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Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call ‘Organ-On-VascularNet’. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting., The transient reactivation of ETV2 in adult human endothelial cells reprograms these cells to become adaptable vasculogenic endothelia that in three-dimensional matrices self-assemble into vascular networks that can transport blood and physiologically arborize organoids and decellularized tissues.

Background Although interest in frailty has expanded among cardiology experts over the past 2 decades, its integration, as part of cardiovascular disease management, is still lacking, above all in the acute cardiac care setting. The Clinical Frailty Scale (CFS) is a brief guided tool to assess frailty in hospital settings without specialist equipment. Purpose Our objective was to test the performance of the CFS in an older, unselected population, admitted to an Intensive Cardiac Care Unit (ICCU) during the year 2019. Methods The study sample included 431 patients ≥65 years old, admitted to an ICCU of a tertiary cardiac center in Italy. The CFS ranged from “very fit: 1” to “terminally ill: 9”, but it was considered present at a score ≥5. Our primary endpoint was defined by a combination of severe complications requiring critical care and in-hospital death. The data were collected from the hospital discharge summary and the electronic chart records. Results 158 patients (36.7%) were frail. These individuals had greater comorbidity and higher in-hospital mortality (Table 1). After a multivariable logistic regression analysis, 4 predictors were identified: signs of congestive heart failure (OR: 8.51, 95% Confidence Interval-CI: 4.63–14.6; p Conclusions The CFS is a simple guided frailty tool that may enhance outcome prediction in the acute cardiac care setting. These findings merit evaluation in larger cohorts of unselected patients. Funding Acknowledgement Type of funding sources: None.

In utero transplantation (IUT) of hematopoietic stem cells (HSC) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are sub-therapeutic, likely due to host fetal HSC out-competing their bone-marrow (BM) derived donor equivalents for space in the hematopoietic compartment. In the present study we demonstrate that amniotic fluid stem cells (AFSC; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favourable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly-isolated or cultured AFSC resulted in stable mutli-lineage hematopoietic engraftment, far higher to that achieved with BM-HSC. Intravascular IUT of allogenic AFSC was not successful as recently reported after intraperitoneal IUT. Herein we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSC in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene engineered and in-vitro expanded AFSC could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. SIGNIFICANCE STATEMENT: Amniotic fluid stem cells can be expanded without losing their hematopoietic characteristics In utero transplantation of allogenic AFSC results in adaptive immune response and donor cell rejection, due to failure of timely homing of donor cells to the host fetal thymus and lack of central tolerance induction. Autologous/congenic amniotic fluid stem cells can be transplanted in utero and result in stable, multi-lineage, long-term hematopoietic engraftment that is significantly higher to that achieved with bone marrow-derived cells and could be therapeutic in many inherited disorders of hematopoiesis. © AlphaMed Press 2019.

Background The relevance of laparoscopic resection of intrahepatic cholangiocarcinoma (ICC) remains debated. The aim of this study was to compare laparoscopic (LLR) and open (OLR) liver resection for ICC, with specific focus on textbook outcome and lymph node dissection (LND). Methods Patients undergoing LLR or OLR for ICC were included from two French, nationwide hepatopancreatobiliary surveys undertaken between 2000 and 2017. Patients with negative margins, and without transfusion, severe complications, prolonged hospital stay, readmission or death were considered to have a textbook outcome. Patients who achieved both a textbook outcome and LND were deemed to have an adjusted textbook outcome. OLR and LLR were compared after propensity score matching. Results In total, 548 patients with ICC (127 LLR, 421 OLR) were included. Textbook-outcome and LND completion rates were 22.1 and 48.2 per cent respectively. LLR was independently associated with a decreased rate of LND (odds ratio 0.37, 95 per cent c.i. 0.20 to 0.69). After matching, 109 patients remained in each group. LLR was associated with a decreased rate of transfusion (7.3 versus 21.1 per cent; P = 0.001) and shorter hospital stay (median 7 versus 14 days; P = 0.001), but lower rate of LND (33.9 versus 73.4 per cent; P = 0.001). Patients who underwent LLR had lower rate of adjusted TO completion than patients who had OLR (6.5 versus 17.4 per cent; P = 0.012). Conclusion The laparoscopic approach did not substantially improve quality of care of patients with resectable ICC.

Background & Aims Herein, we aimed to establish benchmark values – based on a composite indicator of healthcare quality – for the performance of laparoscopic left lateral sectionectomy (LLLS) and laparoscopic right hepatectomy (LRH) using data from expert centers. Methods Data from a nationwide multicenter survey including all patients undergoing LLLS and LRH between 2000 and 2017 were analyzed. Textbook outcome (TO) completion was considered in patients fulfilling all 6 of the following characteristics: negative margins, no transfusion, no complication, no prolonged hospital stay, no readmission and no mortality. For each procedure, a cut-off laparoscopic liver resection (LLR) volume by center was associated with TO on multivariable analysis. These cut-offs defined the expert centers. The benchmark values were set at the 75th percentile of median outcomes among these expert centers. Results Among 4,400 LLRs performed in 29 centers, 855 patients who underwent LLLS and 488 who underwent LRH were identified. The overall incidences of TO after LLLS and LRH were 43.7% and 23.8%, respectively. LLR volume cut-offs of 25 LLR/year (odds ratio [OR] 2.45; bootstrap 95% CI 1.65–3.69; p = 0.001) and 35 LLR/year (OR 2.55; bootstrap 95% CI 1.34–5.63; p = 0.003) were independently associated with completion of TO after LLLS and LRH, respectively. Eight centers for LLLS and 6 centers for LRH, including 516 and 346 patients undergoing LLLS/LRH respectively, reached these cut-offs and were identified as expert centers. After LLLS, benchmark values of severe complication, mortality and TO completion were defined as ≤5.3%, ≤1.2% and ≥46.6%, respectively. After LRH, benchmark values of severe complication, mortality and TO completion were ≤20.4%, ≤2.8% and ≥24.2%, respectively. Conclusions This study provides an up-to-date reference on the benchmark performance of LLLS and LRH in expert centers. Lay summary In a nationwide French survey of laparoscopic liver resection, expert centers were defined according to the completion of a textbook outcome, which is a composite indicator of healthcare quality. Benchmark values regarding intra-operative details and outcomes were established using data from 516 patients with laparoscopic left lateral sectionectiomy and 346 patients with laparoscopic right hepatectomy from expert centers. These values should be used as a reference point to improve the quality of laparoscopic resections.

The aim was to analyse the impact of cirrhosis on short-term outcomes after laparoscopic liver resection (LLR) in a multicentre national cohort study.This retrospective study included all patients undergoing LLR in 27 centres between 2000 and 2017. Cirrhosis was defined as F4 fibrosis on pathological examination. Short-term outcomes of patients with and without liver cirrhosis were compared after propensity score matching by centre volume, demographic and tumour characteristics, and extent of resection.Among 3150 patients included, LLR was performed in 774 patients with (24·6 per cent) and 2376 (75·4 per cent) without cirrhosis. Severe complication and mortality rates in patients with cirrhosis were 10·6 and 2·6 per cent respectively. Posthepatectomy liver failure (PHLF) developed in 3·6 per cent of patients with cirrhosis and was the major cause of death (11 of 20 patients). After matching, patients with cirrhosis tended to have higher rates of severe complications (odds ratio (OR) 1·74, 95 per cent c.i. 0·92 to 3·41; P = 0·096) and PHLF (OR 7·13, 0·91 to 323·10; P = 0·068) than those without cirrhosis. They also had a higher risk of death (OR 5·13, 1·08 to 48·61; P = 0·039). Rates of cardiorespiratory complications (P = 0·338), bile leakage (P = 0·286) and reoperation (P = 0·352) were similar in the two groups. Patients with cirrhosis had a longer hospital stay than those without (11 versus 8 days; P = 0·018). Centre expertise was an independent protective factor against PHLF in patients with cirrhosis (OR 0·33, 0·14 to 0·76; P = 0·010).Underlying cirrhosis remains an independent risk factor for impaired outcomes in patients undergoing LLR, even in expert centres.El objetivo de este estudio fue analizar el impacto de la cirrosis en los resultados a corto plazo después de la resección hepática laparoscópica (laparoscopic liver resection, LLR) en un estudio de cohortes multicéntrico nacional. MÉTODOS: Este estudio retrospectivo incluyó todos los pacientes sometidos a LLR en 27 centros entre 2000 y 2017. La cirrosis se definió como fibrosis F4 en el examen histopatológico. Los resultados a corto plazo de los pacientes con hígado cirrótico (cirrhotic liver CL) (pacientes CL) y los pacientes con hígado no cirrótico (non-cirrhotic liver, NCL) (pacientes NCL) se compararon después de realizar un emparejamiento por puntaje de propension del volumen del centro, las características demográficas y del tumor, y la extensión de la resección.Del total de 3.150 pacientes incluidos, se realizó LLR en 774 (24,6%) pacientes CL y en 2.376 (75,4%) pacientes NCL. Las tasas de complicaciones graves y mortalidad en el grupo de pacientes CL fueron del 10,6% y 2,6%, respectivamente. La insuficiencia hepática posterior a la hepatectomía (post-hepatectomy liver failure, PHLF) fue la principal causa de mortalidad (55% de los casos) y se produjo en el 3,6% de los casos en pacientes CL. Después del emparejamiento, los pacientes CL tendieron a tener tasas más altas de complicaciones graves (razón de oportunidades, odds ratio, OR 1,74; i.c. del 95% 0,92-0,41; P = 0,096) y de PHLF (OR 7,13; i.c. del 95% 0,91-323,10; P = 0,068) en comparación con los pacientes NCL. Los pacientes CL estuvieron expuestos a un mayor riesgo de mortalidad (OR 5,13; i.c. del 95% 1,08-48,6; P = 0,039) en comparación con los pacientes NCL. Los pacientes CL presentaron tasas similares de complicaciones cardiorrespiratorias graves (P = 0,338), de fuga biliar (P = 0,286) y de reintervenciones (P = 0,352) que los pacientes NCL. Los pacientes CL tuvieron una estancia hospitalaria más larga (11 versus 8 días; P = 0,018) que los pacientes NCL. La experiencia del centro fue un factor protector independiente de PHLF (OR 0,33; i.c. del 95% 0,14-0,76; P = 0,010) pacientes CL. CONCLUSIÓN: La presencia de cirrosis subyacente sigue siendo un factor de riesgo independiente de peores resultados en pacientes sometidos a resección hepática laparoscópica, incluso en centros con experiencia.

Cancer cells require increased levels of NADPH for increased nucleotide synthesis and for protection from ROS. Recent studies show that increased NADPH is generated in several ways. Activated AKT phosphorylates NAD kinase (NADK), increasing its activity. NADP formed, is rapidly converted to NADPH by glucose 6-phosphate dehydrogenase and malic enzymes, overexpressed in tumor cells with mutant p53. Calmodulin, overexpressed in some cancers, also increases NADK activity. Also, in IDH1/2 mutant cancer, NADPH serves as the cofactor to generate D-2 hydroxyglutarate, an oncometabolite. The requirement of cancer cells for elevated levels of NADPH provides an opportunity to target its synthesis for cancer treatment.

Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), has debilitating long-term effects on infants and children with this condition. Priority of care is to increase the child’s length of functional intestine, jejunum in particular, to improve nutritional independence. Here we report a robust protocol for reconstruction of autologous intestinal mucosal grafts using primary IF patient materials. Human jejunal intestinal organoids derived from paediatric IF patients can be expanded efficiently in vitro with region-specific markers preserved after long-term culture. Decellularized human intestinal matrix with intact ultrastructure is used as biological scaffolds. Proteomic and Raman spectroscopic analyses reveal highly analogous biochemical composition of decellularized human SI and colon matrix, implying that they can both be utilised as scaffolds for jejunal graft reconstruction. Indeed, seeding of primary human jejunal organoids to either SI or colonic scaffolds in vitro can efficiently reconstruct functional jejunal grafts with persistent disaccharidase activity as early as 4 days after seeding, which can further survive and mature after transplantation in vivo. Our findings pave the way towards regenerative medicine for IF patients.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

Liver transplantation remains the treatment of choice for patients with end-stage liver disease. However, allograft availability continues to be a problem, and extending the criteria for organ acceptance is key. Deceased donors after electrical accidents, as well as electricity-traumatized allografts, are not common but should be considered suitable. This study describes 2 cases of heart-beating organ donors with electrical injury to the liver. In 1 case, the electric shock was the cause of death; in the second case, the injury was caused by defibrillation at organ procurement. Both allografts had sustained sizeable electrical injury, and both resulted in excellent early posttransplant outcomes. These cases demonstrate that electrocution is not a contraindication to donation and that electricity-traumatized allografts may remain transplantable after careful assessment. Education of all staff in the management of such donors can optimize utility of such allografts.

Purpose To investigate the effectiveness of a combination of 6-thioguanine (6-TG) and pralatrexate (PDX) in methylthioadenosine phosphorylase (MTAP)-deficient T-cell acute lymphoblastic leukemia (T-cell ALL). Methods CCRF-CEM (MTAP−/−) and Molt4 (MTAP+/+) T-cell ALL cell lines were treated with 6-TG or PDX and evaluated for efficacy 72 h later. NOD/SCID gamma mice bearing CEM or Molt4 xenografts were treated with 6-TG and PDX alone or in combination to evaluate antitumor effects. Results CEM cells were more sensitive to 6-TG and PDX in vitro than Molt4. In vivo, CEM cells were very sensitive to PDX and 6-TG, whereas Molt4 cells were highly resistant to 6-TG. A well-tolerated combination of PDX and 6-TG achieved significant tumor regression in CEM xenografts. Conclusions The loss of MTAP expression may be therapeutically exploited in T-cell ALL. The combination of 6-TG and PDX, with the inclusion of leucovorin rescue, allows for a safe and effective regimen in MTAP-deficient T-cell ALL.

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets., Next generation sequencing allows the identification of oncogenic driver genes in pancreatic cancer. Here, in an effort to identify additional causal genes, the authors develop a high throughput in vivo screen and identify genes that whilst infrequently mutated in pancreatic cancer contribute to tumour formation.

The purpose of the present work was to investigate the in vivo concentrations of sorbic acid and vanillin as markers of the fate of organic acids (OA) and natural identical flavors (NIF) from a microencapsulated mixture and from the same mixture non-microencapsulated, and the possible consequences on the intestinal microbial fermentation. Fifteen weaned pigs were selected from 3 dietary groups and were slaughtered at 29.5 +/- 0.27 kg of BW. Diets were (1) control; (2) control supplemented with a blend of OA and NIF microencapsulated with hydrogenated vegetable lipids (protected blend, PB); and (3) control supplemented with the same blend of OA and NIF mixed with the same protective matrix in powdered form but without the active ingredient coating (non-protected blend, NPB). Stomach, cranial jejunum, caudal jejunum, ileum, cecum, and colon were sampled to determine the concentrations of sorbic acid and vanillin contained in the blend and used as tracers. Sorbic acid and vanillin were not detectable in pigs fed the control, and their concentrations were not different in the stomach of PB and NPB treatments. Pigs fed PB showed a gradual decrease of the tracer concentrations along the intestinal tract, whereas pigs fed NPB showed a decline of tracer concentration in the cranial jejunum and onwards, compared with the stomach concentrations. Sorbic acid and vanillin concentrations along the intestinal tract were greater (P = 0.02) in pigs fed PB compared with pigs fed NPB. Pigs fed PB had lower (P = 0.03) coliforms in the caudal jejunum and the cecum than pigs fed the control or NPB. Pigs fed the control or PB had a greater (P = 0.03) lactic acid bacteria plate count in the cecum than pigs fed NPB, which showed a reduction (P = 0.02) of lactic acid concentrations and greater (P = 0.02) pH values in the caudal jejunum. The protective lipid matrix used for microencapsulation of the OA and NIF blend allowed slow-release of both active ingredients and prevented the immediate disappearance of such compounds upon exiting the stomach.

Summary Background Effects of frequent nocturnal symptoms of gastro-oesophageal reflux disease (GERD-FNS) on health-related quality of life (HRQOL) and work productivity are not well documented. Aim To assess symptom severity, production loss, and HRQOL among employed adults with and without GERD-FNS. Methods Using several validated outcome measures in a web survey design, GERD was pre-specified as GERD Symptom and Medication Questionnaire score >9, and ≥1 episode of heartburn or acid regurgitation during the preceding week. GERD-FNS patients were those reporting ≥2 symptom-nights during the previous week; their outcomes were compared with those of patients having minimal or no nocturnal symptoms (GERD-NNS) and vs. non-GERD controls. Results Data were collected from 1002 GERD patients (476 GERD-FNS, 526 GERD-NNS) and 513 controls. Severe symptoms were more common, sleep abnormalities were more frequent (P

Introduction Postoperative delirium can be a serious consequence of major surgery, associated with longer hospital stays, readmission, cognitive and functional deterioration and mortality. Delirium is an acute, reversible disorder characterised by fluctuating course, inattention, disorganised thinking and altered level of consciousness. Delirium occurring in the hours immediately following anaesthesia and delirium occurring in the postoperative period of 1–5 days have been described as distinct clinical entities. This protocol describes an observational study with the aim of determining if delirium in the first hour following tracheal tube removal is a predictor of delirium in the 5 subsequent postoperative days. Improved understanding regarding the development of postoperative delirium would improve patient care and allow more effective implementation of delirium prevention measures. Methods and analysis Patients enrolled to the Electroencephalography Guidance of Anesthesia to Alleviate Geriatric Syndromes (ENGAGES) randomised controlled trial will be eligible for this substudy. A validated delirium assessment method, the 3-min Diagnostic Confusion Assessment Method and the Richmond Agitation and Sedation Scale will be used to assess 100 patients for delirium at 30 min and 60 min following tracheal tube removal. Patients will also be assessed for delirium over postoperative days 1–5 using three validated methods, the Confusion Assessment Method (CAM), CAM for the Intensive Care Unit and structured chart review. Logistic regression analysis will then be performed to test whether immediately postoperative delirium independently predicts subsequent postoperative delirium. Ethics and dissemination This observational substudy of ENGAGES has been approved by the ethics board of Washington University School of Medicine. Enrolment began in June 2016 and will continue until June 2017. Dissemination plans include presentations at scientific conferences and scientific publications. Trial registration number NCT02241655.

NAD(+) kinase (NADK) is the only known cytosolic enzyme that converts NAD(+) to NADP(+), which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.

We report an unusual case of an isolated, large, thrombosed hypogastric artery aneurysm presenting as a voluminous pelvic mass with bilateral hydroureteronephrosis. The patient underwent successful surgical removal of the mass. Histology confirmed an aneurysm of the left hypogastric artery.

6Boehnnger Mannheim, Italv Summary Background: Early clinical trials have suggested that glutathione (GSH) offers protection from the toxic effects of cisplatin. Patients and methods: One hundred fifty-one patients with ovarian cancer (stage I-IV) were evaluated in a clinical trial of cisplatin (CDDP) ± glutathione (GSH). The objective was to determine whether GSH would enhance the feasibility of giving six cycles of CDDP at 100 mg/m2 without dose reduction due to toxicity. Results: When considering the proportion of patients receiving six courses of CDDP at any dose, GSH produced a significant advantage over control - 58% versus 39%, (P = 0.04). For these patients there was a significant difference between the reduction in creatinine clearance for GSH treated patients compared with control - 74% versus 62% (P = 0.006). Quality of life scores demonstrated that for patients receiving GSH there was a statistically significant improvement in depression, emesis, peripheral neurotoxicity, hair loss, shortness of breath and difficulty concentrating. As an indication of overall activity, these patients were statistically significantly more able to undertake housekeeping and shopping. Clinically assessed response to treatment demonstrated a trend towards a better outcome in the GSH group (73% versus 62%) but this was not statistically significant (P = 0.25). Conclusions: The results demonstrate that adding GSH to CDDP allows more cycles of CDDP treatment to be administered because less toxicity is observed and the patient's quality of life is improved.

Introduction Human hematopoietic stem cell transplantation (HSCT) has been used to treat a range of hematological and immunological disorders. As a result, the demand for hematopoietic stem cells (HSC) in clinical applications is increasing. Amniotic fluid stem cells (AFSC) serve as a potential alternative cell source for therapy. Amniotic fluid can be derived by amniocentesis or therapeutic amniodrainage. AFSC are multi-potent, have low risk of tumorigenicity, can be expanded and do not have legal or ethical limitations. The significant hematopoietic activity of murine AFSC led us to explore the potential of human AFSC (CD117/c-Kit+) towards hematopoietic differentiation and to reconstitution in vivo. Methods Human AFSC (2nd and 3rd trimester) and cord blood HSC (CB-HSC; control) were selected for CD117 and CD34 respectively using a MoFlo XDP sorter. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NOD-SCID/IL2rγnull (NSG) mice (n=6/group). Hematopoietic engraftment of human cells (% of human CD45+ within total CD45+) and multi-lineage reconstitution (myeloid: CD13, CD14, CD15 and lymphoid: CD3, CD4 and CD8) were assessed at 16 weeks in blood, bone marrow (BM) and spleen by flow cytometry. For subsequent secondary transplants, BM mononuclear cells (MNC) derived from BM harvested from primary recipients of mice were intravenously injected into secondary recipients (1.5x107 MNC in 200μl PBS). Hematopoietic engraftment was assessed at 16 weeks post-transplantation (n=6/group). For further analysis of human donor cell engraftment, Q-PCR was performed on spleen samples harvested from primary and secondary recipients using oligonucleotide primers specific for human ALU repeat sequences; Immunohistochemistry was carried out using anti-human CD45 antibody and detected with a commercially available kit (Dako EnVision Plus, Dako). Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results Human AFSC engrafted the hematopoietic system of NSG mice at levels similar to the ones achieved with CB-HSC (blood: AFSC 7.5±1.3% vs. CB-HSC 6.1±2.2%, p=0.6; BM: AFSC 46.3±7.9% vs. CB-HSC 38.3±8.2%, p=0.6; spleen: AFSC 39.6±9.3% vs. CB-HSC 34.7±10.5%, p=0.7). Similarly, at 16 weeks following secondary transplantation, human donor cell engraftment was comparable between groups in blood (AFSC 11.5 ± 3.9% vs. CB-HSC 16.9 ± 3.9%, p=0.3) and other hematopoietic tissues. Q-PCR and immunohistochemistry confirmed donor cell engraftment in AFSC and CB-HSC groups. Importantly, there were no differences between groups in multi-lineage differentiation at 16 weeks post primary and secondary transplantation. Conclusion Human CD117/c-Kit+ AFSC have functional, multi-lineage hematopoietic potential that is similar to the current "gold-standard" stem cell source for hematopoietic transplantation. The ease of isolation during early gestation, as well as their gene-engineering and expansion potential make human AFSC a novel autologous fetal cell source for pre- and post-natal therapy of inherited hematological disorders. Disclosures No relevant conflicts of interest to declare.

Introduction Castleman's disease (CD) is a rare benign disorder that may grow in any area where lymphoid tissue is normally present but the most frequent site is mediastinum (67%); the abdomen is rarely affected by this pathology. In the pathogenesis of CD an important role could be played by HHV-8. Diagnosis is difficult and the diagnostic certainty is obtained only by histological examination. For this disease there is no gold-standard treatment. Case report We report the case of a woman of 33 years of age affected by a rare form of abdominal nodal localization of Castleman's Disease for which the patient underwent surgery. Conclusion CD must be included in the differential diagnosis of retroperitoneal tumors even though the definitive diagnosis can only be achieved by histological and immunohistochemical examination. In the case of single location, the treatment of choice is surgical removal.

Next generation sequencing (NGS) technologies are rapidly being incorporated into the clinic to facilitate decisions on cancer patient care. However, successful translation of NGS data requires knowledge on which DNA aberrations represent actionable events, either for development or re-positioning of approved agents to target their activated pathways. Recognizing this, large-scale tumor profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are cataloging genomic aberrations across major cancer lineages. These efforts have revealed an extraordinary level of genome complexity made up of not only key “driver” events critical to pathogenesis, but also numerous biologically-neutral “passengers” that accompany unstable tumor genomes. The challenge now is to find ways to identify functional driver aberrations, as targeting such events or their activated pathways has great potential for improving patient outcomes. To do this, we have developed high-throughput approaches to construct molecularly-barcoded versions of gene aberrations for functional screens. Specifically, we developed technologies that include (1) high-throughput, accurate modeling of somatic DNA mutations (somatic missense mutations and small insertions/deletions) using our robotics-driven platform of >35,000 sequenced-verified open reading frame (ORF) clones, (2) a molecular barcoding strategy that permits rapid DNA tagging of wild-type and mutant ORFs, (3) multi-fragment recombineering methodologies allowing construction of cancer fusion genes, and (4) combining the use of these reagents for individual or pooled functional screens in vitro and in vivo using human and mouse systems. We are using these technologies, which are widely applicable to all cancer types, to identify the highest priority targets to enroll in deep mechanistic studies and drug discovery programs. We have scaled our pipelines to functionalize thousands of cancer gene aberrations. Importantly, we are now constructing entire somatically-mutated exomes from individual patients sequenced in the clinic. As an example of our “Personalized Functionalization” approach, we screened individual pancreatic ductal adenocarcinoma (PDAC) patient-derived aberration libraries for mutations capable of promoting tumorigenesis in vivo using a mouse xenograft model engineered with regulatable KRASG12D, an oncogene active in the majority of PDAC patient tumors. These studies revealed potent aberration drivers that are active as individual drivers as well as those that are contextual and are only active in the presence of KRASG12D. Based on these results, we have chosen NAD Kinase (NADK) for deep mechanistic studies and drug discovery programs. NADK catalyzes the conversion of cytoplasmic NAD+ to NADP+/NADPH and thus aids other modes of cellular NADPH production. Our validation studies indicate that the NADK mutation results in robust gain-of-function kinase activity leading to its hyper-phosphorylation of NAD+ accompanied by reduced accumulation or reactive oxygen species and increased tumor formation and growth. Interestingly, recent work by others report other mechanisms by which KRAS rewires PDAC tumors to maximize energy production and promote NADPH accumulation to maintain redox state and tumor growth. Even though NADK is mutated at low frequency in PDAC, its selection demonstrates that the discovery of rare, functional aberrations may intersect or otherwise lead us to important pathways and potential therapeutic liabilities. Our ultimate goal is to functionally annotate thousands of somatic aberrations in cancer, the vast majority of which have not been previously recognized or assayed for clinical relevance. These systems will reveal high priority edited targets to enroll in deep mechanistic biology studies, drug discovery and development programs ultimately leading to personalized treatment strategies. Citation Format: Kenneth Scott, Yiu Huen Tsang, Turgut Dogruluk, Philip Tedeschi, Joseph Bertino, Gordon Mills. Functional prioritization of rare gene aberration drivers of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR06.

BACKGROUND. Prostatic small cell carcinoma is increasing in frequency in patients resistant to all forms of androgen ablation. It is a high-grade malignant neoplasm with neuroendocrine features. There are no suitable cellular models to study this particular type of carcinoma. A recent study showed that the PC-3 cell line has neuroendocrine characteristics (1). This report prompted us to determine if this cell line is enriched in tumor initiating cells, and to use this cell line as a model to determine sensitivity to second generation anti androgen treatments and chemotherapeutic agents. METHODS. To determine the percentage of tumor initiating cells (TICs) marker expression comparing LNCaP with PC-3 cells, flow cytometry analysis was used. Functional assays such as migration, invasion, colony formation and sphere formation were used to determine metastatic potential. Secondary and tertiary spheroid assays were used to determine the self-renewal ability. The CellTiter 96® AQueous non-radioactive cell proliferation assay was used to identify response to hormone deprivation and chemotherapeutic drugs. RESULTS. The growth rate of PC-3 was similar to LNCaP cells while the TIC marker CD44 was only expressed on PC-3 cells. Both LNCaP and PC-3 cells expressed α2β1 with the highest expression detected in PC-3 cells. No expression of CD133 was detected in either of the cell lines. PC-3, as compared to LNCaP cells, displayed an increase in 5-minute collagen-I attachment, cell migration, invasion, colony formation and sphere formation in an anchorage-independent environment. PC-3 cells were capable of self-renewal in the secondary and tertiary spheroid assays and expressed neuroendocrine markers such as chromogranin A and EMT marker, Twist. PC-3 cells were able to also grow in androgen-deprived media, and as compared to LNCaP cells, demonstrated resistance to anti-androgen drugs that included enzalutamide and abiraterone and chemotherapy drugs such as doxorubicin and taxotere. Surprisingly, PC-3 cells were more sensitive to methotrexate than LNCaP or DU145 cells. PC3 cells had lower levels of MTAP (methylthioadenosine phosphorylase) and hence may be the reason for their sensitivity to denovo purine synthesis inhibitors such as methotrexate. CONCLUSIONS. The novel findings presented here provide evidence that castrate resistant PC-3 cells are CD44+/ α2β1+/CD24low/CD133_ and possess stem cell properties and may serve as a useful model to study the role of new treatments for prostate cancer with neuroendocrine features. Reference: 1) Prostate. 2011 Nov; 71(15): 1668-79; PC3 is a cell line characteristic of prostatic small cell carcinoma; Tai S, Sun Y, Squires JM, Zhang H, Oh WK, Liang CZ, Huang J Citation Format: Nitu Bansal, Lisa Wu, Nadine Johnson Farley, Philip Tedeschi, Joseph R. Bertino. The castrate resistant PC-3 cell line with neuroendocrine features is enriched in tumor initiating cells and is resistant to enzalutamide and abiraterone but sensitive to antimetabolites. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4208. doi:10.1158/1538-7445.AM2015-4208

In T-cell acute lymphoblastic leukemia (ALL), the genes CDKN2A (p16 and p14) and MTAP are homozygously co-deleted, with a frequency of ∼35 %. MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine, to adenine and 5-methylthioribose-1-phosphate, which are converted to adenine nucleotides and methionine. Cells lacking MTAP are unable to salvage adenine through this pathway and rely on the de novo pathway for purine synthesis. This variation between normal MTAP+ cells and tumor MTAP-/- cells led to several studies showing that MTAP -/- cells are more sensitive to inhibitors of de novo purine synthesis. Pralatrexate (PDX) is a second generation antifolate that targets dihydrofolate reductase and via conversion to polyglutamylates, inhibits de novo purine synthesis. 6-Thioguanine (6TG), an antimetabolite analog of guanine, also target de novo purine synthesis. MTAP-deficient ALL cells should be more sensitive to these agents, remarkably so in combination. We tested single agent PDX and 6TG as well as a combination treatment in MTAP+ (Molt4) and MTAP-/- (CEM) ALL xenografts. CEM xenografts were more sensitive to PDX treatment than MOLT4 xenografts. CEM xenografts were also much more sensitive to 6TG, while Molt4 xenografts were resistant to 6TG. Early results of the combination of PDX and 6TG in vivo show promise as an effective therapy of MTAP-/- T-cell ALL. Citation Format: Philip M. Tedeschi, Yamini K. Kathari, Iqra Farooqi, Joseph R. Bertino. Methylthioadenosine phosphorylase (MTAP)-deficient T-cell ALL xenografts are sensitive to pralatrexate and 6-thioguanine alone and in combination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2014-843

Pralatrexate is a second generation antifolate that targets dihydrofolate reductase. It was found to have a higher affinity to reduced folate carrier type 1 (RFC-1), the primary folate transporter, and has high intracellular retention via enhanced polyglutamylation. Mucositis, rather than bone marrow toxicity, is the dose limiting toxicity (DLT) in patients. In this study, we examine whether the use of leucovorin (folinic acid) can reduce pralatrexate DLT when used in mouse xenografts, and possibly enable high dose pralatrexate treatment. Nude mice bearing H2052 mesothelioma xenografts were treated with pralatrexate at normal and toxic doses with or without leucovorin administration 24 hours later. Tumors were measured three times weekly and toxicity monitored through weight loss. Leucovorin was found to abrogate pralatrexate toxicities at high doses without sacrificing efficacy. These results demonstrate that high dose pralatrexate with subsequent leucovorin rescue may be a viable treatment strategy in mesothelioma and other cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A266. Citation Format: Philip M. Tedeschi, Iqra Farooqi, Yamini Kathari, Joseph R. Bertino. Leucovorin rescue of high dose pralatrexate in mesothelioma xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A266.

The bioequivalence of two megestrol acetate formulations, 160-mg "tablets" and 160-mg "sachets," was investigated in a single-dose, open-label, balanced-for-sequence cross-over study involving 12 advanced-cancer patients. The observed plasma megestrol-acetate time course obtained with both formulations was consistent with the literature data. The main source of variability in the pharmacokinetic parameters was intersubject variability; drug formulation played only a minor (and nonsignificant) role. The width of the 90% confidence interval of the area-under-the-curve (AUC) ratio (sachets: tablets) computed according to Schuirmann (0.9-1.4) was mainly due to the presence of a single outlier, showing an AUC ratio of 2.7. The trend to higher bioavailability of the new formulation was not significant, especially as compared with the dose-response data reported in the literature.

The kinetics of platinum (Pt) was studied in 12 patients suffering from non-small-cell lung cancer or pleural mesothelioma. Each subject received an infusion of cisplatin (CDDP, 80 mg/m2), and six patients were pretreated with glutathione (GSH, 2.5 g given i.v.) at 15 min prior to the cisplatin infusion. After a 3- to 4-week interval, all patients were given a second course of treatment on the same schedule. A biexponential model was fitted to plasma concentrations of total and ultrafilterable Pt. The excretion of Pt in urine was evaluated during the first 48 h after the CDDP infusion. Following the administration of CDDP alone or with GSH pretreatment, the pharmacokinetic parameters of Pt did not significantly differ between the treatments. Also, the unbound fraction determined at each sampling time did not vary significantly between the treatments. However, it is noteworthy that the mean values obtained for the terminal half0life, the volume of distribution, the renal clearance, the percentage of the dose excreted in the urine, and the mean residence time of total Pt were higher in patients who had been pretreated with GSH, suggesting that SGH might increase both the rate of Pt elimination and the extent of Pt distribution and, as a consequence of the latter, might prolong the residence time of Pt in the body. In addition, the unbound fraction of Pt from the 4th to the 48th h was higher following the first dose of CDDP+GSH than after treatment with CDDP alone. Because of the rather high variability in the values of the parameters obtained, further work is planned using a larger number of patients.

Megestrol acetate (MA) was reported to induce weight gain and increase of appetite in cancer patients. Methods Out-patients of the “Divisione Terapia del Dolore” of the “Istituto Nazionale per la Cura dei Tumori” (Milano) with advanced non hormone-responsive tumors and loss or absence of appetite, who didn’t assume any corticosteroid were randomized for a Phase III trial, that consisted of two consecutive phases: a double-blind placebo controlled phase of 14 days (phase A) and an open phase of 76 days (phase B). During phase A patients were treated with MA 2 tablets × 160 mg/day (a low dose for this indication) or placebo. In phase B all patients received different dosages of MA according to the response to treatment. A patient was considered responsive if his appetite, evaluated by means of a categoric-numeric scale, increased by 2 or more points. Other parameters investigated were: food intake, body weight, Perfonnance Status (Karnofsky), mood state (POMS) and pain. Results Forty-two patients were enrolled in this trial. Thirty-three patients were evaluable for efficacy: 13/16 MA patients were responsive for appetite vs 5/17 placebo patients. This result is clinically and statistically significant ( P Conclusions MA showed a remarkable effect on appetite with a low dose (2 tablets × 160 mg/die) already after 14 days, without side-effects of relief. This result is of great importance considering the relevance of the quality of life and the low life expectancy of these patients and the low daily therapy costs of this treatment.

Carotid angiography showed a lesion in more than two-thirds of this series of ischaemic carotid strokes; two-thirds of the lesions shown were in the neck vessels. Carotid narrowing (incomplete occlusion) in the neck was much commoner after the age of 50 years and was frequently associated wth a temporary stroke. Complete carotid occlusion in the neck did not become commoner with increasing age as did carotid narrowing; nor was it associated with previous temporary strokes (as was carotid narrowing). It is therefore doubtful whether complete occlusion is usually the end-result of narrowing. Persisting strokes were frequently preceded by one or more temporary strokes; these ‘warning strokes’ were more frequent in patients with carotid narrowing. Middle cerebral artery lesions accounted for two-thirds of all intracranial lesions; they were particularly common in association with pregnancy and rarely caused temporary strokes.

Purpose To investigate the ability of leucovorin (LV) to abrogate dose-limiting toxicities of pralatrexate (PDX) while maintaining efficacy, in vivo. Methods H2052 mesothelioma cells were treated with the antifolates methotrexate (MTX), PDX and pemetrexed, with and without LV rescue 24 h later. Cell killing was evaluated 48 h later. Female nude mice bearing H2052 xenografts were treated with varying doses and schedules of the antifolate PDX and LV. Results In vitro, H2052 cells were more sensitive to PDX as compared to MTX and pemetrexed. Administration of LV 24 h after antifolate treatment reduced efficacy of antifolates MTX and pemetrexed, but not PDX. In vivo, LV was found to reduce toxicity of PDX at the maximum tolerated dose without sacrificing efficacy. Lethal doses of PDX were rescued by LV, and mice bearing the H2052 tumor demonstrated prolonged and enhanced tumor regression. Conclusions High-dose PDX with subsequent LV rescue may be a viable treatment strategy in mesothelioma and other cancers. The inclusion of LV rescue into new and existing PDX treatment protocols should be explored as a way to expand the tolerability and effectiveness of PDX in the clinic.

Clinical and biochemical hormonal changes observed in a 27-year-old man with Cushing's syndrome treated by implantation of the pituitary with yttrium90 are reported. Eight months after implantation a complete clinical regression of the syndrome was obtained. Hormonal determinations indicated relative adrenal insufficiency, whereas thyroidal and gonadal functions werenormal.

The mode circulation time was measured using intravenously injected technetium 99(m) and a collimation system devised to discriminate between the right and left sides of the head. The results in 21 normal men were used as a basis of assessing the findings in 205 patients, made up of five diagnostic groups (ischaemia, haematoma, subarachnoid haemorrhage, intracranial tumour, and head injury). The average mode circulation time in the affected hemisphere for the three groups with vascular disease was increased, but even in these groups half the patients had a mode circulation time within 1 SD of normal mean; similar results were found for asymmetry between the hemispheres. In serial measurements in 45 patients no correlation was found between change in mode circulation time and the clinical state. That so many results are within the normal range limits the value of this method.

The relationship between the velocity of the cerebral circulation and the cerebral blood flow was explored at varying levels of PaCO(2), systemic arterial pressure, intracranial pressure, and perfusion pressure, using radioisotope techniques in baboons. Only at low flow rates did velocity increase with flow, and then non-linearly; at high rates velocity increased progressively less. Changes in flow are reflected by changes in velocity in such restricted circumstances that mean circulation time is a very unreliable indication of cerebral blood flow.

e11073 Background: NCT has been shown to improve disease-free and overall survival in BC patients. Predictive markers of therapeutic response need to be carefully studied in order to distinguish sp...

Background Although cells require nutrients to proliferate, most nutrient exchange rates of the NCI60 panel of cancer cell lines correlate poorly with their proliferation rate. Here, we provide evidence indicating that this inconsistency is rooted in the variability of cell size. Results We integrate previously reported data characterizing genome copy number variations, gene expression, protein expression and exchange fluxes with our own measurements of cell size and protein content in the NCI60 panel of cell lines. We show that protein content, DNA content, and protein synthesis per cell are proportional to the cell volume, and that larger cells proliferate slower than smaller cells. We estimate the metabolic fluxes of these cell lines and show that their magnitudes are proportional to their protein synthesis rate and, after correcting for cell volume, to their proliferation rate. At the level of gene expression, we observe that genes expressed at higher levels in smaller cells are enriched for genes involved in cell cycle, while genes expressed at higher levels in large cells are enriched for genes expressed in mesenchymal cells. The latter finding is further corroborated by the induction of those same genes following treatment with TGFβ, and the high vimentin but low E-cadherin protein levels in the larger cells. We also find that aromatase inhibitors, statins and mTOR inhibitors preferentially inhibit the in vitro growth of cancer cells with high protein synthesis rates per cell. Conclusions The NCI60 cell lines display various metabolic activities, and the type of metabolic activity that they possess correlates with their cell volume and protein content. In addition to cell proliferation, cell volume and/or biomarkers of protein synthesis may predict response to drugs targeting cancer metabolism.

Background Alterations in cell proliferation and tissue remodeling are major hallmarks of cancer. Given that these two physiological processes require a significant investment of metabolic resources, we hypothesize that the metabolism of cancer cells is, in a first approximation, determined by the magnitude of cell proliferation and tissue remodeling. Materials and methods To address this question in further detail we have analyzed in vitro data characterizing cancer cell lines and gene expression profiles of human cancers. Results We show that the metabolism of cancer cells is strongly correlated with cell proliferation and tissue remodeling. Based on in vitro data, cancer cell lines align from an extreme of highly proliferating cells of relatively small size to another extreme of slowly proliferating cells with large size and mesenchymal properties [1]. Similarly, in human cancers we detect a strong negative correlation between gene signatures associated with cell proliferation and tissue remodeling [2]. The stratification of human cancers based on the cell-proliferation/ tissue-remodeling signatures results in divergence in the survival plots as significant as those obtained using current site-specific classifications. Furthermore, in vitro data suggest that these different subtypes have a differential sensitivity to diverse drug classes targeting metabolism [1]. Conclusions We conclude that human cancers can be subject to a universal cell-proliferation/tissue-remodeling classification independent of their site of origin that can guide personalized treatment targeting cancer metabolism.