ectopic overexpression of miR-34a by transfection with synthetic miR-34a mimics markedly enhances IR-induced senescence, whereas inhibition of miR-34a by transfection with a synthetic miR-34a inhibitor attenuates IR-induced senescence. Clonogenic assays reveal that treatment with miR-34a mimics augments IR-induced cell killing in human NSCLC cells. Mechanistically, we found that the senescence-promoting effect of miR-34a is associated with a dramatic down-regulation of c-Myc (Myc) expression, suggesting that miR-34a may promote IR-induced senescence via targeting Myc. In agreement with this suggestion, knockdown of Myc expression by RNAi recapitulates the senescence-promoting effect of miR-34a and enhances IR-induced cell killing in NSCLC cells. Collectively, these results demonstrate a previously unrecognized role for miR-34a in modulating IR-induced senescence in human NSCLC cells and suggest that pharmacological intervention of miR-34a expression may represent a new therapeutic strategy for improving the efficacy of lung cancer radiotherapy.

Acute complications of MammoSite brachytherapy: a single institution's initial clinical experience.

PURPOSE: To report the acute complications incurred by the initial 37 patients who underwent accelerated partial breast irradiation with the MammoSite balloon breast brachytherapy applicator at the Medical University of South Carolina. METHODS AND MATERIALS: Between May 2002 and March 2003, 37 patients with ductal carcinoma in situ or invasive carcinoma had MammoSite brachytherapy catheters successfully place after lumpectomy by one of four surgeons and were deemed eligible for high-dose-rate brachytherapy. An open technique was used in 32 implants and the scar entry technique was used in 5 implants. Patients had Stage pTis-pT2N1 with negative margins. A dose of 34 Gy was prescribed to 1 cm from the balloon surface using (192)Ir high-dose-rate brachytherapy and was delivered in 10 fractions twice daily. CT was used to confirm that the balloon surface was adherent to the lumpectomy cavity and to measure the balloon surface to skin surface distance. CT images and daily fluoroscopic simulations were used for treatment planning. Patients were assessed for acute toxicity on the day of therapy completion and 4 weeks after therapy by the radiation oncologist. In addition, all available data from radiation, surgical, and medical oncology were retrospectively reviewed for documentation of complications. All patients in this series had a minimal follow-up of 3 months; the mean follow-up for all patients was 7 months. RESULTS: The acute complications were categorized as operative wound complications, infections, skin toxicity, seromas, or catheter failures. Operative wound complications occurred in 3 patients (8%). Radiation Therapy Oncology Group Grade 2 and Grade 3 toxicity occurred in 2 (5.4%) and 1 (2.7%) patient, respectively. Six (16.2%) developed wound infections and 12 (32.4%) seromas. Catheter failures due to leak occurred in 2 patients (5.4%) and rupture in 3 (8%). CONCLUSION: The types of complications in this experience were similar to those in the Phase I trial of the MammoSite brachytherapy applicator. However, catheter failure due to leak occurred in our experience and was not described in the Phase I trial. The incidence of complications was greater in our series than in the Phase I trial; however, differences in toxicity scoring and the length of follow-up between the two series impeded direct comparisons. The incidences of complications over time reflect the steep learning curve for accelerated partial breast irradiation using the MammoSite brachytherapy applicator. Finally, radiation recall dermatitis developed in 1 patient treated after this review was completed.

Catalase inhibits ionizing radiation-induced apoptosis in hematopoietic stem and progenitor cells.

Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme that coverts hydrogen peroxide into hydrogen and water. In this study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells and hematopoietic progenitor cells. Subsequent colony-forming cell and cobble-stone area-forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments, whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism by which catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.

Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence.

Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated ß-glactosidase (SA-ß-gal) staining, decreased BrdU incorporation and elevated expression of p16(INK4a) (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53-p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53-p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.

Conformal high dose external radiation therapy, 80.5 Gy, alone for medically inoperable non-small cell lung cancer: a retrospective analysis.

BACKGROUND: Retrospective analysis of patients with medically inoperable non-small cell lung cancer treated with continuous high-dose external beam radiation therapy at the Medical University of South Carolina. METHODS: We identified 35 patients with non-small cell lung cancer treated 1998-2002. None were candidates for resection for reasons including: pulmonary function (n = 23), previous cancer (n = 9), other co-morbidities (n = 2), and refusal of surgery (n = 1). Median percent predicted forced expiratory volume in 1 second was 41.5%. Median age was 71 years. Five patients had more than one primary tumor: three were concurrently treated, two were sequentially treated. Lesion sizes were <3 cm (n = 24); 3-5 cm (n = 12), and >5 cm (n = 5). Nodal stage was as follows: N0 (n = 33) and N1 (n = 2). Radiation therapy was administered once daily: median dose was 80.5 Gy/35 fx/2.3 Gy/fx. The clinical target volume was tumor plus nodes > or =1.0 cm. V20 data were available for 12 patients, with a mean value of 15.7%. RESULTS: Thirty-four patients completed treatment. Median follow-up was 23.0 months. There were 26 deaths: 19 died from non-small cell lung (73%) and seven died from co-morbid illness (27%). Median survival was 24 months (95% CI, 18.0-31.9 months). Four patients were alive with disease, and five were alive disease-free at 10- and 68-month follow-ups. Of 41 lesions, local failure occurred in 15 lesions (37%) of which 3 local failure patients (9%) failed concomitantly in untreated regional lymph nodes. There were no isolated nodal recurrences. Distant progression: 10 patients (29%) of which 6 distant progression without local failure. Two patients who both had prior lobectomies experienced grade 5 toxicities. CONCLUSION: Continuous high-dose external beam radiation therapy 80.5 Gy administered in 35 fractions was tolerated. Treatment-related death was rare (6%) and isolated to patients with prior lobectomies in an extremely high-risk population. Most mortality was lung cancer-related. The dose of 80.5 Gy in 7 weeks is supported for patients with single lesions and no prior lobectomy. Local failure dominates and higher effective doses should be explored.