A Randomised Phase II Clinical Trial Comparing the Deliverability and Acute Toxicity of Wide Tangent versus Volumetric Modulated Arc Therapy to the Breast and Internal Mammary Chain.

AIMS: Inclusion of the internal mammary chain in the radiotherapy target volume (IMC-RT) improves disease-free and overall survival in higher risk breast cancer patients, but increases radiation doses to heart and lungs. Dosimetric data show that either modified wide-tangential fields (WT) or volumetric modulated arc therapy (VMAT) together with [AQ1]voluntary deep inspiration breath hold (vDIBH) keep mean heart doses below 4 Gy in most patients. However, the impact on departmental resources has not yet been documented. This phase II clinical trial compared the time taken to deliver IMC-RT using either WT and vDIBH or VMAT and vDIBH, together with planning time, dosimetry, set-up reproducibility and toxicity. MATERIALS AND METHODS: Left-sided breast cancer patients requiring IMC-RT were randomised to receive either WT(vDIBH) or VMAT radiotherapy. The primary outcome was treatment time, powered to detect a minimum difference of 75 min (5 min/fraction) between techniques. The population mean displacement, systematic error and random error for cone beam computed tomography chest wall matches in three directions of movement were calculated. Target volume and organ at risk doses were compared between groups. Side-effects, including skin (Radiation Therapy Oncology Group), lung and oesophageal toxicity (Common Terminology Criteria for Adverse Events v 4.03) rates, were compared between the groups over 3 months. Patient-reported outcome measures, including shoulder toxicity at baseline, 6 months and 1 year, were compared. RESULTS: Twenty-one patients were recruited from a single UK centre between February 2017 and January 2018. The mean (standard deviation) total treatment time per fraction for VMAT treatments was 13.2 min (1.7 min) compared with 28.1 min (3.3 min) for WT(vDIBH). There were no statistically significant differences in patient set-up errors in between groups. The average mean heart dose for WT(vDIBH) was 2.6 Gy compared with 3.4 Gy for VMAT(vDIBH) (P = 0.13). The mean ipsilateral lung V17Gy was 32.8% in the WT(vDIBH) group versus 34.4% in the VMAT group (P = 0.2). The humeral head (mean dose 16.8 Gy versus 2.8 Gy), oesophagus (maximum dose 37.3 Gy versus 20.1 Gy) and thyroid (mean dose 22.0 Gy versus 11.2 Gy) all received a statistically significantly higher dose in the VMAT group. There were no statistically significant differences in skin, lung or oesophageal toxicity within 3 months of treatment. Patient-reported outcomes of shoulder toxicity, pain, fatigue, breathlessness and breast symptoms were similar between groups at 1 year. CONCLUSION: VMAT(vDIBH) and WT(vDIBH) are feasible options for locoregional breast radiotherapy including the IMC. VMAT improves nodal coverage and delivers treatment more quickly, resulting in less breath holds for the patient. This is at the cost of increased dose to some non-target tissues. The latter does not appear to translate into increased toxicity in this small study.

Evaluation of a Novel Field-placement Algorithm for Locoregional Breast Cancer Radiotherapy Including the Internal Mammary Chain.

AIMS: Irradiation of the internal mammary chain (IMC) is increasing following recently published data, but the need for formal delineation of lymph node volumes is slowing implementation in some healthcare settings. A field-placement algorithm for irradiating locoregional lymph nodes including the IMC could reduce the resource impact of introducing irradiation of the IMC. This study describes the development and evaluation of such an algorithm. MATERIALS AND METHODS: An algorithm was developed in which six points representing lymph node clinical target volume borders (based on European Society for Radiotherapy and Oncology consensus nodal contouring guidelines) were placed on computed tomography-defined anatomical landmarks and used to place tangential and nodal fields. Single-centre testing in 20 cases assessed the success of the algorithm in covering planning target volumes (PTVs) and adequately sparing organs at risk. Plans derived using the points algorithm were also compared with plans generated following formal delineation of nodal PTVs, using the Wilcoxon signed rank test. Timing data for point placement were collected. Multicentre testing using the same methods was then carried out to establish whether the technique was transferable to other centres. RESULTS: Single-centre testing showed that 95% of cases met the nodal PTV coverage dose constraints (binomial probability confidence interval 75.1-99.9%) with no statistically significant reduction in mean heart dose or ipsilateral lung V17Gy associated with formal nodal delineation. In multicentre testing, 69% of cases met nodal PTV dose constraints and there was a statistically significant difference in IMC PTV coverage using the points algorithm when compared with formally delineated nodal volumes (P < 0.01). However, there was no difference in axillary level 1-4 PTV coverage (P = 0.11) with all cases meeting target volume constraints. CONCLUSIONS: The optimal strategy for breast and locoregional lymph node radiotherapy is target volume delineation. However, use of this novel points-based field-placement algorithm results in dosimetrically acceptable plans without the need for formal lymph node contouring in a single-centre setting and for the breast and level 1-4 axilla in a multicentre setting. Further quality assurance measures are needed to enable implementation of the algorithm for irradiation of the IMC in a multicentre setting.

Physiological and emotional effects of pentatonic live music played for preterm neonates and their mothers in the Newborn Intensive Care Unit: A randomized controlled trial.

OBJECTIVE: Despite attempts to increase calmness in the Newborn Intensive Care Unit (NICU), preterm neonates still experience stress. The question arises how to further promote the infants' wellbeing. Therefore, the immediate effects of pentatonic live music on preterm infants and their mothers were examined. DESIGN AND METHODS: In a two-centre randomized controlled trial with crossover design preterm infants were exposed sequentially to two conditions: live pentatonic harp music (LPHM) used in Anthroposophic Medicine or standard care. The order of the conditions was randomized within each subject. The primary outcome was change of the number of oxygen desaturations < 90%/h, whereas secondary outcomes were: heart rate, respiratory rate, oxygen saturation, heart rate variability (HRV), the perfusion index, pulse-transit-time and maternal anxiety and others not reported on in this article. RESULTS: 21 preterm infants were randomized (14 girls), mean gestational age at measurement 35 + 0 weeks (SD 1 week). The primary outcome parameter showed no significant changes. Regarding the secondary outcomes the comparison of the pre-post-differences between the conditions showed significant effects for the HRV parameters pNN50 (ΔpNN50 = 1.46%, z = -2.47, p = .001) and SDNN (ΔSDNN=-0.06 ms, z = -2.25, p = .002). The music intervention significantly increased the values of pNN50 (Mdn 1.2% vs. 2.6%, p = 0.04) and marginally those of SDNN (Mdn 31.7 ms vs. 36.4 ms, p = 0.05). No changes were found in the other parameters. CONCLUSIONS: While the use of music in the NICU had no effect on the number of oxygen desaturations, it increased two HRV parameters indicative of infants' parasympathetic tone.

A Dosimetric Comparison of Breast Radiotherapy Techniques to Treat Locoregional Lymph Nodes Including the Internal Mammary Chain.

AIMS: Radiotherapy target volumes in early breast cancer treatment increasingly include the internal mammary chain (IMC). In order to maximise survival benefits of IMC radiotherapy, doses to the heart and lung should be minimised. This dosimetry study compared the ability of three-dimensional conformal radiotherapy, arc therapy and proton beam therapy (PBT) techniques with and without breath-hold to achieve target volume constraints while minimising dose to organs at risk (OARs). MATERIALS AND METHODS: In 14 patients' datasets, seven IMC radiotherapy techniques were compared: wide tangent (WT) three-dimensional conformal radiotherapy, volumetric-modulated arc therapy (VMAT) and PBT, each in voluntary deep inspiratory breath-hold (vDIBH) and free breathing (FB), and tomotherapy in FB only. Target volume coverage and OAR doses were measured for each technique. These were compared using a one-way ANOVA with all pairwise comparisons tested using Bonferroni's multiple comparisons test, with adjusted P-values ≤ 0.05 indicating statistical significance. RESULTS: One hundred per cent of WT(vDIBH), 43% of WT(FB), 100% of VMAT(vDIBH), 86% of VMAT(FB), 100% of tomotherapy FB and 100% of PBT plans in vDIBH and FB passed all mandatory constraints. However, coverage of the IMC with 90% of the prescribed dose was significantly better than all other techniques using VMAT(vDIBH), PBT(vDIBH) and PBT(FB) (mean IMC coverage ± 1 standard deviation = 96.0% ± 4.3, 99.8% ± 0.3 and 99.0% ± 0.2, respectively). The mean heart dose was significantly reduced in vDIBH compared with FB for both the WT (P < 0.0001) and VMAT (P < 0.0001) techniques. There was no advantage in target volume coverage or OAR doses for PBT(vDIBH) compared with PBT(FB). CONCLUSIONS: Simple WT radiotherapy delivered in vDIBH achieves satisfactory coverage of the IMC while meeting heart and lung dose constraints. However, where higher isodose coverage is required, VMAT(vDIBH) is the optimal photon technique. The lowest OAR doses are achieved by PBT, in which the use of vDIBH does not improve dose statistics.

Pregnancy in type 2B VWD: a case series.

Type 2B von Willebrand disease (VWD) is a rare, inherited bleeding disorder resulting from a qualitative defect in von Willebrand factor (VWF). There is very little published information on how to quantify bleeding risk and manage haemostasis in type 2B VWD patients during pregnancy. This article presents the changes in VWF parameters and details of patient management and delivery outcomes for four pregnancies in three women with two different mutations causing type 2B VWD. We report an unexpected rise in the VWF:Ag at 37 weeks gestation in two sisters with R1306W associated with significant thrombocytopenia. These patients were supported with platelet transfusions as well as intermediate purity VWF-FVIII plasma concentrates during the peri- and postpartum periods. No thrombocytopenia was observed in our third case with a mutation encoding an R1308C substitution; haemostatic support was with intermediate purity VWF-FVIII plasma concentrates alone. No adverse bleeding events occurred and in all cases a live healthy infant was delivered. One patient was readmitted post partum with bleeding symptoms due to retained placenta; no further haemostatic support was given at this time. This case series is the first to detail the progression of laboratory parameters, management and outcomes of pregnancy in patients with type 2B VWD. The cases illustrate some of the challenges posed by the increased production of a VWF variant with a gain-of-function effect. The rapid coagulation changes observed in this series illustrate the need for continual monitoring of VWF parameters and platelet count throughout pregnancy in women with type 2B VWD.

Spontaneous spinal epidural hematoma in an 11-month-old girl.

An 11-month-old girl was admitted for a 2-month history of regression of motor milestones and lateralizing neurologic findings. Tests for hypercoagulability were all within normal limits except for a moderately elevated fibrinogen level. Urine and blood cultures were negative for growth. Complete MRI of the spine revealed a well-defined extradural inhomogeneous signal intensity extending from C(4) to T(3) suggestive of a subacute hematoma with evidence of evolving hemorrhagic change. There was no evidence of underlying arterial venous fistula or malformation. She underwent a cervicothoracic laminoplasty and evacuation of the hematoma. The clot was confirmed to be a hematoma on pathologic investigation. She made an uneventful recovery with gradual return of neurologic function. This case represents the longest known duration between initial symptoms and definitive management of spontaneous spinal epidural hematoma.

Histone H3 phosphorylation in the rat pineal gland: adrenergic regulation and diurnal variation.

In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

Mouse models of cell death.

Cell death is critical for the development and orderly maintenance of cellular homeostasis in metazoans. Developmental genetics in model systems, including Caenorhabditis elegans and Drosophila melanogaster, have helped to identify and order the components of cell-death pathways. An even more complex network of apoptotic pathways has evolved in higher organisms that possess homologs within each set of cell-death regulators. Whereas biochemical studies provide details of molecular mechanisms, genetic models reveal the essential physiologic roles. Transgenic and gene-ablated mice have helped to elucidate mammalian apoptotic pathways and identify the principal effect of each cell death regulator. Here, we review the details of the apoptotic machinery as revealed by mice deficient in critical components of cell-death pathways; we concentrate on cell-death regulators classified as members of the caspase and Bcl2 families or, broadly, as adaptors and mitochondrial released factors.

NFATc1 and NFATc2 together control both T and B cell activation and differentiation.

NFAT transcription factors play critical roles in gene transcription during immune responses. To investigate further the two most prominent NFAT family members, NFATc1 and NFATc2, we generated mice bearing lymphoid systems devoid of both. Doubly deficient T cells displayed cell surface markers of activation yet were significantly deficient in the development of multiple effector functions, including Th cytokine production, surface effector molecule expression, and cytolytic activity. Nevertheless, doubly deficient B cells were hyperactivated, as evidenced by extremely elevated serum IgG1 and IgE, as well as plasma cell expansion and infiltration of end organs. Thus, in T cells, NFATc1 and NFATc2 are dispensable for inflammatory reactivity but are required for effector differentiation, while in B cells, NFATs regulate both normal homeostasis and differentiation.

The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis.

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.

Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development.

Nuclear factor of activated T cells (NFAT) is a critical regulator of early gene transcription in response to TCR-mediated signals. Here, we show that mice lacking both NFATp and NFAT4 develop a profound lymphoproliferative disorder likely due to a lowered threshold for TCR signaling coupled with increased resistance to apoptosis secondary to defective FasL expression. NFAT mutant mice also have allergic blepharitis, interstitial pneumonitis, and a 10(3) to 10(4) fold increase in serum IgG1 and IgE levels, secondary to a dramatic and selective increase in Th2 cytokines. This phenotype may be ascribed to unopposed occupancy of the IL-4 promoter by NFATc. Our data demonstrate that lymphoid homeostasis and Th2 activation require a critical balance among NFAT family members.

The transcription factor NF-ATc is essential for cardiac valve formation.

Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc.

The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.

Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway.

Ligation of the cell surface receptor Fas/APO-1 (CD95) by its specific ligand or by anti-Fas antibodies rapidly induces apoptosis in susceptible cells. To characterize the molecular events involved in Fas-induced apoptosis, we examined the contribution of two subgroups of the mitogen-activated protein (MAP) kinase family, the Jun kinases or stress-activated protein kinases (JNKs/SAPKs) and the extracellular signal-regulated kinases (ERKs), in a Fas-sensitive neuroblastoma cell line. Here we show that both JNK and ERK protein kinases were activated upon Fas crosslinking through a Ras-dependent mechanism. Interference with either the JNK or ERK pathway by ectopic expression of dominant-interfering mutant proteins blocked Fas-mediated apoptosis. ERK activation was transient and associated with induced expression of the Fas receptor. In contrast, JNK activation was sustained and correlated with the onset of apoptosis. These data indicate that the ERK and the JNK groups of MAP kinases cooperate in the induction of cell death by Fas. Inhibition of Fas killing by an interleukin 1beta-converting enzyme (ICE)-like protease inhibitor peptide did not modify Fas-induced JNK activation upon Fas ligation. In contrast, changes in Bcl-2 level due to expression of sense and antisense vectors influenced the sensitivity to Fas killing and Fas-induced JNK activation.

Single amino acid mutations in the murine MHC class II A beta cytoplasmic domain abrogate antigen presentation.

Class II MHC molecules are heterodimeric transmembrane glycoproteins that function in the presentation of Ag to CD4+ T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-chains has previously been shown to diminish Ag presentation and abrogate rejection of class II-transfected tumor cells. To examine the contributions of individual amino acid residues of the A beta cytoplasmic domain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the five conserved amino acid residues, Q224, K225, L235, L236, and Q237 (delta5). In addition, cDNA were produced in which alanine was individually substituted for A beta(k) cytoplasmic domain residues 224 through 237 or doubly substituted at residues G226 and P227 or L235 and L236. These mutant cDNAs were individually cotransfected with wild-type A alpha cDNA into the class II-negative M12.C3 B lymphoma and Sal sarcoma cell lines. As was previously reported for transfectants lacking the entire A beta(k) cytoplasmic domain, the delta5 M12.C3 transfectant could not effectively present Ag to an autoreactive Ak-restricted T cell hybrid, and the delta5 Sal transfectant was not rejected when inoculated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrogated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domain in Ag presentation both in vitro and in vivo can be disturbed by mutation of single amino acid residues.

B7-2 (CD86) is essential for the development of IL-4-producing T cells.

The CD28/CTLA-4 ligands, B7-1 (CD80) and B7-2 (CD86), provide a co-stimulatory signal necessary for optimal T cell activation. We have examined the effect of blocking B7-1 and B7-2 in an in vitro system using ovalbumin-specific T cells from alpha beta TCR-transgenic mice. This system allowed us to examine the interaction of B7 co-stimulators on physiologic antigen-presenting cells (APC) with antigen-specific T helper precursor (ThP) cells. We report that blocking Thp/B7-1 or B7-2 interactions in a primary response differentially affects the cytokine profile observed in a secondary stimulation, even in the absence of additional anti-B7 antibody. Engagement of B7-2 in the primary stimulation was found to be essential for production of the Th2 cytokine, IL-4, but not the Th1 cytokines, IL-2 and IFN-gamma, in a secondary stimulation. Conversely, inclusion of the anti-B7-1 mAb in cultures using highly purified naive T cells increased levels of IL-4 and significantly depressed levels of IFN-gamma, upon re-stimulation. The effect of the anti-B7-2 mAb in reducing IL-4 production could be overcome by the addition of recombinant IL-4 in the primary stimulation. The effects of the anti-B7-2 mAb appear to be due to blocking and not cross-linking, as F(ab) fragments mimicked the intact antibody. Taken together, our data demonstrate that the interaction between Thp and B7-2 favors the development of Th2 cells.

Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice.

NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response.

B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy.

CD4 T helper precursor cells mature along two alternative pathways, Th1 and Th2. Here we show that these pathways are differentially activated by two costimulatory molecules, B7-1 and B7-2. Using anti-B7 antibodies, this developmental step was manipulated both in vitro and in vivo in experimental allergic encephalomyelitis (EAE). Anti-B7-1 reduced the incidence of disease while anti-B7-2 increased disease severity. Neither antibody affected overall T cell induction but rather altered cytokine profile. Administration of anti-B7-1 at immunization resulted in predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease. Since co-treatment with anti-IL-4 antibody prevented disease amelioration, costimulatory molecules may directly affect initial cytokine secretion. Thus, interaction of B7-1 and B7-2 with shared counterreceptors CD28 and CTLA-4 results in very different outcomes in clinical disease by influencing commitment of precursors to a Th1 or Th2 lineage.