Significance of hematopoietic surface antigen CD34 in neuroblastoma prognosis and the genetic landscape of CD34-expressing neuroblastoma CSCs.

High-risk neuroblastoma (HR-NB) is branded with hematogenous metastasis, relapses, and dismal long-term survival. Intensification of consolidation therapy with tandem/triple autologous stem cell (SC) rescue (with bone marrow [BM]/peripheral blood [PB] CD34+ selection) after myeloablative chemotherapy has improved long-term survival. However, the benefit is limited by the indication of NB cells in CD34+ PBSCs, CD34 expression in NB cells, and the risk of reinfusing NB cancer stem cells (NB CSCs) that could lead to post-transplant relapse. We investigated the association of CD34 surface expression (92 patients) with NB evolution/clinical outcomes. CD34 gene-level status in NB was assessed through RNA-Seq data mining (18 cohorts, n, 3324). Genetic landscape of CD34-expressing NB CSCs (CD133+CD34+) was compared with CD34- CSCs (CD133+CD34-). RNA-seq data revealed equivocal association patterns of CD34 expression with patient survival. Our immunohistochemistry data revealed definite, but rare (mean, 0.73%; range 0.00-7.87%; median, 0.20%) CD34 positivity in NB. CD34+ significantly associated with MYCN amplification (p, 0.003), advanced disease stage (p, 0.016), and progressive disease (PD, p < 0.0009) after clinical therapy. A general high-is-worse tendency was observed in patients with relapsed disease. High CD34+ correlated with poor survival in patients with N-MYC-amplified HR-NB. Gene expression analysis of CD34+-NB CSCs identified significant up (4631) and downmodulation (4678) of genes compared with NB CSCs that lack CD34. IPA recognized the modulation of crucial signaling elements (EMT, stemness maintenance, differentiation, inflammation, clonal expansion, drug resistance, metastasis) that orchestrate NB disease evolution in CD34+ CSCs compared with CD34- CSCs. While the function of CD34 in NB evolution requires further in-depth investigation, careful consideration should be exercised for autologous stem cell rescue with CD34+ selection in NB patients. Graphical abstract.

Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples.

BACKGROUND: MYCN amplification directly correlates with the clinical course of neuroblastoma and poor patient survival, and serves as the most critical negative prognostic marker. Although fluorescence in situ hybridization (FISH) remains the gold standard for clinical diagnosis of MYCN status in neuroblastoma, its limitations warrant the identification of rapid, reliable, less technically challenging, and inexpensive alternate approaches. METHODS: In the present study, we examined the concordance of droplet digital PCR (ddPCR, in combination with immunohistochemistry, IHC) with FISH for MYCN detection in a panel of formalin-fixed paraffin-embedded (FFPE) human neuroblastoma samples. RESULTS: In 112 neuroblastoma cases, ddPCR analysis demonstrated a 96-100% concordance with FISH. Consistently, IHC grading revealed 92-100% concordance with FISH. Comparing ddPCR with IHC, we observed a concordance of 95-98%. CONCLUSIONS: The results demonstrate that MYCN amplification status in NB cases can be assessed with ddPCR, and suggest that ddPCR could be a technically less challenging method of detecting MYCN status in FFPE specimens. More importantly, these findings illustrate the concordance between FISH and ddPCR in the detection of MYCN status. Together, the results suggest that rapid, less technically demanding, and inexpensive ddPCR in conjunction with IHC could serve as an alternate approach to detect MYCN status in NB cases, with near-identical sensitivity to that of FISH.

Targeting acquired oncogenic burden in resilient pancreatic cancer: a novel benefit from marine polyphenols.

The upsurge of marine-derived therapeutics for cancer treatment is evident, with many drugs in clinical use and in clinical trials. Seaweeds harbor large amounts of polyphenols and their anti-cancer benefit is linear to their anti-oxidant activity. Our studies identified three superlative anti-cancer seaweed polyphenol drug candidates (SW-PD). We investigated the acquisition of oncogenic burden in radiation-resilient pancreatic cancer (PC) that could drive tumor relapse, and elucidated the efficacy of SW-PD candidates as adjuvants in genetically diverse in vitro systems and a mouse model of radiation-residual disease. QPCR profiling of 88 oncogenes in therapy-resilient PC cells identified a 'shared' activation of 40 oncogenes. SW-PD pretreatment inflicted a significant mitigation of acquired (shared) oncogenic burden, in addition to drug- and cell-line-specific repression signatures. Tissue microarray with IHC of radiation-residual tumors in mice signified acquired cellular localization of key oncoproteins and other critical architects. Conversely, SW-PD treatment inhibited the acquisition of these critical drivers of tumor genesis, dissemination, and evolution. Heightened death of resilient PC cells with SW-PD treatment validated the translation aspects. The results defined the acquisition of oncogenic burden in resilient PC and demonstrated that the marine polyphenols effectively target the acquired oncogenic burden and could serve as adjuvant(s) for PC treatment.

Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells.

BACKGROUND: MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes. RESULTS: Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs' targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKß, Birc1, Birc2, Bric5, ß-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3ß, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1ß, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs' targets corresponded strongly with poor overall and relapse-free survival. CONCLUSIONS: For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.

Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes.

BACKGROUND: Determining the driving factors and molecular flow-through that define the switch from favorable to aggressive high-risk disease is critical to the betterment of neuroblastoma cure. METHODS: In this study, we examined the cytogenetic and tumorigenic physiognomies of distinct population of metastatic site- derived aggressive cells (MSDACs) from high-risk tumors, and showed the influence of acquired genetic rearrangements on poor patient outcomes. RESULTS: Karyotyping in SH-SY5Y and MSDACs revealed trisomy of 1q, with additional non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Array CGH analysis of individual clones of MSDACs revealed genetic alterations in chromosomes 1, 7, 8, and 22, corresponding to a gain in the copy numbers of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, and MAL2, and a loss in ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT, and TTLL1. QPCR analysis and immunoblotting showed a definite association between DNA-copy number changes and matching transcriptional/translational expression in clones of MSDACs. Further, MSDACs exert a stem-like phenotype. Under serum-free conditions, MSDACs demonstrated profound tumorosphere formation ex vivo. Moreover, MSDACs exhibited high tumorigenic capacity in vivo and prompted aggressive metastatic disease. Tissue microarray analysis coupled with automated IHC revealed significant association of RALYL to the tumor grade in a cohort of 25 neuroblastoma patients. Clinical outcome association analysis showed a strong correlation between the expression of CFHR3, CSMD3, MDFIC, FOXP2, RALYL, POLDIP3, SLC25A17, SERHL, MGAT3, TTLL1, or LOC400927 and overall and relapse-free survival in patients with neuroblastoma. CONCLUSION: Together, these data highlight the ongoing acquired genetic rearrangements in undifferentiated tumor-forming neural crest cells, and suggest that these alterations could switch favorable neuroblastoma to high-risk aggressive disease, promoting poor clinical outcomes.

Late Effects of Radiation Therapy in Pediatric Cancer Survivors.

The overall survival rates of many pediatric cancers continue to improve with each decade due to new advances in therapy. As this trend continues, the focus and importance of minimizing acute and long-term toxicity associated with treatment is paramount. While significant research regarding many of the late responses of normal tissues associated with radiation exposure has been established, future endeavors must be directed toward the identification of therapy related factors including radiation total dose, dose rate, exposure, and target treatment volumes. Awareness of short and long-term health risks of these patients is important and careful follow-up of long-term survivors is essential. In this report, we review some selected late adverse effects including the development of secondary malignancies, cardiotoxicity, physiological changes to glandular tissue, hormonal and reproductive changes to germ cells, and neurocognitive changes. Furthermore, we compared the differences regarding late effects of normal tissues associated with the use of proton versus photon radiotherapy, a topic that has received a great deal of attention in pediatric cancer and is increasing in utilization in the United States and world-wide.

Changes in Non-Small Cell Lung Cancer Tumor Location Secondary to Gastric Distension, Implications in the Context of Stereotactic Body Radiation Therapy.

OBJECTIVE: Stereotactic body radiation therapy (SBRT) facilitates highly conformal dose distributions to a targe tumor volume. Accurate tumor localization is extremely important, and lung tumors pose a unique challenge due to respiratory motion. Patients are required to fast before PET/CT but not before CT simulation and daily treatment, introducing potential variability from gastric distension. METHODS: A case was reviewed involving a patient with early-stage NSCLC which was simulated and treated with SBRT. PET/CT performed while fasting showed an isolated left lower lobe lesion. Following CT simulation, CT and PET/CT images were superimposed for comparison and treatment planning. RESULTS: Tumor location variation was apparent following image superimposition. Simulation CT showed significant gastric distension compared to PET/CT. The patient was resimulated while fasting, resulting in accurate and reproducible tumor localization for treatment planning. CONCLUSIONS: Gastric distension can alter tumor location and treatment volumes for radiotherapy planning, possibly resulting in inaccurate treatment administration.

Quantitative assessment by measurement and modeling of mobile target elongation in cone-beam computed tomographic imaging.

The purpose of this study was to assess quantitatively elongation of mobile targets in cone-beam CT (CBCT) imaging by measurement and modeling. A mathematical model was derived that predicts the measured lengths of mobile targets and its dependence on target size and motion patterns in CBCT imaging. Three tissue-equivalent targets of differing sizes were inserted in an artificial thorax phantom to simulate lung lesions. Respiratory motion was mimicked with a mobile phantom that moves in one-dimension along the superior-inferior direction at a respiration frequency of 0.24 Hz for eight different amplitudes in the range 0-40 mm. A mathematical model was derived to quantify the variations in target lengths and its dependence on phantom motion parameters in CBCT. Predictions of the model were verified by measurement of the lengths of mobile targets in CBCT images. The model predicts that target lengths increased linearly with increase in speed and amplitude of phantom motion in CBCT. The measured lengths of mobile targets imaged with CBCT agreed with the calculated lengths within half-slice thickness spatial resolution. The maximal length of a mobile target was independent of the frequency and phase of motion. Elongation of mobile targets was similar in half-fan and full-fan CBCT for similar motion patterns, as long as the targets remained within the imaging view. Mobile targets elongated linearly with phantom speed and motion amplitude in CBCT imaging. The model introduced in this work assessed quantitatively the variation in target lengths induced by motion, which may be a useful tool to consider elongations of mobile targets in CBCT applications in diagnostic imaging and radiotherapy.

Radiation-triggered tumor necrosis factor (TNF) alpha-NFkappaB cross-signaling favors survival advantage in human neuroblastoma cells.

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.

Ocular malignancies treated with iodine-125 low dose rate (LDR) brachytherapy at a single high-volume institution: A retrospective review.

The aim of our study is to document our cases of choroidal melanoma treated with low dose rate (LDR) brachytherapy and to correlate the dosimetry and radiobiology with clinical effects and oncologic outcomes. Data from 157 patients treated from 2014 to 2018 with LDR brachytherapy were used for this investigation. Treatments used a collaborative ocular melanoma study eye plaque and Iodine-125 radioactive seeds. The seeds activities were chosen to deliver 85 Gy to the tumor apex or to a prescription point (if the apex < 5 mm). The plaque sizes used were 10, 12, 14, 16, 18, 20, and 22 mm including notched or deep notched. The plaques were modeled in Varian BrachyVision version 11.6 (Varian Medical Systems) with seed coordinates from the AAPM Task Group 129. The Task Group 43 from AAPM was used for brachytherapy dose planning. Dose data were extracted for the apex, prescription point, sclera, retina opposite to the implant, lens, macula, and optic disc. The radiobiological dosimetry were calculated using appropriate α/ß ratios found in the literature and then correlated to clinical side effects. Average biologically effective dose for associated organs at risk were calculated in cases where toxicity occurred. These included: radiation cataract (70.66 Gy), disc atrophy (475.49 Gy), foveal atrophy (263.07 Gy), radiation papillopathy (373.45 Gy), radiation maculopathy (213.62 Gy), vitreous hemorrhage (1437.68 Gy), vascular occlusion (1080.93 Gy), nonproliferative retinopathy (1066.89 Gy), proliferative retinopathy (1590.71 Gy), exudative retinal detachment (1364.32 Gy), and rhegmatogenous retinal detachment (2265.54 Gy). Average biologically effective dose was higher in patients who developed radiation induced long term side effects than in the whole patient population except for radiation maculopathy. In spite of the small patient population and short-term follow-up, it is of interest to correlate the radiation induced effects and create a guideline for the improvement of the treatment of patients treated with LDR brachytherapy.

Cerebral microvascular rarefaction induced by whole brain radiation is reversible by systemic hypoxia in mice.

Whole brain radiation therapy (WBRT) leads to cognitive impairment in 40-50% of brain tumor survivors following treatment. Although the etiology of cognitive deficits post-WBRT remains unclear, vascular rarefaction appears to be an important component of these impairments. In this study, we assessed the effects of WBRT on the cerebrovasculature and the effects of systemic hypoxia as a potential mechanism to reverse the microvascular rarefaction. Transgenic mice expressing green fluorescent protein driven by the Acta2 (smooth muscle actin) promoter for blood vessel visualization were randomly assigned to control or radiated groups. Animals received a clinical series of 4.5 Gy WBRT two times weekly for 4 wk followed by 1 mo of recovery. Subsequently, mice were subjected to 11% (hypoxia) or 21% (normoxia) oxygen for 1 mo. Capillary density in subregions of the hippocampus revealed profound vascular rarefaction that persisted despite local tissue hypoxia. Nevertheless, systemic hypoxia was capable of completely restoring cerebrovascular density. Thus hippocampal microvascular rarefaction post-WBRT is not capable of stimulating angiogenesis and can be reversed by chronic systemic hypoxia. Our results indicate a potential shift in sensitivity to angiogenic stimuli and/or the existence of an independent pathway of regulating cerebral microvasculature.

Low-dose γ-radiation-induced oxidative stress response in mouse brain and gut: regulation by NFκB-MnSOD cross-signaling.

Radiation-induced amplification of reactive oxygen species (ROS) may be a sensing mechanism for activation of signaling cascades that influence cell fate. However, the regulated intrinsic mechanisms and targets of low-dose ionizing radiation (LDIR) are still unclear. Accordingly, we investigated the effects of LDIR on NFκB signal transduction and manganese superoxide dismutase (SOD2) activity in mice brain and gut. LDIR resulted in both dose-dependent and persistent NFκB activation in gut and brain. QPCR displayed a dose- and tissue-dependent differential modulation of 88 signaling molecules. With stringent criteria, a total of 15 (2cGy), 43 (10cGy) and 19 (50cGy) genes were found to be commonly upregulated between brain and gut. SOD2 immunostaining showed a LDIR-dose dependent increase. Consistent with the NFκB results, we observed a persistent increase in SOD2 activity after LDIR. Moreover, muting of LDIR-induced NFκB attenuated SOD2 transactivation and cellular localization. These results imply that exposure of healthy tissues to LDIR results in induced NFκB and SOD2 activity and transcriptional activation of NFκB-signal transduction/target molecules. More importantly, the results suggest that NFκB initiates a feedback response through transcriptional activation of SOD2 that may play a key role in the LDIR-induced oxidative stress response and may control the switch that directs cell fate.

An algorithm to extract three-dimensional motion by marker tracking in the kV projections from an on-board imager: four-dimensional cone-beam CT and tumor tracking implications.

The purpose of this work is to extract three-dimensional (3D) motion trajectories of internal implanted and external skin-attached markers from kV cone-beam projections and reduce image artifact from patient motion in cone-beam computed tomography (CBCT) from on-board imager. Cone beam radiographic projections were acquired for a mobile phantom and liver patients with internal implanted and external skin-attached markers. An algorithm was developed to automatically find the positions of the markers in the projections. It uses normalized cross-correlation between a template image of a metal seed marker and the projections to find the marker position. From these positions and time-tagged angular views, the marker 3D motion trajectory was obtained over a time interval of nearly one minute, which is the time required for scanning. This marker trajectory was used to remap the pixels of the projections to eliminate motion. Then, the motion-corrected projections were used to reconstruct CBCT. An algorithm was developed to extract 3D motion trajectories of internal and external markers from cone-beam projections using a kV monoscopic on-board imager. This algorithm was tested and validated using a mobile phantom and patients with liver masses that had radio-markers implanted in the tumor and attached to the skin. The extracted motion trajectories were used to investigate motion correlation between internal and external markers in liver patients. Image artifacts from respiratory motion were reduced in CBCT reconstructed from cone-beam projections that were preprocessed to remove motion shifts obtained from marker tracking. With this method, motion-related image artifacts such as blurring and spatial distortion were reduced, and contrast and position resolutions were improved significantly in CBCT reconstructed from motion-corrected projections. Furthermore, correlated internal and external marker 3D-motion tracks obtained from the kV projections might be useful for 4DCBCT, beam gating and tumor motion monitoring or tracking.

Linear accelerator based intracranial and extracranial stereotactic radiation therapy at the University of Oklahoma.

PURPOSE: Review our institutional outcomes with linear accelerator based stereotactic radiation therapy at the University of Oklahoma. METHODS: We retrospectively reviewed all patients treated in our department with linear accelerator based stereotactic radiation therapy since we implemented this modality in 2008. Thirty-seven patients have been treated with a mean follow-up of 8.3 months. Seventeen patients had tumors near critical structures; ten had treatment sites not suited for gamma knife therapy. Outcomes are reviewed for efficacy and toxicity. RESULTS: Acute and long term complications reported are minimal. Stabilization of treatment sites was achieved in 96% of patients on follow-up imaging. Thirteen patients (35%) have died, of which eleven died to systemic disease progression outside of the treatment site. No treatment related deaths occurred. CONCLUSIONS: Stereotactic radiation therapy is a successful treatment modality to achieve local disease control with minimal toxicity. We plan to expand its use and applications in the future.

Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage.

PURPOSE: Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-ß, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. METHODS: MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). RESULTS: Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 µM and 5 µM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. CONCLUSIONS: Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.

Effect of black raspberry extract in inhibiting NFkappa B dependent radioprotection in human breast cancer cells.

Black raspberry extracts (RSE) have been shown to inhibit cancer cell growth and stimulate apoptosis. Also, studies have demonstrated that RSE inhibits transcriptional regulators including NFkappa B. Accordingly, we investigated the effect of RSE in inhibiting radiation (IR) induced NFkappa B mediated radioprotection in breast adenocarcinoma cells. MCF-7 cells were exposed to IR (2Gy), treated with RSE (0.5, 1.0, 2.0 micro g/ml) or treated with RSE (1.0 micro g/ml) followed by IR exposure, and harvested after 1, 3, 6, 24, 48, and 72 h. NFkappa B DNA-binding activity was measured by EMSA and phosphorylated Ikappa Balpha by immunoblotting. Expression of IAP1, IAP2, XIAP and survivin were measured by QPCR and immunoblotting. Cell survival was measured using MTT assay and cell death using Caspase-3/7 activity. Effect of RSE on IR induced MnSOD, TNFalpha, IL-1alpha and MnSOD activity was also determined. RSE inhibited NFkappa B activity in a dose-dependent manner. Also, RSE inhibited IR-induced sustained activation of NFkappa B, and NFkappa B regulated IAP1, IAP2, XIAP, and survivin. In addition, RSE inhibited IR-induced TNFalpha, IL-1alpha, and MnSOD levels and MnSOD activity. RSE suppressed cell survival and enhanced cell death. These results suggest that RSE may act as a potent radiosensitizer by overcoming the effects of NFkappa B mediated radioprotection in human breast cancer cells.

Evaluation of the setup accuracy of a stereotactic radiotherapy head immobilization mask system using kV on-board imaging.

The purpose of this study was to evaluate setup accuracy and quantify random and systematic errors of the BrainLAB stereotactic immobilization mask and localization system using kV on-board imaging. Nine patients were simulated and set up with the BrainLAB stereotactic head immobilization mask and localizer to be treated for brain lesions using single and hypofractions. Orthogonal pairs of projections were acquired using a kV on-board imager mounted on a Varian Trilogy machine. The kV projections were then registered with digitally-reconstructed radiographs (DRR) obtained from treatment planning. Shifts between the kV images and reference DRRs were calculated in the different directions: anterior-posterior (A-P), medial-lateral (R-L) and superior-inferior (S-I). If the shifts were larger than 2mm in any direction, the patient was reset within the immobilization mask until satisfying setup accuracy based on image guidance has been achieved. Shifts as large as 4.5 mm, 5.0 mm, 8.0 mm in the A-P, R-L and S-I directions, respectively, were measured from image registration of kV projections and DRRs. These shifts represent offsets between the treatment and simulation setup using immobilization mask. The mean offsets of 0.1 mm, 0.7 mm, and -1.6 mm represent systematic errors of the BrainLAB localizer in the A-P, R-L and S-I directions, respectively. The mean of the radial shifts is about 1.7 mm. The standard deviations of the shifts were 2.2 mm, 2.0 mm, and 2.6 mm in A-P, R-L and S-I directions, respectively, which represent random patient setup errors with the BrainLAB mask. The Brain-LAB mask provides a noninvasive, practical and flexible immobilization system that keeps the patients in place during treatment. Relying on this system for patient setup might be associated with significant setup errors. Image guidance with the kV on-board imager provides an independent verification technique to ensure accuracy of patient setup. Since the patient may relax or move during treatment, uncontrolled and undetected setup errors may be produced with patients that are not well-immobilized. Therefore, the combination of stereotactic immobilization and image guidance achieves more controlled and accurate patient setup within 2mm in A-P, R-L and S-I directions.

Stereotactic body radiation therapy (SBRT) and respiratory gating in lung cancer: dosimetric and radiobiological considerations.

The purpose of this study was to assess the impact of respiratory gating on tumor and normal tissue dosimetry in patients treated with SBRT for early stage non-small cell lung cancer (NSCLC). Twenty patients with stage I NSCLC were studied. Treatment planning was performed using four-dimensional computed tomography (4D CT) with free breathing (Plan I), near-end inhalation (Plan II), and near-end exhalation (Plan III). The prescription dose was 60 Gy in three fractions. The tumor displacement was most pronounced for lower peripheral lesions (average 7.0 mm, range 4.1-14.3 mm) when compared to upper peripheral (average 2.4mm, range 1.0-5.1 mm) or central lesions (average 2.9 mm, range 1.0-4.1 mm). In this study, the pencil beam convolution (PBC) algorithm with modified Batho power law for tissue heterogeneity was used for dose calculation. There were no significant differences in tumor and normal tissue dosimetry among the three gated plans. Tumor location however, significantly influenced tumor doses because of the necessity of respecting normal tissue constraints of centrally located structures. For plans I, II and III, average doses to central lesions were lower as compared with peripheral lesions by 4.88 Gy, 8.24 Gy and 6.93 Gy for minimum PTV and 0.98, 1.65 and 0.87 Gy for mean PTV dose, respectively. As a result, the mean single fraction equivalent dose (SFED) values were also lower for central compared to peripheral lesions. In addition, central lesions resulted in higher mean doses for lung, esophagus, and ipsilateral bronchus by 1.24, 1.93 and 7.75 Gy, respectively. These results indicate that the tumor location is the most important determinant of dosimetric optimization of SBRT plans. Respiratory gating proved unhelpful in the planning of these patients with severe COPD.

Quantitative evaluation of increase in surface dose by immobilization thermoplastic masks and superficial dosimetry using Gafchromic EBT film and Monte Carlo calculations.

PURPOSE: To investigate the increase in surface dose under immobilization thermoplastic masks by measurements and calculation in the build-up region using Gafchromic films and Monte Carlo simulation. MATERIALS AND METHODS: Surface doses were measured underneath three thermoplastic masks in open fields using 6 and 18 MV photon beams. These masks are used to immobilize patients for head and neck (H&N), pelvis and thoracic treatment. Gafchromic EBT films were placed on the top of the flat surface of a phantom partially underneath the mask and exposed in open 10 x 10 cm2 photon fields. The depth doses were calculated using BEAMnrc Monte Carlo code for water-equivalent film detectors with different layers of thickness ranging from 50 microm to 2.5 mm and compared with film measurements. RESULTS: Surface dose increased by a factor of 3 to 4 underneath the mask relative to the open areas and 6 MV beam delivers more skin dose than 18 MV. H&N mask increased surface dose by a factor of 3 using 18 MV and a factor of 4 using 6 MV. In addition, increase in surface dose depended on the type of the mask, the size of openings, and the amount of stretching performed during the mask preparation. The measured depth doses were compared with BEAMnrc Monte Carlo calculation for water-equivalent detectors using different sizes. The calculated depth dose depended significantly on the thickness of film detector and varies by more than 15% using layer thickness of 2.5 mm compared to 50 microm. Surface doses measured by Gafchromic EBT films agreed within 3% with the Monte Carlo calculations using a small detector layer of 50 microm. CONCLUSION: Thermoplastic masks used for patient immobilization can significantly increase skin doses by up to a factor of 4 more than that without the mask using 6 MV beams. The skin reactions resulting from thermoplastic masks should be monitored and corrective measures should be taken during treatment such as partially removing the mask over skin areas with complications and optimizing the skin dose in IMRT planning. Gafchromic EBT films provide accurate skin dosimetry which agrees within 3% with Monte Carlo calculations. Gafchromic EBT film makes an excellent tool for measuring depth doses in the buildup region and these data can be applied for treatment planning calculations and IMRT optimization.

Emerging therapeutic targets for neuroblastoma.

INTRODUCTION: Neuroblastoma (NB) is the prime cancer of infancy, and accounts for 9% of pediatric cancer deaths. While children diagnosed with clinically stable NB experience a complete cure, those with high-risk disease (HR-NB) do not recover, despite intensive therapeutic strategies. Development of novel and effective targeted therapies is needed to counter disease progression, and to benefit long-term survival of children with HR-NB. AREAS COVERED: Recent studies (2017-2020) pertinent to NB evolution are selectively reviewed to recognize novel and effective therapeutic targets. The prospective and promising therapeutic targets/strategies for HR-NB are categorized into (a) targeting oncogene-like and/or reinforcing tumor suppressor (TS)-like lncRNAs; (b) targeting oncogene-like microRNAs (miRs) and/or mimicking TS-miRs; (c) targets for immunotherapy; (d) targeting epithelial-to-mesenchymal transition and cancer stem cells; (e) novel and beneficial combination approaches; and (f) repurposing drugs and other strategies in development. EXPERT OPINION: It is highly unlikely that agents targeting a single candidate or signaling will be beneficial for an HR-NB cure. We must develop efficient drug deliverables for functional targets, which could be integrated and advance clinical therapy. Fittingly, the looming evidence indicated an aggressive evolution of promising novel and integrative targets, development of efficient drugs, and improvised strategies for HR-NB treatment.