SPOP Deregulation Improves the Radiation Response of Prostate Cancer Models by Impairing DNA Damage Repair.

Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is the most commonly mutated gene in prostate cancer (PCa). Recent evidence reports a role of SPOP in DNA damage response (DDR), indicating a possible impact of SPOP deregulation on PCa radiosensitivity. This study aimed to define the role of SPOP deregulation (by gene mutation or knockdown) as a radiosensitizing factor in PCa preclinical models. To express WT or mutant (Y87N, K129E and F133V) SPOP, DU145 and PC-3 cells were transfected with pMCV6 vectors. Sensitivity profiles were assessed using clonogenic assay and immunofluorescent staining of γH2AX and RAD51 foci. SCID xenografts were treated with 5 Gy single dose irradiation using an image-guided small animal irradiator. siRNA and miRNA mimics were used to silence SPOP or express the SPOP negative regulator miR-145, respectively. SPOP deregulation, by either gene mutation or knockdown, consistently enhanced the radiation response of PCa models by impairing DDR, as indicated by transcriptome analysis and functionally confirmed by decreased RAD51 foci. SPOP silencing also resulted in a significant downregulation of RAD51 and CHK1 expression, consistent with the impairment of homologous recombination. Our results indicate that SPOP deregulation plays a radiosensitizing role in PCa by impairing DDR via downregulation of RAD51 and CHK1.

Application of failure mode and effects analysis to optimization of linac quality controls protocol.

PURPOSE: To apply Failure Mode and Effects Analysis (FMEA) to optimize linac quality control (QC) protocol in order to ensure patient safety and treatment quality, taking maximum advantage of the available resources. MATERIAL AND METHODS: Each parameter tested by the QC was considered as a potential failure mode (FM). For each FM, likelihood of occurrence (O), severity of effect (S), and lack of detectability (D) were evaluated and corresponding Risk Priority Number (RPN) was calculated from the product of three indexes. The scores were assigned using two methods: (a) A survey submitted to the medical physicists; (b) A semi-quantitative analysis (SQA) performed through: simulation of FMs in the treatment planning system; studies reported in literature; results obtained by the QC data analysis. A weighted RPN for all FMs was calculated taking into account both the methods. For each linac, the tests were then sorted by their frequency and the RPN value. RESULTS: A high variability was found in the scores of the survey, although in many it was reduced in RPN values, highlighting the more relevant tests as on beam output and imaging system. Integrating these results with those obtained by SQA, the RPN-based ranking of tests has been provided considering the specific use of the accelerator: for example, more accurate tests on dose modulation and multileaf collimator speed were required in linacs where intensity-modulated treatment is performed, while, more specific tests on couch and jaw position indicators were necessary where treatments with multiple isocenters and/or junctions between adjacent fields were often delivered. CONCLUSIONS: Failure Mode and Effects Analysis is a useful tool to prioritize the linac QCs, taking into account the specific equipment and clinical practice. The integration of SQA and survey results reduces subjectivity of the FMEA scoring and allows to optimize linac QCs without "losing" the expertise and experience of medical physicists and clinical staff.

miR-205 enhances radiation sensitivity of prostate cancer cells by impairing DNA damage repair through PKCε and ZEB1 inhibition.

BACKGROUND: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. METHODS: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. RESULTS: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect. CONCLUSIONS: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.

Development of a Ready-to-Use Graphical Tool Based on Artificial Neural Network Classification: Application for the Prediction of Late Fecal Incontinence After Prostate Cancer Radiation Therapy.

PURPOSE: This study was designed to apply artificial neural network (ANN) classification methods for the prediction of late fecal incontinence (LFI) after high-dose prostate cancer radiation therapy and to develop a ready-to-use graphical tool. MATERIALS AND METHODS: In this study, 598 men recruited in 2 national multicenter trials were analyzed. Information was recorded on comorbidity, previous abdominal surgery, use of drugs, and dose distribution. Fecal incontinence was prospectively evaluated through self-reported questionnaires. To develop the ANN, the study population was randomly split into training (n = 300), validation (n = 149), and test (n = 149) sets. Mean grade of longitudinal LFI (ie, expressed as the average incontinence grade over the first 3 years after radiation therapy) ≥1 was considered the endpoint. A suitable subset of variables able to better predict LFI was selected by simulating 100,000 ANN configurations. The search for the definitive ANN was then performed by varying the number of inputs and hidden neurons from 4 to 5 and from 1 to 9, respectively. A final classification model was established as the average of the best 5 among 500 ANNs with the same architecture. An ANN-based graphical method to compute LFI prediction was developed to include one continuous and n dichotomous variables. RESULTS: An ANN architecture was selected, with 5 input variables (mean dose, previous abdominal surgery, use of anticoagulants, use of antihypertensive drugs, and use of neoadjuvant and adjuvant hormone therapy) and 4 hidden neurons. The developed classification model correctly identified patients with LFI with 80.8% sensitivity and 63.7% ± 1.0% specificity and an area under the curve of 0.78. The developed graphical tool may efficiently classify patients in low, intermediate, and high LFI risk classes. CONCLUSIONS: An ANN-based model was developed to predict LFI. The model was translated in a ready-to-use graphical tool for LFI risk classification, with direct interpretation of the role of the predictors.

Predicting Late Fecal Incontinence Risk After Radiation Therapy for Prostate Cancer: New Insights From External Independent Validation.

PURPOSE: This study aimed to validate a previously published predictive model for late fecal incontinence (FI) in a contemporary population of prostate cancer patients treated with radical radiation therapy. METHODS AND MATERIALS: The validation included patients treated with intensity-modulated radiation therapy (IMRT) (2010-2014). Prescribed dose range was 65-80 Gy, including conventional and moderate hypo-fractionated treatments. Rectal toxicity was scored using LENT/SOMA, a minimum 2-year follow up was considered. We chose to validate the model published by Rancati et al for predicting chronic FI, developed on a 3-dimensional conformal radiation therapy (3DCRT) population. It considered a longitudinal endpoint defined as the average toxicity grade during the follow up. This continuous endpoint was dichotomized using a cut-off value of mean FI grade >1. The model included mean rectal dose (Dmean), previous diseases of the colon (COLO) and previous abdominal surgery (SURG). Doses were corrected to 2 Gy/fraction using the linear-quadratic model and applying alpha/beta ratio = 4.8 Gy. RESULTS: 228 patients constituted the validation population. A mean FI grade >1 was scored in 25 patients (11%). Logistic regression confirmed risk factors reported in the literature, with similar odds ratios (ORs) for Dmean (1.04 ± 0.03 vs 1.06 ± 0.04) and SURG (1.9 ± 1.7 vs 1.6 ± 1.45); COLO was not confirmed. Consequently, the predictive models including Dmean/Dmean + SURG were evaluated using calibration plots. Both showed a clear discriminative trend, but the absolute observed toxicity rates were underestimated (ie, absolute predicted rates were always lower than corresponding absolute observed rates). This result was consistent with an unexpected effect of hypofractionation (OR = 2.20, conventional = 8.1% vs hypofractionated = 17.4%) beyond the standard correction using linear-quadratic model. Nevertheless, the FI rate in the conventionally treated group was almost double the rate observed in the previously studied cohort (4.3% vs 8.1%). CONCLUSIONS: The study confirms previously published results indicating that abdominal surgery and rectal mean dose are risk factors for late FI. Calibration plots highlight a possible role of hypofractionation beyond linear-quadratic correction.

miR-875-5p counteracts epithelial-to-mesenchymal transition and enhances radiation response in prostate cancer through repression of the EGFR-ZEB1 axis.

Radiotherapy is one of the main treatment choices for non-metastatic prostate cancer (PCa), although development of radioresistance limits its effectiveness. Mounting evidence supports the ability of microRNAs to interfere with different radioresistance-associated pathways, suggesting their potential as radiosensitizers. Here, we demonstrate that reconstitution of miR-875-5p, whose expression is down-regulated in PCa clinical samples and directly correlates with that of E-cadherin, was able to enhance radiation response in PCa cell lines and xenografts through EGFR direct targeting. Consistent with the established role of EGFR in sustaining epithelial-to-mesenchymal transition (EMT) and promoting DNA repair following radiation-induced nuclear translocation, we found that miR-875-5p reconstitution in PCa cells counteracted EMT and impaired DNA lesion clearance. Down-regulation of the EMT-inducing transcription factor ZEB1, which also plays a role in homologous recombination-mediated repair of DNA lesions by regulating CHK1 expression, was found to be a major determinant of miR-875-5p-induced radiosensitization, as confirmed by phenocopy experiments showing that siRNA-mediated ZEB1 knock-down was able to reproduce the microRNA radiosensitizing effect. Overall, our data support the clinical interest in developing a novel therapeutic approach based on miR-875-5p reconstitution to increase PCa response to radiotherapy.

Radiotherapy for unresectable sinonasal cancers: dosimetric comparison of intensity modulated radiation therapy with coplanar and non-coplanar volumetric modulated arc therapy.

BACKGROUND AND PURPOSE: To compare volumetric modulated arc therapy (VMAT) and intensity modulated radiation therapy (IMRT) plans for treatment of unresectable paranasal sinuses cancers (PNSCs) with different clinical presentations. MATERIAL AND METHODS: Four patients treated for primary target volume only (group 1), four requiring elective nodal irradiation (group 2) and four with positive nodes in macroscopic disease (group 3) were selected. For each patient were generated 7 fields IMRT, coplanar VMAT (c-VMAT) and non-coplanar VMAT (nc-VMAT) treatment plans. Total doses were 70Gy and 54Gy to high dose planning target volume (HD-PTV) and low-dose-PTV, respectively. Dose-volume histogram, conformity and homogeneity index (CI and HI), and monitor units (MUs) per Gy were evaluated. RESULTS: VMAT provided significantly better target coverage, in terms of V100% (Volume encompassed by the isodose 100%), than IMRT, in particular when nc-VMAT was used. In general, organ at risk sparing is similar with the three approaches, although nc-VMAT can allow a statistically significant reduction of dose to contralateral parotid gland and cochlea for all three groups. CONCLUSIONS: VMAT can offer significant improvement of treatment for all unresectable PNSCs over existing IMRT techniques. In particular, nc-VMAT may be a further advantage for those patients with sinonasal cancers and involvement of the nodes in whom large volumes and complex/irregular shape have to be irradiated, even if clinical benefits should be established in the future.

Set-up errors analyses in IMRT treatments for nasopharyngeal carcinoma to evaluate time trends, PTV and PRV margins.

INTRODUCTION: the aims of this study were to analyze the systematic and random interfractional set-up errors during Intensity Modulated Radiation Therapy (IMRT) in 20 consecutive nasopharyngeal carcinoma (NPC) patients by means of Electronic Portal Images Device (EPID), to define appropriate Planning Target Volume (PTV) and Planning Risk Volume (PRV) margins, as well as to investigate set-up displacement trend as a function of time during fractionated RT course. MATERIAL AND METHODS: before EPID clinical implementation, an anthropomorphic phantom was shifted intentionally 5 mm to all directions and the EPIs were compared with the digitally reconstructed radiographs (DRRs) to test the system's capability to recognize displacements observed in clinical studies. Then, 578 clinical images were analyzed with a mean of 29 images for each patient. RESULTS: phantom data showed that the system was able to correct shifts with an accuracy of 1 mm. As regards clinical data, the estimated population systematic errors were 1.3 mm for left-right (L-R), 1 mm for superior-inferior (S-I) and 1.1 mm for anterior-posterior (A-P) directions, respectively. Population random errors were 1.3 mm, 1.5 mm and 1.3 mm for L-R, S-I and A-P directions, respectively. PTV margin was at least 3.4, 3 and 3.2 mm for L-R, S-I and A-P direction, respectively. PRV margins for brainstem and spinal cord were 2.3, 2 and 2.1 mm and 3.8, 3.5 and 3.2 mm for L-R, A-P and S-I directions, respectively. Set-up error displacements showed no significant changes as the therapy progressed (p>0.05), although displacements >3 mm were found more frequently when severe weight loss or tumor nodal shrinkage occurred. DISCUSSION: these results enable us to choose margins that guarantee with sufficient accuracy the coverage of PTVs and organs at risk sparing. Collected data confirmed the need for a strict check of patient position reproducibility in case of anatomical changes.

Neoplastic transformation induced by carbon ions.

PURPOSE: The objective of this experiment was to compare the oncogenic potential of carbon ion beams and conventional photon beams for use in radiotherapy. METHODS AND MATERIALS: The HeLa X human skin fibroblast cell line CGL1 was irradiated with carbon ions of three different energies (270, 100, and 11.4 MeV/u). Inactivation and transformation data were compared with those for 15 MeV photons. RESULTS: Inactivation and transformation frequencies for the 270 MeV/u carbon ions were similar to those for 15-MeV photons. The maximal relative biologic effectiveness (RBE(alpha)) values for 100MeV/u and 11.4 MeV/u carbon ions, respectively, were as follows: inactivation, 1.6 +/- 0.2 and 6.7 +/- 0.7; and transformation per surviving cell, 2.5 +/- 0.6 and 12 +/- 3. The curve for dose-transformation per cell at risk exhibited a maximum that was shifted toward lower doses at lower energies. CONCLUSIONS: Transformation induction per cell at risk for carbon ions in the entrance channel was comparable to that for photons, whereas for the lower energies, 100 MeV/u and 11 MeV/u, which are representative of the energies delivered to the tumor margins and volume, respectively, the probability of transformation in a single cell was greater than it was for photons. In addition, at isoeffective doses with respect to cell killing, the 11.4-MeV/u beam was more oncogenic than were photons.

Postoperative radiotherapy for synovial sarcoma of the head and neck during pregnancy: clinical and technical management and fetal dose estimates.

AIMS AND BACKGROUND: In vivo and phantom dosimetry is reported to estimate the fetal dose and evaluate the effectiveness of a special shielding device to reduce fetal exposure in a woman undergoing postoperative radiation therapy for synovial oral cavity sarcoma at the 30th week of pregnancy. METHODS: In vivo measurements were performed by placing thermoluminescent dosimeters on 3 points for fetal dose estimation: uterine fundus, umbilicus and pubis. A Rando anthropomorphic phantom was used to simulate radiotherapy. We also performed off-axis dose measurements for wedged beams to estimate the dose contribution of this accessory used in the treatment. RESULTS: The special shielding device reduced the fetal dose by 70% on average, despite the presence of wedges, which increased the dose by a factor of about 2.5. Before delivery the patient received 48 Gy, and from the in vivo measurements a fetal dose of 8.5, 1.7 and 0.7 cGy was estimated to the uterine fundus, umbilicus and pubis, respectively. CONCLUSIONS: Pre-treatment simulation in the same irradiation conditions is the only reliable approach to predict the fetal dose. By using a special shielding device, radiotherapy can be optimized while keeping the fetal exposure below the risk of deterministic damage.

Target coverage in head and neck cancer treated with intensity-modulated radiotherapy: a comparison between conventional and conformal techniques.

AIMS AND BACKGROUND: We designed a comparative planning study aimed at quantifying the advantages of intensity-modulated radiotherapy (IMRT) over the conventional 3-field technique (3FT) and a 5-field conformal technique (5FCT) for head and neck (HN) cancer. METHODS: We selected 9 patients treated at our institution with curative radiotherapy for a HN cancer. For all cases 4 plans were generated: 2 plans using the "standard" techniques (3FT and 5FCT), a third plan using IMRT, and a fourth "mixed" plan using IMRT followed by a conventional boost. RESULTS: Our study confirmed literature data on the ability of IMRT to significantly decrease the dose received by organs at risk, compared with previous techniques. Target coverage was systematically better with 5FCT and IMRT than with 3FT. However, the increase in coverage of both PTV2 and PTV1 was only about 3-5% and this was achieved at the price of a similar increase in maximum dose (D1%). Volumetric parameters (V100%, V95%) were much more sensitive in detecting the improvement with IMRT. CONCLUSIONS: The improvement of target coverage attained by IMRT, as compared with conventional and conformal techniques, might be overestimated by data currently available in the medical literature. If treatment with conventional techniques is planned using all tools provided by currently available fully 3-D planning systems, excellent target coverage can be obtained.

Age- and irradiation-associated loss of bone marrow hematopoietic function in mice is reversed by recombinant human growth hormone.

OBJECTIVE: The aim of this story was to evaluate the activity of recombinant human (rh) growth hormone (GH) in restoring bone marrow progenitor cell growth as well as cytokine-elicited stem cell mobilization in aged BALB/c mice with impaired marrow hematopoietic function and reduced stem cell mobilizing capacity. MATERIALS AND METHODS: BALB/c mice included in this study were either naturally aged (group I) or aged after having been used for radioprotective assays (group II). Mice were treated for 5 weeks with either rhGH [2.5 mg/kg/day intraperitoneally (IP)] or phosphate-buffered saline (PBS). Subsequently, colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were evaluated. In addition, progenitor cell mobilization elicited by granulocyte colony-stimulating factor (rhG-CSF) was analyzed. RESULTS: Compared with young controls, the growth of marrow CFCs and LTC-ICs was significantly reduced (P < or = 0.05) in group I and II mice. Treatment with rhGH significantly enhanced marrow hematopoiesis in mice of both groups, as demonstrated by a complete restoration of marrow cellularity, and CFC and LTC-IC growth. To further evaluate the hematopoietic potential of rhGH, aged mice treated with rhGH or PBS were mobilized with rhG-CSF (10 microg/day IP for 5 days). Compared with PBS-injected mice, rhGH-treated mice showed a significant improvement of rhG/CSF-elicited stem cell mobilization, with significant increases of white blood cell counts (5633 vs 8133, P < or = 0.05), frequency of circulating CFCs per 10(5) mononuclear cells (36 vs 67, P < or = 0.009), as well as absolute numbers per mL of blood of circulating CFCs (783 vs 2288, P < or = 0.001) and LTC-IC (21 vs 64, P < or = 0.001). CONCLUSION: Our data demonstrate in mice that a 5-week treatment with rhGH restores age- and irradiation-associated loss of marrow primitive and committed progenitors.

Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.