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Introduction This study investigates the optimal target delineation protocol stratified by treatment planning technique in patients undergoing whole breast radiotherapy after breast conservation surgery. Materials and Methods Target delineation using Tangent (RTOG 0413 Whole Breast Irradiation Protocol), European SocieTy for Radiotherapy and Oncology (ESTRO), and Radiation Therapy & Oncology Group (RTOG) guidelines was performed on 10 randomly selected treatment planning computed tomography datasets of patients with left-sided breast cancer. An objective plan quality metric (PQM) scoring schema was defined and communicated to the medical physicist prior to commencement of treatment planning. Treatment planning was performed using field-in-field (FiF) intensity modulated radiotherapy technique (IMRT), inverse IMRT, and volumetric modulated arc therapy (VMAT), for each type of target. Two-way repeated measures, analysis of variance was utilized to compare the total PQM scores and dosimetric variables, stratified by treatment planning method. Results Total PQM score of plans for FiF, IMRT, and VMAT revealed that Tangent and ESTRO delineations were equivalent regardless of planning technique (Tangent vs. ESTRO for FiF, p = 0.099; Tangent vs. ESTRO for IMRT, p = 0.029; Tangent vs. ESTRO for VMAT, p = 0.438). Both delineation protocols were significantly superior to RTOG for all treatment planning techniques. Conclusion For all treatment planning techniques, ESTRO and Tangent delineation were equivalent and both achieved significantly higher scores than RTOG delineation.

TO THE EDITOR: Successful engraftment of hematopoietic stem cells (HSCs) involves overcoming nonimmunologic barriers that hinder access to the HSC niches in the bone marrow (BM).[1][1],[2][2] Cytotoxic chemotherapy and/or radiation are currently used to reduce these barriers in clinical

Patient selection for critical care admission must balance patient safety with optimal resource allocation. This study aimed to determine the relationship between critical care admission, and postoperative mortality after abdominal surgery.This prespecified secondary analysis of a multicentre, prospective, observational study included consecutive patients enrolled in the DISCOVER study from UK and Republic of Ireland undergoing major gastrointestinal and liver surgery between October and December 2014. The primary outcome was 30-day mortality. Multivariate logistic regression was used to explore associations between critical care admission (planned and unplanned) and mortality, and inter-centre variation in critical care admission after emergency laparotomy.Of 4529 patients included, 37.8% (n=1713) underwent planned critical care admissions from theatre. Some 3.1% (n=86/2816) admitted to ward-level care subsequently underwent unplanned critical care admission. Overall 30-day mortality was 2.9% (n=133/4519), and the risk-adjusted association between 30-day mortality and critical care admission was higher in unplanned [odds ratio (OR): 8.65, 95% confidence interval (CI): 3.51-19.97) than planned admissions (OR: 2.32, 95% CI: 1.43-3.85). Some 26.7% of patients (n=1210/4529) underwent emergency laparotomies. After adjustment, 49.3% (95% CI: 46.8-51.9%, P0.001) were predicted to have planned critical care admissions, with 7% (n=10/145) of centres outside the 95% CI.After risk adjustment, no 30-day survival benefit was identified for either planned or unplanned postoperative admissions to critical care within this cohort. This likely represents appropriate admission of the highest-risk patients. Planned admissions in selected, intermediate-risk patients may present a strategy to mitigate the risk of unplanned admission. Substantial inter-centre variation exists in planned critical care admissions after emergency laparotomies.

Objective: This study compares planning techniques stratified by consensus delineation guidelines in patients undergoing whole-breast radiotherapy based on an objective plan quality assessment scale. Methods: 10 patients with left-sided breast cancer were randomly selected, and target delineation for intact breast was performed using Tangent (RTOG 0413), ESTRO, and RTOG guidelines. Consensus Plan Quality Metric (PQM) scoring was defined and communicated to the physicist before commencing treatment planning. Field-in-field IMRT (FiF), inverse IMRT (IMRT) and volumetric modulated arc therapy (VMAT) plans were created for each delineation. Statistical analyses utilised a two-way repeated measures analysis of variance, after applying a Bonferroni correction. Results: Total PQM score of plans for Tangent and ESTRO were comparable for FiF and IMRT techniques (FiF vs IMRT for Tangent, p = 0.637; FiF vs IMRT for ESTRO, p = 0.304), and were also significantly higher compared to VMAT. Total PQM score of plans for RTOG revealed that IMRT planning achieved a significantly higher score compared to both FiF and VMAT (IMRT vs FiF, p < 0.001; IMRT vs VMAT, p < 0.001). Conclusions: Total PQM scores were equivalent for FiF and IMRT for both Tangent and ESTRO delineations, whereas IMRT was best suited for RTOG delineation. Advances in knowledge: FiF and IMRT planning techniques are best suited for ESTRO or Tangent delineations. IMRT also yields better results with RTOG delineation.

ObjectivesTo analyse the survival of patients with malignant esophagorespiratory fistulas (ERF) and perform a comparative effectiveness analysis of ERF-directed interventions.MethodsFifty-five patients met our inclusion criteria, and data on ERF-directed interventions (stent placement, surgical repair and best supportive care) and their outcomes, along with clinical and treatment details, were recorded. The primary endpoints were overall survival (OS) and survival after developing ERF (F-OS).ResultsThe median OS and F-OS for the entire cohort was 299 days (SE=23.2) and 123 days (SE=11.63), respectively. On univariable analysis, the American Joint Committee on Cancer (AJCC) stage (I–III vs IV) influenced both OS (403 vs 171 days; p=0.006) and F-OS (129 vs 67 days; p=0.034). Proximal location of ERF influenced OS favourably (494 vs 285 days; p=0.021), whereas patients developing ERF late in their disease course experienced inferior F-OS (96 vs 232 days; p=0.03). On multivariable analysis, the AJCC stage (IV vs I–III, HR=3.03 (1.41–6.50)), time to developing ERF from diagnosis (greater than vs within 3 months, HR=5.82 (1.84–18.36)) and location of ERF (distal vs proximal, HR=2.47 (1.14–5.34)) had a significant impact on F-OS. The comparative efficacy (OS and F-OS) of best supportive care was statistically equivalent to any intervention irrespective of AJCC stage and success/failure of initial or subsequent intervention(s).ConclusionsThe survival of patients with ERF is dismal, and our analysis suggests that best supportive care results in equivalent OS and F-OS when compared with any intervention.

SummaryHematopoietic stem cell (HSC) transplantation can replace diseased blood systems with a healthy one, thereby treating or curing genetic blood and immune disorders including autoimmune diseases and immunodeficiencies. However, toxic chemotherapy or radiation is necessary to ablate an animal’s existing blood system prior to HSC transplantation, leading to significant morbidity. To accomplish safer blood-system replacement we developed a combination of six monoclonal antibodies to safely and specifically deplete the HSCs, T cells and NK cells of immune-competent mice. Remarkably, immunologically-foreign (allogeneic) HSCs mismatched at half or all the MHC genes could engraft these antibody-treated mice, generating donor blood systems that stably co-existed with host blood cells. These chimeric immune systems were immunologically tolerant to heart tissue from the HSC donor, providing a safe platform for HSC transplantation as a means to solid organ transplantation. The ability to transplant MHC-mismatched HSCs without chemotherapy or radiation has significant ramifications for regenerative medicine.

Engineering cytokine-receptor pairs Interleukin-2 (IL-2) is an important cytokine that helps T cells destroy tumors and virus-infected cells. IL-2 has great therapeutic promise but is limited by toxic side effects and its capacity to both activate and repress immune responses. Sockolosky et al. set out to improve IL-2–based immunotherapy by engineering synthetic IL-2–receptor pairs (i.e., IL-2 and its receptor, IL-2R) (see the Perspective by Mackall). Engineered complexes transmitted IL-2 signals but only interacted with each other and not with endogenous IL-2/IL-2R. Treatment of mice with IL-2 improved the ability of engineered T cells to reject tumors with no obvious side effects. This type of approach may provide a way to mitigate toxicities associated with some cytokine-based immunotherapies. Science , this issue p. 1037 ; see also p. 990

International audience; Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Summary Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient’s existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, T cells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.

Summary The placenta provides the interface for gas and nutrient exchange between the mother and the fetus. Despite its critical function in sustaining pregnancy, the stem/progenitor cell hierarchy and molecular mechanisms responsible for the development of the placental exchange interface are poorly understood. We identified an Epcam hi labyrinth trophoblast progenitor (LaTP) in mouse placenta that at a clonal level generates all labyrinth trophoblast subtypes, syncytiotrophoblasts I and II, and sinusoidal trophoblast giant cells. Moreover, we discovered that hepatocyte growth factor/c-Met signaling is required for sustaining proliferation of LaTP during midgestation. Loss of trophoblast c-Met also disrupted terminal differentiation and polarization of syncytiotrophoblasts, leading to intrauterine fetal growth restriction, fetal liver hypocellularity, and demise. Identification of this c-Met-dependent multipotent LaTP provides a landmark in the poorly defined placental stem/progenitor cell hierarchy and may help us understand pregnancy complications caused by a defective placental exchange.

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

Hematopoietic stem cell (HSC) transplantation can cure diverse diseases of the blood system, including hematologic malignancies, anemias, and autoimmune disorders. However, patients must undergo toxic conditioning regimens that use chemotherapy and/or radiation to eliminate host HSCs and enable donor HSC engraftment. Previous studies have shown that anti-c-Kit monoclonal antibodies deplete HSCs from bone marrow niches, allowing donor HSC engraftment in immunodeficient mice. We show that host HSC clearance is dependent on Fc-mediated antibody effector functions, and enhancing effector activity through blockade of CD47, a myeloid-specific immune checkpoint, extends anti-c-Kit conditioning to fully immunocompetent mice. The combined treatment leads to elimination of >99% of host HSCs and robust multilineage blood reconstitution after HSC transplantation. This targeted conditioning regimen that uses only biologic agents has the potential to transform the practice of HSC transplantation and enable its use in a wider spectrum of patients.

Maturation of hematopoietic stem cells (HSCs) from fetal to adult state and differentiation to progenitors are thought to follow a one-way street. In this issue of Genes & Development, He and colleagues (pp. 1613–1627) show that overexpression of Sox17 can convert adult multipotential progenitors to self-renewing HSCs that possess fetal properties. These findings challenge the irreversibility of hematopoietic development, and open up new perspectives to understand the different forms of HSC self-renewal at distinct stages of ontogeny and during transformation.

The placenta is a highly vascularized organ that mediates fetal-maternal exchange during pregnancy and is thereby vital for the survival and growth of the developing embryo. In addition to having this well-established role in supporting pregnancy, the placenta was recently shown to function as a hematopoietic organ. The placenta is unique among other fetal hematopoietic organs, as it is capable of both generating multipotential hematopoietic cells de novo and establishing a major hematopoietic stem cell (HSC) pool in the conceptus, while protecting HSCs from premature differentiation. The mouse placenta contains two distinct vascular regions that support hematopoiesis: the large vessels in the chorionic plate where HSCs/progenitors are thought to emerge and the labyrinth vasculature where nascent HSCs/progenitors may colonize for expansion and possible functional maturation. Defining how this cytokine- and growth factor rich organ supports HSC generation, maturation and expansion may ultimately help to establish culture protocols for HSC expansion or de novo generation from pluripotent cells.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

SummaryThe placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.Video Abstract

Bone marrow transplantation (BMT) is an efficacious therapy for many otherwise incurable hematologic malignancies and disorders that affect production of hematopoietic cells, including life-threatening anemias, and immunodeficiency syndromes. In a successful transplantation, clearance of bone-marrow niches must be achieved for donor hematopoietic stem cell (HSC) to engraft. Current methods to clear niche space rely on radiation and/or chemotherapy, which can impart toxic adverse effects that greatly limit the potential clinical utility of BMT. Thus, there is a major clinical need for safer conditioning regimens. To this end, a monoclonal antibody against CD117 (ACK2) has been shown to transiently deplete HSCs in immunocompromised mice and enable donor cell engraftment. However, this regimen has proved ineffective in immunocompetent animals. We hypothesized that HSC-depletion by this antibody may occur, in part, through Fc-mediated effector functions, such as antibody-dependent cellular phagocytosis (ADCP). We thus sought to enhance the ADCP activity of anti-CD117 antibodies by blocking the inhibitory CD47-SIRPα “don’t eat me” pathway. Consistent with previous reports, we found treatment of wild-type mice (C57BL/6) with ACK2 alone resulted in only modest and transient depletion of hematopoietic stem and progenitor cells (HSPCs). Strikingly, the addition of the CD47-antagonist CV1 (Weiskopf & Ring, 2013) in combination with ACK2 resulted in rapid and prolonged depletion of bone marrow HSPCs as well as clearance of the bone marrow niche. This depletion was accompanied by profound reductions in hematocrit and blood leukocyte counts not observed in mice treated with ACK2 alone. In support of the role of Fc receptors in the treatment regimen, depletion of HSCs was not observed in mice lacking functional Fc receptors, nor in animals treated with high-doses F(ab) fragments of ACK2. Finally, high levels of granulocyte chimerism (~70%) were achieved post-BMT in mice receiving the combination of ACK2 and CV1 as compared to no chimerism in animals treated with ACK2 alone (p-value Disclosures Ring: Stanford University: PCT/US2013/021937 Patents & Royalties. Weiskopf:Stanford University: PCT/US2013/021937 Patents & Royalties. Weissman:Stanford University: PCT/US2013/021937 Patents & Royalties.

Abstract 1559 The goals of hematopoiesis during embryogenesis are two-fold, to rapidly produce red blood cells to support the survival and development of the embryo, and to establish a pool of undifferentiated hematopoietic stem cells (HSC) for postnatal life. These goals are achieved by segregation of fetal hematopoiesis in multiple waves that are executed in distinct anatomical sites and microenvironmental niches. However, the microenvironmental cues that promote “stemness” vs. differentiation remain poorly understood. The placenta is a recently discovered hematopoietic organ that supports HSC generation and expansion without promoting their differentiation. The placental HSC pool is thought to reside in the placental labyrinth, which is comprised of an intricate vascular network surrounded by trophoblasts. So far, it has been unknown how a disruption of the integrity of the placental vascular labyrinth affects fetal hematopoiesis. The structure of the placental labyrinth is compromised in embryos that lack components of PDGF-B signaling. PDGF-B-/- and PDGF-Rβ-/- (receptor for PDGF-B) embryos display dilation of fetal blood vessels and reduction of trophoblast cells in the placental labyrinth. Our studies revealed that loss of PDGF-B signaling alters the unique placental hematopoietic microenvironment, resulting in active erythropoiesis in the placenta. The unexpected erythropoiesis in the placenta exhibited the same hallmarks of normal definitive erythropoiesis observed in the fetal liver as confirmed by flow cytometry for erythroid markers, morphological analysis and association of erythroblasts with macrophages. Interestingly, deletion of PDGF-Rβ in hematopoietic cells by using a Tie2-Cre strain did not induce the differentiation of placental hematopoietic progenitors into erythroid cells, implying that the ectopic definitive erythropoiesis results from the lack of PDGF-B signaling in the placental microenvironment rather than in the hematopoietic cells themselves. Our studies revealed that the erythroid differentiation in placentas of PDGF-B-/- embryos was induced by marked upregulation of Epo in placental trophoblasts. Strikingly, lentiviral overexpression of Epo specifically in the placental trophoblasts was sufficient to convert the placenta into an ectopic erythropoietic organ. These data reveal a critical function of PDGF-B signaling in protecting the integrity of the placental microenvironment that is required for preventing hematopoietic progenitors from differentiation during their residence in the placenta. Furthermore, these studies highlight the placenta as a versatile hematopoietic organ that supports HSC development during normal pregnancy but can be recruited as a site for definitive erythropoiesis during pathological conditions. Disclosures: No relevant conflicts of interest to declare.

Abstract 698 Hematopoietic stem cells (HSCs) are required for continuous blood cell production throughout life. HSCs emerge only within a short developmental time window during embryogenesis. Mounting evidence posits that HSCs arise directly from hemogenic endothelial cells during midgestation within the large arteries of the conceptus, which include the dorsal aorta, the umbilical and vitelline arteries and the chorioallantoic vessels of the placenta. However, the microenvironmental signals that mediate this temporally regulated process remain unclear. Here we examine, by using Ncx1−/− embryos that lack heartbeat and circulation, how blood flow imparts instructive cues that ensure proper HSC development. Immunostaining revealed that CD41+ hematopoietic cells, although present, were markedly decreased in Ncx1-/-placentas as compared to wild-type controls. Furthermore, mutant placentas evidenced large clusters of round CD31+ cells protruding into the lumens of the chorioallantoic vessels. Based on these data, we hypothesized that lack of blood flow may impede the generation of hematopoietic stem and progenitor cells (HS/PCs) and that the endothelial clusters represent hemogenic intermediates. FACS analysis and colony forming assays confirmed a dramatic reduction in the number of clonogenic progenitors in the placenta and the embryo proper of Ncx mutants, while the yolk sac was unaffected. However, HS/PC generation in the placenta and embryo could be rescued by culturing explants on OP9 stroma before plating in colony forming assays, verifying intact hematopoietic potential. To determine if the rescue observed was due to expansion of existing progenitors or generation of new HS/PCs, we sorted CD41medckit+hematopoietic progenitors and CD31+CD41− endothelial cells from hematopoietic tissues and co-cultured them on stroma. These experiments demonstrated that endothelial cells from placenta, embryo proper and yolk sac can generate HS/PCs following stroma stimulation, confirming the presence of hemogenic endothelium in these organs. Immunostaining of Ncx−/− placentas revealed that although the development of the arterio-venous vascular network was impaired, Notch1 signaling, required for both arterial specification and HSC development, was robust in cells of the endothelial clusters. Furthermore, positive staining for Runx1 and c-myb indicated that cells in the clusters had activated the hematopoietic program. Interestingly, electron microscopy demonstrated that cells in the clusters were tethered to each other via adherens junctions, a characteristic of endothelial cells. In addition, they also maintained high levels of Flk1, expressed VEGF and were actively proliferating, consistent with exposure to extended hypoxia. These data suggest that although cells in the clusters have initiated hematopoietic commitment, they are unable to down-regulate their endothelial identity and complete hematopoietic emergence, resulting in the formation of clusters of hemogenic intermediates. These results imply that cues imparted via circulation are required to complete the commitment to a hematopoietic fate from hemogenic endothelium. Data from co-culture experiments suggest that prolonged Notch1 signaling impairs hematopoietic emergence from hemogenic endothelial cells, and may account for the HSC emergence defect in the absence of blood flow. Overall, these data suggest that blood flow and circulating primitive red blood cells are critical components of the dynamic microenvironment necessary to both relieve the hypoxia required for the specification and proliferation of hemogenic endothelium and provide important mechanical and/or molecular signals required by HSCs to fully commit to the hematopoietic fate and complete emergence. Disclosures: No relevant conflicts of interest to declare.

The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education [1,2]. In functional genomics, we have found an ideal platform to simultaneously benefit students and contribute to scientific discovery. The sequencing of eukaryotic genomes has facilitated the identification of complete sets of genes in humans and model genetic organisms. This has allowed many forms of high-throughput analyses of transcriptional profiles, protein interactions, structural motifs, and even genome-wide knock-downs in cell lines or in selected organisms. However, one of the best tools to provide functional information about gene action— obtaining in vivo evidence about the phenotype resulting from heritable loss of function—is difficult and less amenable to high-throughput research. We were able to achieve a large-scale in vivo analysis with a significant number of undergraduate students at UCLA, called the UCLA Undergraduate Consortium for Functional Genomics. This work, a practical manifestation of policy positions proposing discoverybased education, is described in summary form here (and in Box 1) and in detail online at http://www.bruinfly.ucla.edu. This effort combines professional-quality research with a strategy for research-based undergraduate education.