Comparative Evaluation of Proton Therapy and Volumetric Modulated Arc Therapy for Brachial Plexus Sparing in the Comprehensive Reirradiation of High-Risk Recurrent Breast Cancer.

Purpose: Recurrent or new primary breast cancer requiring comprehensive regional nodal irradiation after prior radiation therapy (RT) to the supraclavicular area and upper axilla is challenging due to cumulative brachial plexus (BP) dose tolerance. We assessed BP dose sparing achieved with pencil beam scanning proton therapy (PBS-PT) and photon volumetric modulated arc therapy (VMAT). Methods and Materials: In an institutional review board-approved planning study, all patients with ipsilateral recurrent breast cancer treated with PBS-PT re-RT (PBT1) with at least partial BP overlap from prior photon RT were identified. Comparative VMAT plans (XRT1) using matched BP dose constraints were developed. A second pair of proton (PBT2) and VMAT (XRT2) plans using standardized target volumes were created, applying uniform prescription dose of 50.4 per 1.8 Gy and a maximum BP constraint <25 Gy. Incidence of brachial plexopathy was also assessed. Results: Ten consecutive patients were identified. Median time between RT courses was 48 months (15-276). Median first, second, and cumulative RT doses were 50.4 Gy (range, 42.6-60.0), 50.4 Gy relative biologic effectiveness (RBE) (45.0-64.4), and 102.4 Gy (RBE) (95.0-120.0), respectively. Median follow-up was 15 months (5-33) and 18 months for living patients (11-33) Mean BP max was 37.5 Gy (RBE) for PBT1 and 36.9 Gy for XRT1. Target volume coverage of V85% (volume receiving 85% of prescription dose), V90%, and V95% were numerically lower for XRT1 versus PBT1. Similarly, axilla I-III and supraclavicular area coverage were significantly higher for PBT2 than XRT2 at dose levels of V55%, V65%, V75%, V85%, and V95%. Only axilla I V55% did not reach significance (P = .06) favoring PBS-PT. Two patients with high cumulative BPmax (95.2 Gy [RBE], 101.6 Gy [RBE]) developed brachial plexopathy symptoms with ulnar nerve distribution neuropathy without pain or weakness (1 of 2 had symptom resolution after 6 months without intervention). Conclusions: PBS-PT improved BP sparing and target volume coverage versus VMAT. For patients requiring comprehensive re-RT for high-risk, nonmetastatic breast cancer recurrence with BP overlap and reasonable expectation for prolonged life expectancy, PBT may be the preferred treatment modality.

Detection of COVID-19 pulmonary manifestations with radiotherapy simulation CT imaging.

COVID-19 is associated with characteristic lung CT findings. Radiotherapy simulation CT scans may reveal characteristic COVID-19 findings and identify patients with active or prior infection. We reviewed patients undergoing CT simulation at a major cancer center in an early epicenter of the COVID-19 pandemic in the United States. Scans were reviewed by radiation oncologists using established radiographic criteria for COVID-19 pneumonia. Radiographic classifications were compared with available COVID-19 PCR test results. A one-tailed t-test was used to compare the rate of positive COVID-19 tests in radiographically suspicious vs. non-suspicious groups. Scans deemed suspicious were re-reviewed by expert diagnostic radiologists. 414 CT simulation scans were performed on 400 patients. 119 patients had COVID-19 PCR test results available. Radiation oncologists considered 71 scans (17.1%) suspicious for COVID-19. Of these, 23 had corresponding COVID-19 PCR tests, and 3/23 (15.7%) were positive for COVID. 107 non-suspicious scans had corresponding COVID-19 test results, and 9 were positive (8.4%). The difference in positive test results between suspicious and non-suspicious groups was not significant (p = 0.23). Upon re-review by a diagnostic radiologist, 25 (35%) scans deemed suspicious by radiation oncologists were confirmed to meet criteria, while the rest were re-classified as "atypical" for COVID-19. We conclude that radiotherapy simulation CT scans can be reviewed for signs of COVID-19 pneumonia by radiation oncologists. However, suspicious CT simulation was not associated with a higher incidence of COVID infection compared with non-suspicious CT simulation, and there was low concordance between radiation oncologist and diagnostic radiologist classification of scans.

Multimodal single-cell analysis reveals distinct radioresistant stem-like and progenitor cell populations in murine glioma.

Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Chromosomal instability drives metastasis through a cytosolic DNA response.

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Patterns of nodal failure after intensity modulated radiotherapy for nasopharyngeal carcinoma.

OBJECTIVES: To evaluate the sites of nodal failure (NF) of nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). STUDY DESIGN: Retrospective chart review. METHODS: We reviewed the records of 165 patients with nonmetastatic NPC treated with IMRT between July 1998 and April 2011 at our institution. Recurrent nodes were delineated on imaging and coregistered with the original treatment planning computed tomography. Failures were assessed as in-field, out-field, or marginal based on the relative volumes of the recurrent nodes covered by the original dose distribution. RESULTS: Ten patients had NF at a median follow-up of 70.4 months for surviving patients. The 3- and 5-year overall survival and NF rates were 88.7%, 76.0% and 5.8%, 7.7%, respectively. Six of the nodal failures were in-field, of which five occurred in level II; whereas four had out-field failures, all of which were in the protected parotid gland area. There were no recurrences in level 1b despite this region being protected. The cumulative 3- and 5-year failure rates in the parotid gland area were 2.2% and 3.1%, respectively. Three patients with parotid failure initially had subcentimeter, nonspecific nodules in the same locations of the parotid gland as the recurrent nodes. CONCLUSION: Nodal failure is uncommon after IMRT in NPC. Recurrence in the parotid gland region accounts for all of the out-field failures and 40% of NF in our study. Comprehensive assessment of nodules in or around the parotid gland is therefore a key aspect of treatment planning and follow-up. LEVEL OF EVIDENCE: 4. Laryngoscope, 2016 127:377-382, 2017.

A Role for Mitogen- and Stress-Activated Kinase 1 in L-DOPA-Induced Dyskinesia and ∆FosB Expression.

BACKGROUND: Abnormal regulation of extracellular signal-regulated kinases 1 and 2 has been implicated in 3,4-dihydroxy-l-phenylalanine (L-DOPA)-induced dyskinesia (LID), a motor complication affecting Parkinson's disease patients subjected to standard pharmacotherapy. We examined the involvement of mitogen- and stress-activated kinase 1 (MSK1), a downstream target of extracellular signal-regulated kinases 1 and 2, and an important regulator of transcription in LID. METHODS: 6-Hydroxydopamine was used to produce a model of Parkinson's disease in MSK1 knockout mice and in ∆FosB- or ∆cJun-overexpressing transgenic mice, which were assessed for LID following long-term L-DOPA administration. Biochemical processes were evaluated by Western blotting or immunofluorescence. Histone H3 phosphorylation was analyzed by chromatin immunoprecipitation followed by promotor-specific quantitative polymerase chain reaction. RESULTS: Genetic inactivation of MSK1 attenuated LID and reduced the phosphorylation of histone H3 at Ser10 in the striatum. Chromatin immunoprecipitation analysis showed that this reduction occurred at the level of the fosB gene promoter. In line with this observation, the accumulation of ∆FosB produced by chronic L-DOPA was reduced in MSK1 knockout. Moreover, inducible overexpression of ∆FosB in striatonigral medium spiny neurons exacerbated dyskinetic behavior, whereas overexpression of ∆cJun, which reduces ∆FosB-dependent transcriptional activation, counteracted LID. CONCLUSIONS: Results indicate that abnormal regulation of MSK1 contributes to the development of LID and to the concomitant increase in striatal ∆FosB, which may occur via increased histone H3 phosphorylation at the fosB promoter. Results also show that accumulation of ∆FosB in striatonigral neurons is causally related to the development of dyskinesia.

Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens.

BACKGROUND: Increasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies that have intrinsic limitations. RESULTS: We use next generation sequencing methods, RNA-seq and ChIP-seq for RNA polymerase II and several histone methylation marks, to obtain a more complete view of cocaine-induced changes in gene expression and associated adaptations in numerous modes of chromatin regulation in the mouse nucleus accumbens, a key brain reward region. We demonstrate an unexpectedly large number of pre-mRNA splicing alterations in response to repeated cocaine treatment. In addition, we identify combinations of chromatin changes, or signatures, that correlate with cocaine-dependent regulation of gene expression, including those involving pre-mRNA alternative splicing. Through bioinformatic prediction and biological validation, we identify one particular splicing factor, A2BP1(Rbfox1/Fox-1), which is enriched at genes that display certain chromatin signatures and contributes to drug-induced behavioral abnormalities. Together, this delineation of the cocaine-induced epigenome in the nucleus accumbens reveals several novel modes of regulation by which cocaine alters the brain. CONCLUSIONS: We establish combinatorial chromatin and transcriptional profiles in mouse nucleus accumbens after repeated cocaine treatment. These results serve as an important resource for the field and provide a template for the analysis of other systems to reveal new transcriptional and epigenetic mechanisms of neuronal regulation.

In vivo radiation response of proneural glioma characterized by protective p53 transcriptional program and proneural-mesenchymal shift.

Glioblastoma is the most common adult primary brain tumor and has a dismal median survival. Radiation is a mainstay of treatment and significantly improves survival, yet recurrence is nearly inevitable. Better understanding the radiation response of glioblastoma will help improve strategies to treat this devastating disease. Here, we present a comprehensive study of the in vivo radiation response of glioma cells in a mouse model of proneural glioblastoma. These tumors are a heterogeneous mix of cell types with differing radiation sensitivities. To explicitly study the gene expression changes comprising the radiation response of the Olig2(+) tumor bulk cells, we used translating ribosome affinity purification (TRAP) from Olig2-TRAP transgenic mice. Comparing both ribosome-associated and total pools of mRNA isolated from Olig2(+) cells indicated that the in vivo gene expression response to radiation occurs primarily at the total transcript level. Genes related to apoptosis and cell growth were significantly altered. p53 and E2F were implicated as major regulators of the radiation response, with p53 activity needed for the largest gene expression changes after radiation. Additionally, radiation induced a marked shift away from a proneural expression pattern toward a mesenchymal one. This shift occurs in Olig2(+) cells within hours and in multiple genetic backgrounds. Targets for Stat3 and CEBPB, which have been suggested to be master regulators of a mesenchymal shift, were also up-regulated by radiation. These data provide a systematic description of the events following radiation and may be of use in identifying biological processes that promote glioma radioresistance.

Mathematical modeling of PDGF-driven glioblastoma reveals optimized radiation dosing schedules.

Glioblastomas (GBMs) are the most common and malignant primary brain tumors and are aggressively treated with surgery, chemotherapy, and radiotherapy. Despite this treatment, recurrence is inevitable and survival has improved minimally over the last 50 years. Recent studies have suggested that GBMs exhibit both heterogeneity and instability of differentiation states and varying sensitivities of these states to radiation. Here, we employed an iterative combined theoretical and experimental strategy that takes into account tumor cellular heterogeneity and dynamically acquired radioresistance to predict the effectiveness of different radiation schedules. Using this model, we identified two delivery schedules predicted to significantly improve efficacy by taking advantage of the dynamic instability of radioresistance. These schedules led to superior survival in mice. Our interdisciplinary approach may also be applicable to other human cancer types treated with radiotherapy and, hence, may lay the foundation for significantly increasing the effectiveness of a mainstay of oncologic therapy. PAPERCLIP:

Neurobiological sequelae of witnessing stressful events in adult mice.

BACKGROUND: It is well known that exposure to severe stress increases the risk for developing mood disorders. However, most chronic stress models in rodents involve at least some form of physically experiencing traumatic events. METHODS: This study assessed the effects of a novel social stress paradigm that is insulated from the effects of physical stress. Specifically, adult male C57BL/6J mice were exposed to either emotional (ES) or physical stress (PS) for 10 minutes per day for 10 days. The ES mice were exposed to the social defeat of a PS mouse by a larger, more aggressive CD-1 mouse from the safety of an adjacent compartment. RESULTS: Like PS mice, ES mice exhibited a range of depression- and anxiety-like behaviors both 24 hours and 1 month after the stress. Increased levels of serum corticosterone, part of the stress response, accompanied these behavioral deficits. Based on previous work that implicated gene expression changes in the ventral tegmental area (a key brain reward region) in the PS phenotype, we compared genome-wide mRNA expression patterns in this brain region of ES and PS mice using RNA-seq. We found significant overlap between these conditions, which suggests several potential gene targets for mediating the behavioral abnormalities observed. CONCLUSIONS: These findings demonstrate that witnessing traumatic events is a potent stress in adult male mice capable of inducing long-lasting neurobiological perturbations.

BDNF is a negative modulator of morphine action.

Brain-derived neurotrophic factor (BDNF) is a key positive regulator of neural plasticity, promoting, for example, the actions of stimulant drugs of abuse such as cocaine. We discovered a surprising opposite role for BDNF in countering responses to chronic morphine exposure. The suppression of BDNF in the ventral tegmental area (VTA) enhanced the ability of morphine to increase dopamine (DA) neuron excitability and promote reward. In contrast, optical stimulation of VTA DA terminals in nucleus accumbens (NAc) completely reversed the suppressive effect of BDNF on morphine reward. Furthermore, we identified numerous genes in the NAc, a major target region of VTA DA neurons, whose regulation by BDNF in the context of chronic morphine exposure mediated this counteractive function. These findings provide insight into the molecular basis of morphine-induced neuroadaptations in the brain's reward circuitry.

Drug experience epigenetically primes Fosb gene inducibility in rat nucleus accumbens.

ΔFosB, a Fosb gene product, is induced in nucleus accumbens (NAc) and caudate-putamen (CPu) by repeated exposure to drugs of abuse such as cocaine. This induction contributes to aberrant patterns of gene expression and behavioral abnormalities seen with repeated drug exposure. Here, we assessed whether a remote history of cocaine exposure in rats might alter inducibility of the Fosb gene elicited by subsequent drug exposure. We show that prior chronic cocaine administration, followed by extended withdrawal, increases inducibility of Fosb in NAc, as evidenced by greater acute induction of ΔFosB mRNA and faster accumulation of ΔFosB protein after repeated cocaine reexposure. No such primed Fosb induction was observed in CPu; in fact, subsequent acute induction of ΔFosB mRNA was suppressed in CPu. These abnormal patterns of Fosb expression are associated with chromatin modifications at the Fosb gene promoter. Prior chronic cocaine administration induces a long-lasting increase in RNA polymerase II (Pol II) binding at the Fosb promoter in NAc only, suggesting that Pol II "stalling" primes Fosb for induction in this region upon reexposure to cocaine. A cocaine challenge then triggers the release of Pol II from the gene promoter, allowing for more rapid Fosb transcription. A cocaine challenge also decreases repressive histone modifications at the Fosb promoter in NAc, but increases such repressive marks and decreases activating marks in CPu. These results provide new insight into the chromatin dynamics at the Fosb promoter and reveal a novel mechanism for primed Fosb induction in NAc upon reexposure to cocaine.

Subregional, dendritic compartment, and spine subtype specificity in cocaine regulation of dendritic spines in the nucleus accumbens.

Numerous studies have found that chronic cocaine increases dendritic spine density of medium spiny neurons in the nucleus accumbens (NAc). Here, we used single-cell microinjections and advanced 3D imaging and analysis techniques to extend these findings in several important ways: by assessing cocaine regulation of dendritic spines in the core versus shell subregions of NAc in the mouse, over a broad time course (4 h, 24 h, or 28 d) of withdrawal from chronic cocaine, and with a particular focus on proximal versus distal dendrites. Our data demonstrate subregion-specific, and in some cases opposite, regulation of spines by cocaine on proximal but not distal dendrites. Notably, all observed density changes were attributable to selective regulation of thin spines. At 4 h after injection, the proximal spine density is unchanged in the core but significantly increased in the shell. At 24 h, the density of proximal dendritic spines is reduced in the core but increased in the shell. Such downregulation of thin spines in the core persists through 28 d of withdrawal, whereas the spine density in the shell returns to baseline levels. Consistent with previous results, dendritic tips exhibited upregulation of dendritic spines after 24 h of withdrawal, an effect localized to the shell. The divergence in regulation of proximal spine density in NAc core versus shell by cocaine correlates with recently reported electrophysiological data from a similar drug administration regimen and might represent a key mediator of changes in the reward circuit that drive aspects of addiction.

Paternal transmission of stress-induced pathologies.

BACKGROUND: There has been recent interest in the possibility that epigenetic mechanisms might contribute to the transgenerational transmission of stress-induced vulnerability. Here, we focused on possible paternal transmission with the social defeat stress paradigm. METHODS: Adult male mice exposed to chronic social defeat stress or control nondefeated mice were bred with normal female mice, and their offspring were assessed behaviorally for depressive- and anxiety-like measures. Plasma levels of corticosterone and vascular endothelial growth factor were also assayed. To directly assess the role of epigenetic mechanisms, we used in vitro fertilization (IVF); behavioral assessments were conducted on offspring of mice from IVF-control and IVF-defeated fathers. RESULTS: We show that both male and female offspring from defeated fathers exhibit increased measures of several depression- and anxiety-like behaviors. The male offspring of defeated fathers also display increased baseline plasma levels of corticosterone and decreased levels of vascular endothelial growth factor. However, most of these behavioral changes were not observed when offspring were generated through IVF. CONCLUSIONS: These results suggest that, although behavioral adaptations that occur after chronic social defeat stress can be transmitted from the father to his male and female F1 progeny, only very subtle changes might be transmitted epigenetically under the conditions tested.

Hippocampal-dependent antidepressant-like activity of histone deacetylase inhibition.

Chronic social defeat stress in mice significantly decreases subsequent social interactions and induces other depression-like behaviors. Here we measured and manipulated levels of acetylated histone H3 (acH3), a chromatin mark of transcriptional activation, in the hippocampus and amygdala after ten continuous days of social defeat stress in male C57/Bl6J mice. This form of social stress causes a transient increase, followed by a persistent decrease, in the levels of acH3 in hippocampus. By comparison, increased acH3 in amygdala was more robust but also highly transient. The persistent decrease in acH3 in hippocampus may be pathological, since it is reversed by chronic fluoxetine administration. Consistent with this hypothesis, infusion of a histone deacetylase (HDAC) inhibitor MS-275 (100 µM) into hippocampus reverses a defeat-induced deficit in sucrose preference, although it does not restore social interaction behavior. Next, different forms of social enrichment were examined with or without hippocampal infusion of MS-275. After social stress, simple pair-housing with another male C57, or female C57, mouse does not reverse social avoidance. However, when HDAC inhibitors are infused into hippocampus during social housing with another male, social avoidance is attenuated. Interestingly, social avoidance is reversed when MS-275 is infused directly into amygdala. Together, these findings further support the antidepressant potential of HDAC inhibitors, and indicate that temporally overlapping environmental and molecular events are required to optimally reverse specific stress-induced behavioral symptoms.