Low-Efficacy Mu Opioid Agonists as Candidate Analgesics: Effects of Novel C-9 Substituted Phenylmorphans on Pain-Depressed Behavior in Mice.

Low efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO2 were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ~1hr. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics. Significance Statement This study used a novel set of mu opioid receptor (MOR)-selective opioids with graded MOR efficacies to examine the lower boundary of MOR efficacy sufficient to relieve pain-related behavioral depression in mice. Two novel low-efficacy opioids (JL-2-39, DC-1-76.1) produced effective antinociception with improved safety relative to higher- or lower-efficacy opioids, and results support further consideration of these and other low-efficacy opioids as candidate analgesics.

Role of mu opioid receptor (MOR) agonist efficacy as a determinant of opioid antinociception in a novel assay of pain-depressed behavior in female and male mice.

Introduction: Intermediate efficacy mu opioid receptor (MOR) agonists have potential to retain analgesic effectiveness while improving safety, but the optimal MOR efficacy for effective and safe opioid analgesia is unknown. Preclinical assays of pain-depressed behavior can assess effects of opioids and other candidate analgesics on pain-related behavioral depression, which is a common manifestation of clinically relevant pain and target of pain treatment. Accordingly, the present study goal was to validate a novel assay of pain-depressed locomotor behavior in mice and evaluate the role of MOR efficacy as a determinant of opioid analgesic effects and related safety measures. Methods: Male and female ICR mice were tested in a locomotor chamber consisting of 2 compartments connected by a doorway that contained a 1-inch-tall barrier. Dependent measures during 15-min behavioral sessions included crosses between compartments (which required vertical activity to surmount the barrier) and total movement counts (which required horizontal activity to break photobeams in each compartment). Results and Discussion: Intraperitoneal injection of lactic acid (IP acid) produced a concentration- and time-dependent depression of both endpoints. Optimal blockade of IP acid-induced behavioral depression with minimal motor impairment was achieved with intermediate-efficacy MOR treatments that also produced less gastrointestinal-transit inhibition and respiratory depression than the high-efficacy MOR agonist fentanyl. Sex differences in treatment effects were rare. Overall, these findings validate a novel procedure for evaluating opioids and other candidate analgesic effects on pain-related behavioral depression in mice and support continued research with intermediate-efficacy MOR agonists as a strategy to retain opioid analgesic effectiveness with improved safety.

Engineered bacteria detect tumor DNA.

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Macrophage protease-activated receptor 2 regulates fetal liver erythropoiesis in mice.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

Use of radiomics for the prediction of local control of brain metastases after stereotactic radiosurgery.

BACKGROUND: Local response prediction for brain metastases (BM) after stereotactic radiosurgery (SRS) is challenging, particularly for smaller BM, as existing criteria are based solely on unidimensional measurements. This investigation sought to determine whether radiomic features provide additional value to routinely available clinical and dosimetric variables to predict local recurrence following SRS. METHODS: Analyzed were 408 BM in 87 patients treated with SRS. A total of 440 radiomic features were extracted from the tumor core and the peritumoral regions, using the baseline pretreatment volumetric post-contrast T1 (T1c) and volumetric T2 fluid-attenuated inversion recovery (FLAIR) MRI sequences. Local tumor progression was determined based on Response Assessment in Neuro-Oncology‒BM criteria, with a maximum axial diameter growth of >20% on the follow-up T1c indicating local failure. The top radiomic features were determined based on resampled random forest (RF) feature importance. An RF classifier was trained using each set of features and evaluated using the area under the receiver operating characteristic curve (AUC). RESULTS: The addition of any one of the top 10 radiomic features to the set of clinical features resulted in a statistically significant (P < 0.001) increase in the AUC. An optimized combination of radiomic and clinical features resulted in a 19% higher resampled AUC (mean = 0.793; 95% CI = 0.792-0.795) than clinical features alone (0.669, 0.668-0.671). CONCLUSIONS: The increase in AUC of the RF classifier, after incorporating radiomic features, suggests that quantitative characterization of tumor appearance on pretreatment T1c and FLAIR adds value to known clinical and dosimetric variables for predicting local failure.

Evaluation of multi-institutional end-to-end testing for post-operative spine stereotactic body radiation therapy.

BACKGROUND AND PURPOSE: Post-operative spine stereotactic body radiation therapy (SBRT) represents a significant challenge as there are many restrictions on beam geometry to avoid metal hardware as it surrounds the target volume. In this study, an international multi-institutional end-to-end test using an in-house spine phantom was developed and executed. The aim was to evaluate the impact of titanium spine hardware on planned and delivered dose for post-operative spine SBRT. MATERIALS AND METHODS: Five centers performed simulation, planning and irradiation of the spine phantom, with/without titanium metal hardware (MB/B), following our pre-specified protocol. The doses were calculated using the centers' treatment planning system (TPS) and measured with radiophotoluminescent glass dosimeters (RPLDs) embedded within each phantom. RESULTS: The dose differences between the RPLD measured and calculated doses in the target region were within ±â€¯5% for both phantoms studied. Differences greater than 5% were observed for the spinal cord and the out-of-the target regions due to steeper dose gradient regions that are created in these plans. Dose measurements within ±â€¯3% were observed between RPLDs that were embedded in MB and B inserts. For the spinal cord and the out-of-target regions surrounded by metal hardware, the dose measured using RPLDs was within 3% different near the titanium screws compared to the dose measured near only the metal rods. CONCLUSION: We have successfully performed the first multi-institutional end-to-end dose analysis using an in-house phantom built specifically for post-operative spine SBRT. The differences observed between the measured and planned doses in the presence of metal hardware were clinically insignificant.

Postoperative Stereotactic Body Radiotherapy for Spinal Metastases and the Impact of Epidural Disease Grade.

BACKGROUND: Postoperative stereotactic body radiotherapy (pSBRT) is an emerging indication for spinal metastases (SM). OBJECTIVE: To report our experience with pSBRT for SM. METHODS: A retrospective chart review was performed for prospectively collected data of patients treated between September 2008 to December 2015 with pSBRT and followed with serial spinal MRIs every 2 to 3 mo until death or last follow-up. Univariate and multivariable analyses were performed to identify predictive factors. RESULTS: A total of 83 spinal segments in 47 patients treated with a median dose of 24 Gy in 2 fractions were included, with mostly lung and breast primaries. A total of 59.3% had preoperative high-grade epidural disease (ED) and 39.7% were unstable. The 12-mo cumulative incidence of local failure was 17% for all segments, and 33.3%, 21.8%, and 0% in segments with postoperative high-grade, low-grade, and no ED, respectively. Downgrading preoperative ED was predictive of better local control (P = .03). The grade of postoperative ED was also predictive for local control (P < .0001), as was a longer interval between prior radiotherapy and pSBRT in those previously irradiated (P = .004). The 12-mo overall survival rate was 55%. One case of radiculopathy, 3 vertebral compression fractures, and no cases of myelopathy, hardware failure, or skin breakdown were observed. CONCLUSION: pSBRT is an effective and safe treatment. The association between downgrading preoperative ED and better local control following pSBRT is confirmed and supports the concept of separation surgery.

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

OBJECTIVE: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. DESIGN: We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. RESULTS: Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. CONCLUSION: We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

Stereotactic Body Radiotherapy for Spinal Metastases at the Extreme Ends of the Spine: Imaging-Based Outcomes for Cervical and Sacral Metastases.

BACKGROUND: The unique anatomy and biomechanical features of the cervical spine and sacrum may impact treatment outcomes following spine stereotactic body radiotherapy (SBRT). Current data for spine metastases are not specific for these locations. OBJECTIVE: To report imaging-based SBRT outcomes to cervical and sacral metastases. METHODS: We retrospectively reviewed our prospective spine SBRT database for cervical and sacral metastases. Patients were followed at 2- to 3-mo intervals with a clinical visit and full spine magnetic resonance imaging (MRI) and we report overall survival (OS), vertebral compression fracture (VCF), and MR imaging-based local control (LC) rates. RESULTS: Fifty-two patients and 93 treated spinal segments were identified. Fifty-six segments were within the cervical spine and 37 within the sacrum, the median follow-up was 14.4 and 19.5 mo, and the median total dose/number of fractions was 24 Gy/2, respectively. Cumulative LC at 1 and 2 yr were 94.5% and 92.7% for the cervical cohort, and 86.5% and 78.7% in the sacral cohort, respectively. Lack of posterior spinal element involvement in the cervical spine (P < .0001) and absence of epidural disease (hazard ratio 0.275, 95% confidence interval 0.076-0.989, P = .048) in the sacral cohort predicted LC. Median OS was 16.3 and 28.5 mo in the cervical spine and sacrum cohorts, respectively. Two cases of sacral VCF, 1 brachial plexopathy, and 1 lumbar-sacral plexopathy were observed. CONCLUSION: Although high rates of LC were observed, strategies specific to the sacrum may require further optimization.

Imaging-Based Outcomes for 24 Gy in 2 Daily Fractions for Patients with de Novo Spinal Metastases Treated With Spine Stereotactic Body Radiation Therapy (SBRT).

PURPOSE: We report mature outcomes for a cohort of patients with no prior radiation (de novo) to the spine treated with 24 Gy in 2 daily fractions for metastases, which represents the same stereotactic body radiation therapy (SBRT) regimen under evaluation in the current Symptom Control-24 phase 3 randomized trial (NCT02512965). METHODS AND MATERIALS: The cohort consisted of 279 de novo spinal metastases in 145 consecutive patients treated with 24 Gy in 2 SBRT fractions, identified from a prospective single-institution database. The endpoints were overall survival (OS), imaging-based local failure (LF), and cumulative risk of vertebral compression fractures (VCF). RESULTS: The median follow-up per treated metastasis was 15.0 months (range, 0.1-71.6). The 1-year and 2-year OS rates were 73.1% and 60.7%, respectively. Presence of epidural disease (P < .0001), lung (P = .0415), and renal cell (P < .0001) primary histologies and baseline diffuse metastases (P = .0034) were significant prognostic factors for OS. The 1-year and 2-year LF rates were 9.7% and 17.6%, respectively, and the median time to LF was 9.2 month (range, 0.4-31.3 months). Only the presence of epidural disease predicted for LF (P < .0001). The cumulative risk of VCF at 1 and 2 years was 8.5% and 13.8%, respectively. Lytic (P = .0143) or mixed lytic/blastic (P = .0214) lesions, spinal malalignment (P = .0121), and the dose to 90% of the planning target volume (P = .0085) were significant predictors for VCF. CONCLUSIONS: Twenty-four Gray in 2 daily fractions is safe and effective in achieving high tumor control rates for de novo spinal metastases. These outcomes will serve as a benchmark for the ongoing Symptom Control-24 randomized trial comparing 24 Gy in 2 SBRT fractions to 20 Gy delivered in 5 daily conventional fractions.

Dosimetric feasibility of the hybrid Magnetic Resonance Imaging (MRI)-linac System (MRL) for brain metastases: The impact of the magnetic field.

BACKGROUND AND PURPOSE: We aimed to investigate the suitability of treating patients with single brain metastases using stereotactic radiosurgery (SRS) with the MRL and to characterize the dosimetric impact at tissue-air interfaces resulting primarily from the electron return effect (ERE). MATERIAL AND METHODS: 24 patients treated for intact single brain metastases were analyzed. Three radiotherapy plans with the same prescribed dose were generated for each case: (1) noncoplanar volumetric modulated arc therapy (VMAT), (2) coplanar step-and-shoot intensity modulated radiotherapy (IMRT) on the MRL in the absence (MRLB=0), and (3) in the presence of the transverse magnetic field (MRLB=1.5). The plans were evaluated using cumulative dose-volume histograms and by calculation of Paddick conformity index (CI), V100%, V12Gy minus gross tumor volume (V12Gy - GTV), and V2Gy. At tissue-air boundaries, the dosimetric impact of the magnetic field was quantified using a 5 mm rim of tissue. RESULTS: All plans met the target coverage and organs-at-risk planning objectives. Differences between all investigated dosimetric parameters significantly favored the VMAT plans as compared to the MRLB=0 and MRLB=1.5 plans, except for V2Gy. The mean V12Gy - GTV and V2Gy marginally favored the MRLB=0 plans compared to the MRLB=1.5 plans (mean difference: 0.45 cm3, p = 0.0019 and 16.46 cm3, p < 0.0001, respectively). The presence of the magnetic field resulted in a statistically significant but small increase in mean dose and D2cc in the skin (0.08 Gy, p < 0.0001 and 0.6 Gy, p < 0.0001, respectively) and around air cavities (0.07 Gy, p = 0.0092 and 0.3 Gy, p = 0.0004, respectively). CONCLUSIONS: It is feasible to generate stereotactic radiation plans that satisfy clinical requirements using the MRL in the setting of single brain metastases. The dosimetric impact of the magnetic field including the ERE at tissue-air boundaries is minor and does not negatively impact target conformity or dose gradient.

PIF4 Promotes Expression of LNG1 and LNG2 to Induce Thermomorphogenic Growth in Arabidopsis.

Arabidopsis plants adapt to high ambient temperature by a suite of morphological changes including elongation of hypocotyls and petioles and leaf hyponastic growth. These morphological changes are collectively called thermomorphogenesis and are believed to increase leaf cooling capacity by enhancing transpiration efficiency, thereby increasing tolerance to heat stress. The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) has been identified as a major regulator of thermomorphogenic growth. Here, we show that PIF4 promotes the expression of two homologous genes LONGIFOLIA1 (LNG1) and LONGIFOLIA2 (LNG2) that have been reported to regulate leaf morphology. ChIP-Seq analyses and ChIP assays showed that PIF4 directly binds to the promoters of both LNG1 and LNG2. The expression of LNG1 and LNG2 is induced by high temperature in wild type plants. However, the high temperature activation of LNG1 and LNG2 is compromised in the pif4 mutant, indicating that PIF4 directly regulates LNG1 and LNG2 expression in response to high ambient temperatures. We further show that the activities of LNGs support thermomorphogenic growth. The expression of auxin biosynthetic and responsive genes is decreased in the lng quadruple mutant, implying that LNGs promote thermomorphogenic growth by activating the auxin pathway. Together, our results demonstrate that LNG1 and LNG2 are directly regulated by PIF4 and are new components for the regulation of thermomorphogenesis.

Spine Stereotactic Body Radiotherapy: Indications, Outcomes, and Points of Caution.

STUDY DESIGN: A broad narrative review. OBJECTIVES: The objective of this article is to provide a technical review of spine stereotactic body radiotherapy (SBRT) planning and delivery, indications for treatment, outcomes, complications, and the challenges of response assessment. The surgical approach to spinal metastases is discussed with an overview of emerging minimally invasive techniques. METHODS: A comprehensive review of the literature was conducted on the techniques, outcomes, and developments in SBRT and surgery for spinal metastases. RESULTS: The optimal management of patients with spinal metastases is complex and requires multidisciplinary assessment from an oncologic team that is familiar with the shifting paradigm as a consequence of evolving techniques in surgery and stereotactic radiation, as well as new developments in systemic agents. The Spinal Instability Neoplastic Score and the epidural spinal cord compression (Bilsky) grading system are useful tools that facilitate communication among oncologic team members and can direct management by providing a baseline assessment of risks prior to therapy. The combined multimodality approach with "separation surgery" followed by postoperative spine SBRT achieves thecal sac decompression, improves tumor control, and avoids complications that may be associated with more extensive surgery. CONCLUSION: Spine SBRT is a highly effective treatment that is capable of delivering ablative doses to the target while sparing the critical organs-at-risk, chiefly the critical neural tissues, within a short and manageable schedule. At the same time, surgery occupies an important role in select patients, particularly with the expanding availability and expertise in minimally invasive techniques. With rapid adoption of spine SBRT in centers outside of the academic setting, it is imperative for the practicing oncologist to understand the relevance and application of these evolving concepts.

Volume of Lytic Vertebral Body Metastatic Disease Quantified Using Computed Tomography-Based Image Segmentation Predicts Fracture Risk After Spine Stereotactic Body Radiation Therapy.

PURPOSE: To determine a threshold of vertebral body (VB) osteolytic or osteoblastic tumor involvement that would predict vertebral compression fracture (VCF) risk after stereotactic body radiation therapy (SBRT), using volumetric image-segmentation software. METHODS AND MATERIALS: A computational semiautomated skeletal metastasis segmentation process refined in our laboratory was applied to the pretreatment planning CT scan of 100 vertebral segments in 55 patients treated with spine SBRT. Each VB was segmented and the percentage of lytic and/or blastic disease by volume determined. RESULTS: The cumulative incidence of VCF at 3 and 12 months was 14.1% and 17.3%, respectively. The median follow-up was 7.3 months (range, 0.6-67.6 months). In all, 56% of segments were determined lytic, 23% blastic, and 21% mixed, according to clinical radiologic determination. Within these 3 clinical cohorts, the segmentation-determined mean percentages of lytic and blastic tumor were 8.9% and 6.0%, 0.2% and 26.9%, and 3.4% and 15.8% by volume, respectively. On the basis of the entire cohort (n=100), a significant association was observed for the osteolytic percentage measures and the occurrence of VCF (P<.001) but not for the osteoblastic measures. The most significant lytic disease threshold was observed at ≥11.6% (odds ratio 37.4, 95% confidence interval 9.4-148.9). On multivariable analysis, ≥11.6% lytic disease (P<.001), baseline VCF (P<.001), and SBRT with ≥20 Gy per fraction (P=.014) were predictive. CONCLUSIONS: Pretreatment lytic VB disease volumetric measures, independent of the blastic component, predict for SBRT-induced VCF. Larger-scale trials evaluating our software are planned to validate the results.

Ectrodactyly and Lethal Pulmonary Acinar Dysplasia Associated with Homozygous FGFR2 Mutations Identified by Exome Sequencing.

Ectrodactyly/split hand-foot malformation is genetically heterogeneous with more than 100 syndromic associations. Acinar dysplasia is a rare congenital lung lesion of unknown etiology, which is frequently lethal postnatally. To date, there have been no reports of combinations of these two phenotypes. Here, we present an infant from a consanguineous union with both ectrodactyly and autopsy confirmed acinar dysplasia. SNP array and whole-exome sequencing analyses of the affected infant identified a novel homozygous Fibroblast Growth Factor Receptor 2 (FGFR2) missense mutation (p.R255Q) in the IgIII domain (D3). Expression studies of Fgfr2 in development show localization to the affected limbs and organs. Molecular modeling and genetic and functional assays support that this mutation is at least a partial loss-of-function mutation, and contributes to ectrodactyly and acinar dysplasia only in homozygosity, unlike previously reported heterozygous activating FGFR2 mutations that cause Crouzon, Apert, and Pfeiffer syndromes. This is the first report of mutations in a human disease with ectrodactyly with pulmonary acinar dysplasia and, as such, homozygous loss-of-function FGFR2 mutations represent a unique syndrome.

Practical dose delivery verification of craniospinal IMRT.

Craniospinal irradiation (CSI) using IMRT allows dose sparing to organs-at-risk (OAR) whilst conforming the dose to the target volume. Due to the complexity of treatment involving several isocenters, the dose distribution created by the inverse-planned segmented fields must be verified prior to treatment. We propose and test methods to verify dose delivery using commonly available dosimetry equipment for commissioning and routine patient-specific dose verification of craniospinal IMRT. Ten patients treated with conventional CSI were retrospectively planned with a 3-isocenter (cranial, upper spine, and lower spine) IMRT technique. The isocenters were placed 25-27 cm away from each other in the longitudinal direction but in the same lateral and anterioposterior positions. The planning target volume (PTV) was defined as the brain with a 0.5 cm margin and spinal canal with a 1.0 cm margin. The plans were prescribed to 36 Gy in 20 fractions to the PTV mean dose. Eleven beams (five cranial, three upper spine, and three lower spine) were optimized simultaneously. The dose delivered by the IMRT plans was then recalculated on several different phantoms and measured using the following methods: 1) ionization chamber inserted in a cylindrical phantom, positioned in the junction regions between cranial/upper-spine isocenters and upper-/lower-spine isocenters; 2) MapCHECK centered in the overlap regions; and 3) ArcCHECK measurement with beams from each isocenter. For 1) ± 3% dose difference and for 2) and 3) ≥ 95% of measured points with a γ-index < 1 for 3% dose difference and 3 mm distance-to-agreement were deemed clinically acceptable. The median (range) planned to measured dose differences for the ionization chamber is 0.4% (-1.5% to 3.0%) for the cranial/upper-spine field and 1.8% (-0.8% to 2.6%) for the upper-/lower-spine field overlap region. The median (range) percentage of MapCHECK diodes with a γ index < 1 for 3%/3 mm criterion is 98.0% (95.3% to 99.7%) for the cranial/upper-spine and 97.3% (95.0% to 99.6%) for the upper-/lower-spine field overlap regions. The median (range) percentage of ArcCHECK diodes with a γ index < 1 for 3%/3 mm criterion is 99.7% (97.1% to 100%). Three different methods of verifying craniospinal IMRT were compared and tested. All techniques offer different benefits and together can be used for 1) commissioning the treatment technique and, separately, for 2) patient-specific pretreatment verification measurements.

The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-ß1-induced growth inhibition by targeting Smad3 linker region.

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-ß1 (TGF-ß1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-ß1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-ß1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-ß1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-ß1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

Salvage Stereotactic Body Radiotherapy (SBRT) Following In-Field Failure of Initial SBRT for Spinal Metastases.

PURPOSE: We report our experience in salvaging spinal metastases initially irradiated with stereotactic body radiation therapy (SBRT), who subsequently progressed with imaging-confirmed local tumor progression, and were re-irradiated with a salvage second SBRT course to the same level. METHODS AND MATERIALS: From a prospective database, 56 metastatic spinal segments in 40 patients were identified as having been irradiated with a salvage second SBRT course to the same level. In addition, 24 of 56 (42.9%) segments had initially been irradiated with conventional external beam radiation therapy before the first course of SBRT. Local control (LC) was defined as no progression on magnetic resonance imaging at the treated segment, and calculated according to the competing risk model. Overall survival (OS) was evaluated for each patient treated by use of the Kaplan-Meier method. RESULTS: The median salvage second SBRT total dose and number of fractions was 30 Gy in 4 fractions (range, 20-35 Gy in 2-5 fractions), and for the first course of SBRT was 24 Gy in 2 fractions (range, 20-35 Gy in 1-5 fractions). The median follow-up time after salvage second SBRT was 6.8 months (range, 0.9-39 months), the median OS was 10.0 months, and the 1-year OS rate was 48%. A longer time interval between the first and second SBRT courses predicted for better OS (P=.02). The crude LC was 77% (43/56), the 1-year LC rate was 81%, and the median time to local failure was 3.0 months (range, 2.7-16.7 months). Of the 13 local failures, 85% (11/13) and 46% (6/13) showed progression within the epidural space and paraspinal soft tissues, respectively. Absence of baseline paraspinal disease predicted for better LC (P<.01). No radiation-induced vertebral compression fractures or cases of myelopathy were observed. CONCLUSION: A second course of spine SBRT, most often with 30 Gy in 4 fractions, for spinal metastases that failed initial SBRT is a feasible and efficacious salvage treatment option.

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Use of volumetric-modulated arc therapy for treatment of Hodgkin lymphoma.

To evaluate volumetric-modulated arc therapy (VMAT) for treatment of Hodgkin lymphoma (HL) in patients where conventional radiotherapy was not deliverable. A planning computed tomography (CT) scan was acquired for a twelve-year-old boy with Stage IIIB nodular sclerosing HL postchemotherapy with positive positron emission tomography scan. VMAT was used for Phase 1 (19.8Gy in 11 fractions) and Phase 2 (10.8Gy in 6 fractions) treatment plans. Single anticlockwise arc plans were constructed using SmartArc (Philips Radiation Oncology Systems, Fitchburg, WI) with control points spaced at 4°. The inverse-planning objectives were to uniformly irradiate the planning target volume (PTV) with the prescription dose while keeping the volume of lung receiving greater than 20Gy (V20Gy) to less than 30% and minimize the dose to the other adjacent organs at risk (OAR). Pretreatment verification was conducted and the treatment delivery was on an MLCi Synergy linear accelerator (Elekta Ltd, Crawley, UK). The planning results were retrospectively confirmed in a further 4 patients using a single PTV with a prescribed dose of 19.8Gy in 11 fractions. Acceptable dose coverage and homogeneity were achieved for both Phase 1 and 2 plans while keeping the lung V20Gy at 22.5% for the composite plan. The beam-on times for Phase 1 and Phase 2 plans were 109 and 200 seconds, respectively, and the total monitor units were 337.2MU and 292.5MU, respectively. The percentage of measured dose points within 3% and 3mm for Phase 1 and Phase 2 were 92% and 98%, respectively. Both plans were delivered successfully. The retrospective planning study showed that VMAT improved PTV dose uniformity and reduced the irradiated volume of heart and lung, although the volume of lung irradiated to low doses increased. Two-phased VMAT offers an attractive option for large volume sites, such as HL, giving a high level of target coverage and significant OAR sparing together with efficient delivery.