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.

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.

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.

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice.

Bruton's Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.

S-Nitrosoglutathione-mediated STAT3 regulation in efficacy of radiotherapy and cisplatin therapy in head and neck squamous cell carcinoma.

S-nitrosoglutathione (GSNO) is an endogenous nitric oxide (NO) carrier that plays a critical role in redox based NO signaling. Recent studies have reported that GSNO regulates the activities of STAT3 and NF-κB via S-nitrosylation dependent mechanisms. Since STAT3 and NF-κB are key transcription factors involved in tumor progression, chemoresistance, and metastasis of head and neck cancer, we investigated the effect of GSNO in cell culture and mouse xenograft models of head and neck squamous cell carcinoma (HNSCC). For the cell culture studies, three HNSCC cell lines were tested (SCC1, SCC14a and SCC22a). All three cell lines had constitutively activated (phosphorylated) STAT3 (Tyr(705)). GSNO treatment of these cell lines reversibly decreased the STAT3 phosphorylation in a concentration dependent manner. GSNO treatment also decreased the basal and cytokine-stimulated activation of NF-κB in SCC14a cells and reduced the basal low degree of nitrotyrosine by inhibition of inducible NO synthase (iNOS) expression. The reduced STAT3/NF-κB activity by GSNO treatment was correlated with the decreased cell proliferation and increased apoptosis of HNSCC cells. In HNSCC mouse xenograft model, the tumor growth was reduced by systemic treatment with GSNO and was further reduced when the treatment was combined with radiation and cisplatin. Accordingly, GSNO treatment also resulted in decreased levels of phosphorylated STAT3. In summary, these studies demonstrate that GSNO treatment blocks the NF-κB and STAT3 pathways which are responsible for cell survival, proliferation and that GSNO mediated mechanisms complement cispaltin and radiation therapy, and thus could potentiate the therapeutic effect in HNSCC.

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.

T-bet is critical for the development of acute graft-versus-host disease through controlling T cell differentiation and function.

T-bet is a master regulator for IFN-γ production and Th1 differentiation. We evaluated the roles of T-bet and IFN-γ in T cell responses in acute graft-versus-host disease (GVHD) and found that T-bet(-/-) T cells induced significantly less GVHD compared with wild-type or IFN-γ(-/-) counterparts in both MHC-mismatched and MHC-matched but minor histocompatibility Ag-mismatched models driven by CD4 T cells. T-bet(-/-), but not IFN-γ(-/-), CD4 T cells had a markedly reduced ability to cause tissue damage in liver and gut. This distinct outcome is reflected by the differential gene expression on donor CD4 T cells deficient for T-bet or IFN-γ. At mRNA and protein levels, we defined several T-bet-dependent molecules that may account for the impaired ability of T-bet(-/-) T cells to migrate into target organs and to produce Th1-related cytokines. Moreover, these molecules were independent of either endogenous IFN-γ, such as CXCR3 and programmed death-1, or systematic IFN-γ, such as NKG2D, I-A(b), and granzyme B. Although both T-bet(-/-) and IFN-γ(-/-) CD4 T cells are prone to differentiate into Th17 cells, polarized Th17 cells deficient for T-bet but not for IFN-γ had a significantly reduced ability to cause GVHD. Finally, T-bet(-/-) T cells had a compromised graft-versus-leukemia effect, which could be essentially reversed by neutralization of IL-17 in the recipients. We conclude that T-bet is required for Th1 differentiation and migration, as well as for optimal function of Th17 cells. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic.

Comparison of radiation treatment delivery for pancreatic cancer: Linac intensity-modulated radiotherapy versus helical tomotherapy.

INTRODUCTION: Intensity-modulated radiotherapy (IMRT) has been shown to reduce dose to organs at risk (OAR) while adequately treating tumour volume. This study quantitatively compares the dosimetric differences from step-and-shoot IMRT compared with helical tomotherapy (HT) for pancreatic head cancer. METHODS: Twelve consecutive patients with non-metastatic, stage T3 or T4, unresectable pancreatic head cancer were planned for step-and-shoot IMRT as well as HT. Radiotherapy was planned to deliver 45.9 Gy to the clinical target volume in 30 fractions with an integrated boost to 54 Gy to the gross tumour volume (planning target volume 5400 including a 1-cm set-up margin). The uniformity index (UI) and conformity index (CI) were used to compare the quality of target coverage, while the quality index (QI) compared the dosimetric performance for OAR. RESULTS: Both methods were effective at covering the tumour with no significant difference in UI or CI. However, HT dosimetry exhibited superior sparing of OAR with significantly less stomach (mean QI(StomV30) = 0.84, P = 0.006) and small bowel dosing (mean small bowel QI(SBV30) = 0.84, P = 0.005). HT reduced dose to the kidney receiving the highest dose but the overall volume of kidney receiving 18 Gy was not significantly different between the two systems, indicating that HT spread the dose more uniformly through the kidneys. CONCLUSIONS: Target coverage is equivalent between the two systems; however, HT shows significantly better sparing of the stomach and small bowel. The decreased dose to OAR with HT is likely to improve the therapeutic ratio in the radiotherapy of pancreatic head cancers.

A ternary Cu-Sn-S cluster complex--(NBu4)[Cu19S28(SnPh)12(PEt2Ph)3].

Reaction of a mixture of CuCl, PhSnCl(3) and PEt(2)Ph with S(SiMe(3))(2) in THF resulted initially in the unexpected synthesis of the ionic, mixed copper-tin sulfide cluster [Li(thf)(4)][Cu(19)S(28)(SnPh)(12)(PEt(2)Ph)(3)] in low yields. However, by adding NBu(4)Cl to the reaction solutions we were able to selectively synthesize the structurally similar cluster ion in (NBu(4))[Cu(19)S(28)(SnPh)(12)(PEt(2)Ph)(3)]. Structural characterization by single crystal X-ray analysis reveals that the cluster anions consist in principle of a copper sulfide core decorated by PhSn(3+) groups. Although additional phosphine ligands are attached to copper atoms the clusters possess an open 'Cu(3)S(3)' face mostly protected by the [Li(thf)(4)](+) and (NBu(4))(+) counterions in the crystal structure. The cluster (NBu(4))[Cu(19)S(28)(SnPh)(12)(PEt(2)Ph)(3)] displays near-infrared, temperature-dependent photoluminescence at ∼820-930 nm in the solid state, which is especially bright at temperatures below ∼100 K.