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Soft tissue sarcoma (STS) is a locally aggressive and infiltrative tumor in dogs. Surgical resection is the treatment of choice for local tumor control. Currently, post-operative pathology is performed for surgical margin assessment. Spectral-domain optical coherence tomography (OCT) has recently been evaluated for its value for surgical margin assessment in some tumor types in dogs. The purpose of this study was to develop an automatic diagnosis system that can assist clinicians in real-time for OCT image interpretation of tissues at surgical margins. We utilized a ResNet-50 network to classify healthy and cancerous tissues. A patch-based approach was adopted to achieve accurate classification with limited training data (80 cancer images, 80 normal images) and the validation set (20 cancer images, 20 normal images). The proposed method achieved an average accuracy of 97.1% with an excellent sensitivity of 94.3% on the validation set; the quadratic weighted κ was 0.94 for the STS diagnosis. In an independent test data set of 20 OCT images (10 cancer images, 10 normal images), the proposed method correctly differentiated all the STS images. Furthermore, we proposed a diagnostic curve, which could be evaluated in real-time to assist clinicians in detecting the specific location of a lesion. In short, the proposed method is accurate, operates in real-time, and is non-invasive, which could be helpful for future surgical guidance. This article is protected by copyright. All rights reserved.

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (KD: 27−182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69−80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans’ clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This work was supported by the Institute of Bioengineering and Bioengineering (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) and the National Research Foundation of Singapore (NRF-CRP17-2017-07).

Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.

Background Myelofibrosis (MF) is a life-shortening complication of myeloproliferative neoplasms associated with ineffective hematopoiesis, splenomegaly, and progressive bone marrow (BM) fibrosis. The oral Janus kinase (JAK) 1/JAK2 inhibitor ruxolitinib has been shown to improve splenomegaly, symptom burden, and overall survival in patients with intermediate-2 or high-risk MF compared with placebo or best available therapy (BAT). Methods The effects of ruxolitinib therapy for up to 66 months on BM morphology in 68 patients with advanced MF with variable BM fibrosis grade were compared with those in 192 matching patients treated with BAT. Available trephine biopsies underwent independent, blinded review by three hematopathologists for consensus-based adjudication of grades for reticulin fibrosis, collagen deposition, and osteosclerosis. Results Ruxolitinib treatment versus BAT was associated with greater odds of BM fibrosis improvement or stabilization and decreased odds of BM fibrosis worsening based on changes from baseline in reticulin fibrosis grade. Generally, these changes were accompanied by a sustained higher level of individual spleen size reduction and regression of leukoerythroblastosis. Patients with more advanced baseline fibrosis showed lower spleen size response. Conclusions The finding that long-term ruxolitinib therapy may reverse or markedly delay BM fibrosis progression in advanced MF suggests that sustained JAK inhibition may be disease-modifying. Trial registration INCB18424-251, ClinicalTrials.gov identifier NCT00509899. Electronic supplementary material The online version of this article (10.1186/s13045-018-0585-5) contains supplementary material, which is available to authorized users.

Background: PRMT5 is overexpressed in certain myeloid malignancies and catalyzes symmetric arginine dimethylation of protein substrates that regulate expression of genes associated with disease pathogenesis (Pastore et al. Cancer Discov. 2020). PRT543 is a potent, selective, oral PRMT5 inhibitor with preclinical antitumor activity in acute myeloid leukemia and myeloproliferative neoplasm models (Bhagwat AACR 2020). An open-label phase 1 study of PRT543 in unselected patients (pts) with advanced solid tumors and hematologic malignancies (NCT03886831) is ongoing. Dose escalation results from pts with myeloid malignancies are presented herein. Materials and Methods: This study assesses the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy per International Working Group (IWG) response criteria for myelodysplastic syndrome (MDS) and myeloproliferative neoplasm of PRT543 administered at varying schedules beginning with twice weekly (BIW) and increasing to once daily (QD) with doses ranging from 5 to 40 mg in 28-day cycles. Dose escalation followed a modified 3+3 design, allowing for accelerated titration at lower doses. Serum symmetric dimethylarginine (sDMA) and intron retention, a marker of PRMT5-mediated mRNA splicing fidelity, were assessed as measures of PRMT5 target engagement and function, respectively. Results: As of 16 July 2021, 23 unselected pts (12 with myelofibrosis [MF] and 11 with MDS) with disease refractory to established therapies had enrolled (5 pts were dosed BIW, 6 pts 3 times per week [TIW], 5 pts 5 times per week, and 7 pts QD). The median number of prior lines of systemic therapies was 2 (range, 0 to 6). Ten of 12 MF pts had prior exposure to ruxolitinib. Median platelet counts at baseline were 176,500/µL for MF pts and 52,000/µL for MDS pts. Three of 23 pts (13%) experienced dose limiting toxicity (DLT) of thrombocytopenia, with a single occurrence at each of 3 different doses/schedules (40 mg TIW, 35 mg 5 times per week, and 20 mg QD). No other DLTs were reported. The most frequent treatment-related adverse events (TRAEs), any grade, in all regimens were nausea (n=5, 22%), thrombocytopenia (n=5, 22%), anemia (n=4, 17%), diarrhea (n=4, 17%), and fatigue (n=3, 13%). Grade ≥3 TRAEs in all regimens were limited to anemia (n=4, 17%) and thrombocytopenia (n=4, 17%). Cytopenias were reversible and managed with dose modifications. Eighteen pts discontinued treatment, primarily due to disease progression or investigator decision. One pt was discontinued due to an AE (performance status decreased/weakness). PRT543 demonstrated dose-dependent increases of C max (nM) and AUC (nM.hr). At the expansion dose (35 mg 5 times per week), half-life (T ½) was 14 hrs, and plasma drug exposures (C max, 1988 nM; AUC, 19813 nM.hr) exceeded those required for preclinical efficacy. Serum sDMA decreased in a dose-dependent manner reaching 58% reduction with the expansion dose. Increased intron retention in select transcripts was observed in peripheral blood mononuclear cells at the expansion dose. Reductions in inflammatory serum markers, including C-reactive protein and serum amyloid A and cytokines interleukin (IL)-6 and IL-8 were observed. Improvement in individual symptoms such as night sweats, fever and pruritis were noted. Of the 5 MF patients with spliceosome mutations, 1 MF pt (SF3B1) with anemia and transfusion history (8 units over the 121 days prior to baseline) experienced a substantial improvement in anemia (hemoglobin increase from 6.0 to ≥11.1 g/dL). This pt has been transfusion-free for over 300 days and remains on study. Three pts with MF exhibited stable disease per IWG for at least 1 year, with 1 MF pt demonstrating improvement in bone marrow reticulin fibrosis (+2-3 to +1-2). Conclusions: PRT543 was well tolerated with a favorable safety profile. Dose-dependent inhibition of target engagement and functional activity of PRMT5 were observed. PRT543 treatment demonstrated reduction in inflammatory markers and improvement in symptoms and anemia in select patients. Prolonged stable disease per IWG was observed in several pts with refractory MF. PRT543 is currently being evaluated as monotherapy in pts with at least one spliceosome mutation (MF and MDS pts with anemia and pts with high-risk myeloid malignancies) and in combination with ruxolitinib in MF patients demonstrating a suboptimal response to ruxolitinib. Disclosures Patel: Vigeo: Research Funding; Portola Pharmaceuticals: Research Funding; Boehringer Ingelheim: Research Funding; AstraZeneca: Research Funding; TopAlliance: Research Funding; Tesaro: Research Funding; Vedanta: Research Funding; Curis: Research Funding; Ciclomed: Research Funding; Klus Pharma: Research Funding; Kymab: Research Funding; Lycera: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Bicycle Therapeutics: Research Funding; Hutchinson MediPharma: Research Funding; Ignyta: Research Funding; Taiho: Research Funding; Loxo Oncology: Research Funding; Millennium Pharmaceuticals: Research Funding; Incyte: Research Funding; Jounce Therapeutics: Research Funding; Prelude Therapeutics: Research Funding; H3 Biomedicine: Research Funding; Revolution Medicines: Research Funding; Hengrui: Research Funding; Cyteir Therapeutics: Research Funding; Ribon Therapeutics: Research Funding; Jacobio: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Evelo Biosciences: Research Funding; Takeda: Research Funding; Forma Therapeutics: Research Funding; Artios Pharma: Research Funding; Aileron Therapeutics: Research Funding; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Verastem: Research Funding; Phoenix Molecular Designs: Research Funding; Macrogenics: Research Funding; Mabspace: Research Funding; Alexion, AstraZeneca Rare Disease: Other: Study investigator; Seven and Eight Biopharmaceuticals: Research Funding; Syndax: Research Funding; Synthorx: Research Funding; GlaxoSmithKline: Research Funding; Gilead: Research Funding; Eli Lilly: Research Funding; EMD Serono: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus: Research Funding; Checkpoint Therapeutics: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Xencor: Research Funding; Daiichi Sankyo: Research Funding; ADC Therapeutics: Research Funding; Acerta Pharma: Research Funding; Florida Cancer Specialists: Research Funding; Qilu Puget Sound Biotherapeutics: Research Funding; ORIC Pharmaceuticals: Research Funding; Stemline Therapeutics: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; BioNTech: Research Funding; ModernaTX: Research Funding; Mirati Therapeutics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Exelixis: Membership on an entity's Board of Directors or advisory committees; LSK Biopartners: Research Funding; Placon Therapeutics: Research Funding; Calithera: Research Funding; Effector Therapeutics: Research Funding; Clovis: Research Funding. Monga: Gunderson Foundation: Other; Rising Tide Foundation: Other; Amgen: Research Funding; Orbus Therapeutics: Research Funding; Prelude Therapeutics: Research Funding; Deciphera: Research Funding; Forma Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. McKean: Ascentage Pharma Group: Research Funding; Bicycle Therapeutics: Research Funding; Dragonfly Therapeutics: Research Funding; Epizyme: Research Funding; Exelixis: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; IDEAYA Biosciences: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ikena Oncology: Research Funding; Infinity Pharmaceuticals: Research Funding; Jacobio Pharmaceuticals: Research Funding; Moderna: Research Funding; NBE Therapeutics: Research Funding; Novartis: Research Funding; Oncorus: Research Funding; Plexxikon: Research Funding; Prelude Therapeutics: Research Funding; Regeneron: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sapience Therapeutics: Research Funding; Seattle Genetics: Research Funding; Tizona Therapeutics: Research Funding; TMUNITY Therapeutics: Research Funding; TopAlliance Biosciences: Research Funding; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; BicycleTX Limited: Membership on an entity's Board of Directors or advisory committees; Castle Biosciences: Membership on an entity's Board of Directors or advisory committees; MedPage Today: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Mauro: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Viscusi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhagwat: Prelude Therapeutics: Current Employment. Moore: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sun: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chiaverelli: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mintah: Prelude Therapeutics: Current Employment. Nath: Actinium: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Stein: PinotBio: Consultancy; Daiichi Sankyo: Consultancy; Syros Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Genentech: Consultancy; Janssen Pharmaceuticals: Consultancy; Syndax Pharmaceuticals: Consultancy; Astellas: Consultancy; Novartis: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy. Palmisiano: Genentech: Research Funding; Takeda: Consultancy; AbbVie: Consultancy, Research Funding; Foundation One: Consultancy. Verstovsek: Ital Pharma: Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding; Genentech: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; Incyte Corporation: Consultancy, Research Funding; CTI BioPharma: Research Funding; PharmaEssentia: Research Funding; Celgene: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy.

Background: In 2 phase III studies comparing the Janus kinase (JAK) 1 and JAK2 inhibitor ruxolitinib with placebo (Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment I [COMFORT-I]) or best available therapy (COMFORT-II), ruxolitinib treatment was associated with rapid and durable reductions in spleen volume, improved myelofibrosis (MF)-related symptoms and quality of life measures, and prolonged survival. However, the key measures of efficacy used in the COMFORT studies—magnetic resonance imaging/computed tomography (MRI/CT) to measure change in spleen volume and the modified Myelofibrosis Symptom Assessment Form v2.0 to assess change in MF-related symptoms—are not commonly used in clinical practice. Therefore, we analyzed the efficacy of ruxolitinib in COMFORT-I using practical, community-based measures of clinical benefit (spleen palpation and the Patient Global Impression of Change [PGIC] questionnaire). Methods: In COMFORT-I, patients (N = 309) with primary MF, post–polycythemia vera MF or post–essential thrombocythemia MF were randomized to receive ruxolitinib (n = 155) or placebo (n = 154). For this analysis, patients were divided into groups by degree of palpable spleen reduction and symptom improvement at week 12 as follows: patients with clinically relevant reductions in palpable spleen as defined by the International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet (ELN) consensus criteria (SPL), patients with “much improved” or “very much improved” symptoms as measured by the PGIC (PGIC), or patients who did not achieve SPL or PGIC (Neither). The Neither group encompassed patients with “minimally improved” symptoms with no worsening in palpable spleen or patients who achieved 10% reduction in palpable spleen with no worsening in symptoms (Minor), and patients who did not achieve any palpable spleen reduction or PGIC-assessed symptom improvement (None). Nonevaluable patients (4/155 for ruxolitinib and 13/154 for placebo) were not included in the analysis. Additional measures of clinical benefit assessed at week 12 included changes in spleen volume and changes in white blood cell (WBC) count. Durability of all measures of clinical benefit with longer-term ruxolitinib therapy was also evaluated. Results: At week 12, the majority (91.4%) of patients randomized to ruxolitinib achieved some clinical benefit as measured by palpable spleen reduction and/or PGIC-assessed symptom improvement, vs 31.9% of those assigned to placebo (Figure 1). Benefits in palpable spleen length reduction and PGIC-assessed symptom improvement achieved at week 12 with ruxolitinib were sustained at week 24. Benefits in palpable spleen length reduction were sustained with longer-term ruxolitinib therapy (median follow-up: 149 weeks). At week 12, ruxolitinib treatment was associated with meaningful reductions in spleen volume by MRI or CT and WBC count in patients who achieved SPL and PGIC criteria, while worsening spleen volume and WBC count was observed with placebo (Figure 2); these effects were durable with longer-term therapy. Patients who achieved neither SPL nor PGIC criteria by week 12 (n = 47; 31.1%) achieved a clinically meaningful reduction in spleen volume by MRI or CT (mean change, −25.6%) and decreased WBC counts (mean change, −7.4%) at week 12 (Figure 2). Patients who did not achieve any palpable spleen reduction or PGIC-assessed symptom improvement (n = 13; 8.6%) were generally older, more likely to have high-risk MF as assessed by the International Prognostic Scoring System, and had higher WBC counts, lower platelet counts, and worse fibrosis grade at baseline compared with those achieving defined palpable spleen length reduction and/or PGIC-assessed symptom improvement with ruxolitinib. Conclusions: Nearly all patients treated with ruxolitinib achieved some clinical benefit as assessed by practical measures of assessing efficacy (ie, palpable spleen reduction or PGIC-assessed symptom improvement) within 12 weeks of treatment initiation. Meaningful reductions in spleen volume and WBC count were observed in patients receiving ruxolitinib whether or not they achieved palpable spleen reduction by IWG-MRT/ELN consensus criteria or PGIC symptom improvement (“much improved” or “very much improved”) at week 12; these reductions were durable with long-term ruxolitinib therapy. Disclosures Miller: Incyte Corporation: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Komrokji:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mesa:CTI: Research Funding; NS Pharma: Research Funding; Gilead: Research Funding; Incyte Corporation: Research Funding; Celgene: Research Funding. Sun:Incyte Corporation: Employment. Montgomery:Incyte Corporation: Employment. Verstovsek:Incyte Corporation: Research Funding.

In the phase III COMFORT-I study, the Janus kinase 1 (JAK1)/JAK2 inhibitor ruxolitinib provided significant improvements in splenomegaly, key symptoms, and quality-of-life measures and was associated with an overall survival benefit relative to placebo in patients with intermediate-2 or high-risk myelofibrosis. This planned analysis assessed the long-term efficacy and safety of ruxolitinib at a median follow-up of 149 weeks. At data cutoff, approximately 50% of patients originally randomized to ruxolitinib remained on treatment whereas all patients originally assigned to placebo had discontinued or crossed over to ruxolitinib. At week 144, mean spleen volume reduction was 34% with ruxolitinib. Previously observed improvements in quality-of-life measures were sustained with longer-term ruxolitinib therapy. Overall survival continued to favor ruxolitinib despite the majority of placebo patients crossing over to ruxolitinib [hazard ratio 0.69 (95% confidence interval: 0.46–1.03); P=0.067]. Exploratory analyses suggest that crossover may have contributed to an underestimation of the true survival difference between the treatment groups. Ruxolitinib continued to be generally well tolerated; there was no pattern of worsening grade ≥3 anemia or thrombocytopenia with longer-term ruxolitinib exposure. These longer-term data continue to support the efficacy and safety of ruxolitinib in patients with myelofibrosis. The study is registered at clinicaltrials.gov: NCT00952289.

Myelofibrosis (MF), including primary myelofibrosis (PMF) and MF secondary to essential thrombocythemia (ET) or polycythemia vera (PV), is a chronic Philadelphia chromosome–negative myeloproliferative neoplasm associated with progressive bone marrow fibrosis.[1][1] Many patients with MF experience

Myelofibrosis (MF) is associated with a variety of burdensome symptoms and reduced survival compared with age-/sex-matched controls. This analysis evaluated the long-term survival benefit with ruxolitinib, a Janus kinase (JAK)1/JAK2 inhibitor, in patients with intermediate-2 (int-2) or high-risk MF. This was an exploratory analysis of 5-year data pooled from the phase 3 COMFORT-I and -II trials. In both trials, patients could cross over to ruxolitinib from the control group (COMFORT-I, placebo; COMFORT-II, best available therapy). All continuing patients in the control groups crossed over to ruxolitinib by the 3-year follow-up. Overall survival (OS; a secondary endpoint in both trials) was evaluated using pooled intent-to-treat data from patients randomized to ruxolitinib or the control groups. OS was also evaluated in subgroups stratified by baseline anemia and transfusion status at week 24. A total of 528 patients were included in this analysis; 301 were originally randomized to ruxolitinib (COMFORT-I, n = 155; COMFORT-II, n = 146) and 227 to control (n = 154 and n = 73, respectively). The risk of death was reduced by 30% among patients randomized to ruxolitinib compared with patients in the control group (median OS, 5.3 vs 3.8 years, respectively; hazard ratio [HR], 0.70 [95% CI, 0.54–0.91]; P = 0.0065). After correcting for crossover using a rank-preserving structural failure time (RPSFT) method, the OS advantage was more pronounced for patients who were originally randomized to ruxolitinib compared with patients who crossed over from control to ruxolitinib (median OS, 5.3 vs 2.3 years; HR [ruxolitinib vs RPSFT], 0.35 [95% CI, 0.23–0.59]). An analysis of OS censoring patients at the time of crossover also demonstrated that ruxolitinib prolonged OS compared with control (median OS, 5.3 vs 2.4 years; HR [ruxolitinib vs censored at crossover], 0.53 [95% CI, 0.36–0.78]; P = 0.0013). The survival benefit with ruxolitinib was observed irrespective of baseline anemia status or transfusion requirements at week 24. These findings support ruxolitinib treatment for patients with int-2 or high-risk MF, regardless of anemia or transfusion status. Further analyses will be important for exploring ruxolitinib earlier in the disease course to assess the effect on the natural history of MF. ClinicalTrials.gov identifiers, NCT00952289 and NCT00934544 .

Patient-reported outcomes (PROs) and spleen size in patients not receiving therapy (N=154) in COMFORT-I, a randomized, double-blind study of the JAK1/JAK2 inhibitor ruxolitinib in patients with intermediate-2 or high-risk myelofibrosis were evaluated. Baseline PROs indicated considerable disease burden. European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 scores, modified Myelofibrosis Symptom Assessment Form v2.0 Total Symptom Score, and Patient Reported Outcome Measurement Information System Fatigue scores worsened from baseline through week 24. At weeks 4 and 24, 18.3 and 40.2% of patients evaluated their condition as having worsened from baseline on the Patient Global Impression of Change questionnaire. Spleen volume and palpable length increased in most patients. These results demonstrate the progressive and debilitating effects of myelofibrosis. The consequences of delayed intervention should be assessed in the management of patients with myelofibrosis and treatment should be considered as clinically indicated for symptomatic relief or splenomegaly control.

Purpose To assess the effects of ruxolitinib on symptom burden and quality of life (QoL) and to evaluate the ability of the modified Myelofibrosis Symptom Assessment Form (MFSAF) v2.0 to measure meaningful changes in myelofibrosis-related symptoms in patients with myelofibrosis. Patients and Methods COMFORT-I (Controlled Myelofibrosis Study With Oral JAK Inhibitor Treatment–I) is a double-blind, placebo-controlled phase III study evaluating ruxolitinib in patients with intermediate-2 or high-risk myelofibrosis. Exploratory analyses were conducted on the following patient-reported outcomes (PROs) assessments: modified MFSAF v2.0 (individual symptoms and Total Symptom Score [TSS]), European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), Patient Reported Outcomes Measurement Information System (PROMIS) Fatigue Scale, and Patient Global Impression of Change (PGIC). Results Patients receiving ruxolitinib experienced improvements in individual myelofibrosis-related symptoms, although patients receiving placebo experienced worsening (P < .001). The majority (91%) of ruxolitinib-treated patients designated as ≥ 50% TSS responders (≥ 50% TSS improvement) self-reported their condition as either “Much improved” or “Very much improved” on the PGIC. These patients achieved significant improvements in the EORTC QLQ-C30 functional domains and Global Health Status/QoL versus patients receiving placebo, who experienced worsening on these measures (P ≤ .0135). Ruxolitinib-treated patients with a lesser degree of symptom improvement (< 50% TSS responders) also achieved improvements over placebo on these measures. The degree of spleen volume reduction with ruxolitinib correlated with improvements in TSS, PGIC, PROMIS Fatigue Scale, and EORTC Global Health Status/QoL. Ruxolitinib-treated patients who achieved a ≥ 35% reduction in spleen volume experienced the greatest improvements in these PROs. Conclusion Ruxolitinib-treated patients achieved clinically meaningful improvements in myelofibrosis-related symptoms and QoL, but patients receiving placebo reported worsening of symptoms and other PROs.

Myelofibrosis (MF) is a rare and life-threatening myeloproliferative neoplasm that can arise de novo [primary MF (PMF)] or evolve from polycythaemia vera (PV), i.e. post-polycythaemia vera MF (PPV-MF), or essential thrombocythaemia (ET), i.e. post-ET MF (PET-MF) (Barosi et al, 2008; Vardiman et al, 2009; Tefferi, 2011). Dysregulated Janus kinase/signal transducers and activators of transcription (JAK-STAT) signalling, resulting from gain- or loss-of-function mutations and/or high circulating levels of inflammatory cytokines, plays a key role in the pathogenesis of MF (Vainchenker et al, 2011). The JAK2 V617F mutation is present in approximately 50–60% of patients with PMF or ET and in over 95% of patients with PV (Nguyen & Gotlib, 2012). Dysregulation of the JAK-STAT signalling pathway in MF is additionally related to mutations in genes such as JAK2 exon 12, MPL exon 10, SH2B3, members of the Casitas B-cell lymphoma family and post-translational modifications of suppressor of cytokine signalling proteins (Vainchenker et al, 2011). Proinflammatory cytokines that have been implicated in MF are known to signal through JAK1 and JAK2 (Vainchenker et al, 2008) and symptoms of MF have been linked to elevated levels of these cytokines (Verstovsek, 2009; Tefferi et al, 2011). An association between elevated cytokines and decreased survival has also been reported (Tefferi et al, 2011). A broad spectrum of disease characteristics exists within the MF patient population (Cervantes et al, 1997, 2009). Patients often experience constitutional symptoms (e.g. fever, night sweats, weight loss) and splenomegaly, which may cause disability and have a profound impact on quality of life (Mesa et al, 2007); however, other MF-related symptoms, such as fatigue (84%), itching (50%) and bone pain (47%), are also common and burdensome to patients (Mesa et al, 2007). Patients with MF have shortened survival, and those with advanced MF have a poor prognosis (Barbui et al, 2011). The major prognostic scoring systems used to categorize risk in patients with MF are the International Prognostic Scoring System (IPSS) (Cervantes et al, 2009), the Dynamic International Prognostic Scoring System (DIPSS) (Passamonti et al, 2010) and DIPSS Plus (Gangat et al, 2011). Risk factors for shorter survival in both the IPSS and DIPSS are age >65 years, presence of constitutional symptoms, haemoglobin 25 × 109/l and peripheral blood blasts ≥1% (Cervantes et al, 2009; Passamonti et al, 2010). The DIPSS Plus adds platelet count

1-(2-Deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB) is a putative bioisostere of iododeoxyuridine (IUdR). The advantages of dRFIB over IUdR for in vivo studies include resistance to both phosphorolytic cleavage of the nucleoside bond and de-iodination. dRFIB was radioiodinated (dRF(123/125)IB) by copper-catalyzed exchange using commercial sodium [(123/125)I]iodide. The in vivo biodistribution of dRF[(125)I]IB in BALBc mice and imaging of dRF[(123)I]IB in Sprague-Dawley rats are reported. In vivo data for rats show rapid clearance of radioactivity from blood (>95%ID in 15 minutes), extensive excretion in urine (56%ID/24 hours), concentration in the hepatobiliary-small intestine system and very little fecal excretion (approximately 3%ID/24 hours). Pharmacokinetic data for dRF[(125)I]IB (i.v. 48.7 ug/kg) in rats (t(1/2)[h] = 0.51 +/- 0.14, AUC(inf)[microg.min/mL] = 3.7 +/- 0.4, Cl[L/kg/h] = 0.75 +/- 0.12, Vss[L/kg] = 0.96 +/- 0.18) confirm previously reported dose-dependent pharmacokinetics. Scintigraphic images of rats dosed with dRF[(123)I]I were compatible with rapid soft-tissue clearance and extensive accumulation of radioactivity in bladder/urine and liver/small intestine. In tumor-bearing mice, thyroid and stomach radioactivity was indicative of moderate deiodination. An unidentified polar radioactive metabolite was detected in serum.

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin produced in the K cells of the intestine and secreted into the circulating blood following ingestion of carbohydrate- and fat-containing meals. GIP contributes to the regulation of postprandial insulin secretion and is essential for normal glucose tolerance. We have established a method of assaying GIP in response to nutrients using the intestinal lymph fistula model. Administration of Ensure, a mixed-nutrient liquid meal, stimulated a significant increase in intestinal lymphatic GIP levels that were approximately threefold those of portal plasma. Following the meal, lymph GIP peaked at 60 min ( P < 0.001) and remained elevated for 4 h. Intraduodenal infusions of isocaloric and isovolumetric lipid emulsions or glucose polymer induced lymph GIP concentrations that were four and seven times the basal levels, respectively. The combination of glucose plus lipid caused an even greater increase of lymph GIP than either nutrient alone. In summary, these findings demonstrated that intestinal lymph contains high concentrations of GIP that respond to both enteral carbohydrate and fat absorption. The change in lymphatic GIP concentration is greater than the change observed in the portal blood. These studies allow the detection of GIP levels at which they exert their local physiological actions. The combination of glucose and lipid has a potentiating effect in the stimulation of GIP secretion. We conclude from these studies that the lymph fistula rat is a novel approach to study in vivo GIP secretion in response to nutrient feeding in conscious rats.

Glucagon-like peptide-1 (GLP-1) is an important incretin produced in the L cells of the intestine. It is essential in the regulation of insulin secretion and glucose homeostasis. Systemic GLP-1 concentrations are typically low in rodents, so it can be difficult to assay physiological levels or detect changes in response to nutrients. We have established a method of assaying GLP-1 in response to nutrients using the intestinal lymph fistula model. Intraduodenal infusion of Intralipid (4.43 kcal/3 ml) induced a significant increase of lymphatic GLP-1 concentration compared with saline control at the peak of 30 min. ( P < 0.001). Isocaloric and isovolumetric treatment with dextrin, a glucose polymer, also caused a significant fourfold increase in peak concentration at 60 min ( P = 0.001). These findings indicate that intestinal lymph contains high concentrations of postprandial GLP-1. Second, they reveal that GLP-1 secretion into lymph occurs in response to both enteral carbohydrate and fat, but the response to dextrin occurs later than to Intralipid with peak times at 60 and 30 min, respectively. Third, the combination of Intralipid plus dextrin demonstrated an additive effect in the stimulation of GLP-1 with peak at 30 min. These results indicate that assessment of levels in lymph is a novel and powerful means of studying the secretion of GLP-1 and potentially other gastrointestinal hormones in vivo. Furthermore, the lymph fistula rat model provides insight into the gut hormone concentrations to which the neurons and cells in the lamina propria of the gut are likely exposed.

Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are peptides that act both peripherally and centrally to reduce food intake by decreasing meal size. The present study examined the effects of intraperitoneally administered bolus doses of recombinant apo AIV, CCK-8, and a combination of subthreshold doses of apo AIV and CCK on 4-h food intake in rats that were fasted overnight. Apo AIV at 100 μg/kg reduced food intake significantly relative to the saline control for 1 h, as did doses of CCK-8 at or above 0.125 μg/kg. Doses of apo AIV (50 μg/kg) or CCK (0.06 μg/kg) alone had no effect on food intake. However, when these subthreshold doses of apo AIV and CCK were administered together, the combination produced a significant inhibition of food intake relative to saline controls ( P < 0.001), and the duration of the effect was longer than that caused by the administration of either apo AIV or CCK alone. The satiation effect produced by CCK-8 + apo AIV was attenuated by lorglumide, a CCK1 receptor antagonist. We conclude that, whereas the intraperitoneal administration of doses of either recombinant apo AIV or CCK at or above threshold levels reduces food intake, the coadministration of subthreshold doses of the two peptides is highly satiating and works via CCK1 receptor.

Cell surface antigens are important targets for monoclonal antibodies, but they are often difficult to work with due to their association with the cell membrane. Phage display is a versatile technique that can be applied to generate binders against difficult targets. Here we used antibody phage display to isolate a binder for a rare and specialized cell, the human corneal endothelial cell. The human corneal endothelium is a medically important cell layer; defects in this layer account for about half of all corneal transplants. Despite its importance, no specific antigens have been found to mark this cell type. By panning a phage library directly on human corneal endothelial cells, we isolated an antibody that bound to these cells and not the other types of corneal cells. Subsequently, we identified the antibody’s putative target to be CD166 by immunoprecipitation and mass spectrometry. This approach can be used to isolate antibodies against other poorly-characterized cell types, such as stem cells or cancer cells, without any prior knowledge of their discriminating markers.

Background In the COMFORT (COntrolled MyeloFibrosis Study with ORal JAK Inhibitor Therapy)-I study, the Janus kinase (JAK)1/JAK2 inhibitor ruxolitinib provided significant reductions in splenomegaly, improvements in myelofibrosis (MF)-related symptoms, and a survival advantage relative to placebo in patients with intermediate-2 or high-risk MF. In this post hoc analysis, we assessed the effects of ruxolitinib treatment on measures of metabolic and nutritional status. Patients and Methods Patients were randomized to receive ruxolitinib (n = 155; 15 or 20 mg twice a day for patients with baseline platelet counts of 100-200 × 10 9 /L or > 200 × 10 9 /L, respectively) or placebo (n = 154). The primary end point was the proportion of patients with a ≥ 35% spleen volume reduction from baseline to week 24. A secondary end point was the proportion of patients with ≥ 50% improvement in Total Symptom Score (TSS) from baseline to week 24, measured using the modified Myelofibrosis Symptom Assessment Form version 2.0. Weight, cholesterol, and albumin were measured at specified time points throughout the study. Results Compared with placebo, ruxolitinib treatment was associated with increased weight (mean change: 3.9 kg vs. −1.9 kg), total cholesterol (mean percentage change: 26.4% vs. −3.3%), and albumin levels (mean percentage change: 5.8% vs. −1.7%) at week 24; sustained improvements were observed with longer-term ruxolitinib therapy. Relative to placebo, increases in mean weight, total cholesterol, and albumin levels were observed with ruxolitinib treatment regardless of the degree of spleen volume and TSS reductions at 24 weeks. Conclusion Treatment with ruxolitinib improved measures of metabolic and nutritional status of patients with intermediate-2 or high-risk MF.

The focus of this article is to review evidence that apolipoprotein A-IV (apo A-IV) acts as a satiety factor. Additionally, information regarding the general involvement of apo A-IV in the regulation of food intake and body weight is stated. Apo A-IV is a glycoprotein synthesized by the human intestine. In rodents, both the small intestine and liver secrete apo A-IV, but the small intestine is the major organ responsible for circulating apo A-IV. There is now solid evidence that the hypothalamus, especially the arcuate nucleus, is another active site of apo A-IV expression. Intestinal apo A-IV synthesis is markedly stimulated by fat absorption and does not appear to be mediated by the uptake or reesterification of fatty acids to form triglycerides. Rather, the local formation of chylomicrons acts as a signal for the induction of intestinal apo A-IV synthesis. Intestinal apo A-IV synthesis is also enhanced by a factor from the ileum, probably peptide tyrosine-tyrosine (PYY). The inhibition of food intake by apo A-IV is mediated centrally. The stimulation of intestinal synthesis and secretion of apo A-IV by lipid absorption are rapid; thus apo A-IV likely plays a role in the short-term regulation of food intake. Other evidence suggests that apo A-IV may also be involved in the long-term regulation of food intake and body weight, as it is regulated by both leptin and insulin. Chronic ingestion of a high-fat diet blunts the intestinal as well as the hypothalamic apo A-IV response to lipid feeding. It also suppresses apo A-IV gene expression in the hypothalamus. Whereas it is tempting to speculate that apo A-IV may play a role in diet-induced obesity, we believe the confirmation of such a proposal awaits further experimental evidence.

SummaryProthrombin has diverse biological functions in addition to its well established role in blood coagulation. In order to study these functions in more detail mouse model systems are needed. Since deficiency of prothrombin in mice results in partial embryonic lethality and neonatal death, alternative approaches are required to study the biology of prothrombin in the adult mouse. The liver is the major site of synthesis of prothrombin and therefore liver-specific promoters were used to express prothrombin in transgenic mice. Mice generated from crosses with these transgenic mice and mice hemizygous for the knock-out allele were used to test whether liver-specific expression is sufficient to correct the phenotype of null mice and whether liver-specific expression is sufficient for the development and survival of mice to adulthood. The mouse albumin promoter/enhancer was used initially for transgene expression without success in obtaining transgene positive, endogenous prothrombin null mice. Two lines of transgene positive, endogenous prothrombin deficient mice were obtained using the mouse transthyretin (TTR) promoter/enhancer driving expression of a human prothrombin cDNA. One line was able to rescue both the embryonic and the neonatal lethality while the other line was only able to correct the embryonic lethality. Expression of prothrombin was restricted to the liver and stomach in one line and to the liver, pancreas, stomach and kidney in the other line of mice. Thrombin activity for one line was determined to be at 5-10% of wildtype levels. These mice developed normally and did not have spontaneous bleeding events unless traumatized. Therefore, transgenic expression of human prothrombin is sufficient for the rescue of the lethality found for prothrombin deficiency in mice.

Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by “foot-print free” reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

Five distinct organic anion transporter cDNAs, hOAT1-5, were isolated from human liver and kidney. hOAT1, 2, and 3 are homologous to their respective rat orthologues OAT1-3, whereas hOAT4 and 5 are novel clones that have not been identified in other species. hOAT1- and hOAT3-transfected cells showed uptake of p-aminohippurate and fluorescein. Cells expressing hOAT2 showed uptake of p-aminohippurate, methotrexate, cAMP, and α-ketoglutarate. Northern blot analysis indicated differential tissue distribution for the transporter transcripts. These results indicate the existence of a family of organic anion transporting proteins in humans distinct from the oatp-like family of transporters.

Background: The phase 3 COMFORT trials demonstrated that the Janus kinase (JAK)1/JAK2 inhibitor RUX reduces spleen volume, prolongs overall survival (OS), and improves MF−related symptoms and measures of quality of life in patients with intermediate-2 or high-risk MF, compared with either placebo (COMFORT-I) or best available treatment (BAT; COMFORT-II). Many patients with MF are anemic or transfusion-dependent; the impact of these features on clinical outcomes is unknown. We evaluated the relationship between transfusion requirement and clinical outcomes in patients treated with RUX in the COMFORT studies. Methods: Analyses of data pooled from COMFORT-I and -II were stratified by baseline anemia status (defined as receiving ≥2 units of red blood cells [RBCs] within the 12 weeks before baseline or baseline hemoglobin [Hb] Results: Overall, 301 patients were randomized to RUX (baseline anemia, n=138 [45.8%]) and 227 to the control group (placebo or BAT; baseline anemia, n=113 [49.8%]). In the RUX group, a greater proportion of patients who were nonanemic at baseline (range, 73.4%-73.8%) achieved transfusion independence compared with those who had anemia at baseline (range, 15.5%-22.4%). Week 24 transfusion independence vs nonindependence status did not significantly affect OS in the RUX group (P=0.1322; Figure A), whereas it was significant (P=0.0004; Figure B) in the control group. Similarly, Week 24 transfusion dependence vs nondependence status did not significantly affect OS in the RUX group (P=0.4547; Figure C), whereas it was significant (P=0.0323; Figure D) in the control group. Median OS was significantly longer in the RUX vs control group for patients who were not transfusion independent (baseline anemia, 200 vs 137 weeks; nonanemic, 271 vs 166 weeks; overall P=0.002) or became transfusion dependent (baseline anemia, 210 vs 127 weeks; nonanemic, 292 vs 90 weeks; overall P=0.0323). Changes from baseline in spleen volume, body weight, and TSS at Week 24 were not affected by transfusion or anemia status in the RUX group; however, TSS worsened in the control group among patients who did not achieve transfusion independence vs those who did. Risk of transfusion dependence decreased after Week 24 in the RUX group. The probability of becoming transfusion independent after 1 year of treatment was similar in both treatment groups (approximately 0.75); median time to transfusion independence for the RUX and control groups was 16.6 and 12.0 weeks, respectively. For patients in the RUX group who became transfusion dependent, the mean monthly units of RBCs peaked at Week 20 (2.82 units), decreasing thereafter to 0.52 units at Week 240. Transfusion dependence did not affect RUX discontinuation rates or dosage. Conclusion: Transfusion requirement had little impact on clinical outcomes or treatment discontinuation within the RUX group but was associated with reduced OS and worsened TSS in the control group. The risk of becoming transfusion dependent, units of RBCs administered, and the monthly proportion of patients requiring transfusions decreased rapidly after 24 weeks of RUX treatment. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Verstovsek:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; Geron: Research Funding; Lilly Oncology: Research Funding; AstraZeneca: Research Funding; Roche: Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding. Paquette:Bristol-Myers Squibb: Research Funding, Speakers Bureau; Ariad: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kiladjian:AOP Orphan: Research Funding; Novartis: Research Funding. Cervantes:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sun:Incyte Corporation: Employment, Equity Ownership. Gao:Incyte Corporation: Employment, Equity Ownership. Langmuir:Incyte Corporation: Employment, Equity Ownership. Gopalakrishna:Novartis Pharma AG: Employment, Equity Ownership. Harrison:Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Incyte Corporation: Honoraria, Speakers Bureau.

Background: MF is a life-shortening complication of myeloproliferative neoplasms associated with ineffective hematopoiesis, splenomegaly, cytopenias, debilitating symptoms, and progressive BM fibrosis The 2 phase 3 COMFORT studies have shown that RUX, an oral Janus kinase (JAK) 1/JAK2 inhibitor, improves splenomegaly, constitutional symptoms, and overall survival in patients with MF. Accumulating evidence suggests that RUX may also modulate the BM microenvironment. Aims: We evaluated the effects of long-term RUX treatment on changes in BM fibrosis in patients with intermediate-2 or high-risk primary MF, post-polycythemia vera MF, or post-essential thrombocythemia MF who were enrolled in the phase 3 COMFORT-I study. Methods: BM biopsies were obtained at baseline (BL), Weeks 48 and 72, and approximately every 48 weeks thereafter for up to 5 years of RUX treatment. Biopsies were reviewed independently in a blinded fashion (blinded for patient and treatment) by 3 hematopathologists (HMK, JT, and CEB-R). The final grading was based on consensus; no disagreements were recorded. The WHO grading system was used to grade BM fibrosis density based on a scale of 0-3 (Thiele et al, Haematologica 2005;90). Other details on the patient population and study design for the COMFORT-I study have been published previously (Verstovsek et al, N Engl J Med 2012;366). Biopsies from 59 patients were included in this exploratory analysis; patients who failed screening or received only 1 BM measurement were excluded. Three subgroups were defined for the analysis: 1) originally randomized to RUX (n=36); 2) randomized to placebo with BM measurements at BL and Week 48 (n=15); and 3) crossover to RUX with BM measurements at BL and ≥1 post-BL measurement after crossover (n=21). Changes from BL in BM fibrosis grades at various time points were categorized for each patient as improvement (-1 to -3), stabilization (0), or worsening (1 to 3). Patients with a BL score of 0 for improvement and 3 for worsening were excluded from the analysis. Patients who received placebo for ≥36 weeks were included in the crossover group, with Week 48 used as the BL BM measurement. RUX and crossover groups were combined for evaluation of RUX effect. Placebo effect in the crossover group was assessed by analyzing change from BL to Week 48. Change from BL was evaluated using a signed rank test. Change from BL to last grade, and time to the first occurrence of a ≥1 grade improvement from BL was assessed for RUX and crossover groups. KM analysis was used to estimate time to improvement in BM fibrosis for a subgroup of patients who had a BM fibrosis grade of ≥1 at BL. Results: BL characteristics for age, gender, International Prognostic Scoring System risk, spleen volume, hemoglobin, and platelet counts were similar between the 3 groups. At BL, of 36 patients originally randomized to RUX, 17% (n=6) presented with WHO-defined fibrosis grade 1, 39% (n=14) with grade 2, and 36% (n=13) with grade 3 (3 patients were grade 0). Of the 15 patients randomized to placebo, 20% (n=3) presented with grade 1, 40% (n=6) with grade 2, and 27% (n=4) with grade 3 WHO-defined fibrosis at BL (2 patients were grade 0). Mean exposure to RUX in the RUX and crossover groups was 136.0 (SD, 67.4) weeks and 129.1 (SD, 67.7) weeks, respectively. The proportion of evaluable patients with an improvement in BM fibrosis from BL to Week 48 was 26% (n=27) in the RUX group and 15.4% (n=13) in the placebo group. When evaluating all patients who received RUX (including placebo crossover), a significant shift was observed from BL to the last change in BM fibrosis grade (P=0.0119; signed rank test). For all RUX-treated patients (n=57), 33% (grade -1, n=11; -2, n=7; -3, n=1) had an improvement, 49% had no change or stabilization, and 18% had a worsening in BM fibrosis from BL to the last grade (Figure). At the final grading, 82% (n=47) of patients had improvement or stabilization while on RUX. Median time to a ≥1 grade improvement in BM fibrosis grade was approximately 3.5 years (95% CI, 2.5 to 4.5; n=51). Conclusions: This analysis from the COMFORT-I study showed that treatment with RUX was associated with improvement and stabilization in WHO-defined BM fibrosis in the majority of patients with MF in this study cohort. These results support evidence from other studies, suggesting that RUX treatment may contribute to disease-modifying effects in MF. The clinical effect of improvement and stabilization in BM fibrosis requires further study. Disclosures Kvasnicka: Novartis: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria; AOP Pharma: Consultancy, Honoraria. Thiele:Novartis: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria. Sun:Incyte Corporation: Employment, Equity Ownership. Naim:Incyte Corporation: Employment, Equity Ownership. Svaraman:Incyte Corporation: Employment, Equity Ownership. Gao:Incyte Corporation: Employment, Equity Ownership. Gotlib:Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gupta:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Dao:Incyte Corporation: Research Funding. Talpaz:Incyte Corporation: Other: Travel expense reimbursement, Research Funding; Novartis: Research Funding; Ariad: Other: Expense reimbursement, travel accomodation expenses, Research Funding; Pfizer: Consultancy, Other: travel accomodation expenses, Research Funding. Winton:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:AstraZeneca: Research Funding; Roche: Research Funding; Celgene: Research Funding; Lilly Oncology: Research Funding; Galena BioPharma: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Geron: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Genentech: Research Funding.

Background:The Janus kinase (JAK) 1/JAK2 inhibitor ruxolitinib has been evaluated for patients with MF in the phase 3 COMFORT studies. In both trials, ruxolitinib prolonged OS, reduced splenomegaly, and improved MF-related symptoms and quality of life compared with controls. Here, we report the results of an exploratory pooled analysis of OS in the COMFORT studies at 5 years of follow-up. Methods: The double-blind COMFORT-I trial and the open-label COMFORT-II trial were randomized phase 3 studies that evaluated the safety and efficacy of ruxolitinib in patients with intermediate-2 (int-2) or high-risk primary MF (PMF), post-polycythemia vera MF (PPV-MF), or post-essential thrombocythemia MF (PET-MF). The comparator was placebo in COMFORT-I and best available therapy (BAT) in COMFORT-II. The ruxolitinib starting dose was 15 or 20 mg twice daily based on baseline platelet counts (100-200 and >200 × 109/L, respectively); dose modifications were permitted for safety and efficacy. Patients were allowed to cross over to ruxolitinib from the control arm for progressive splenomegaly, defined as a ≥25% increase in spleen volume from baseline (COMFORT-I) or study nadir (COMFORT-II), or select protocol-defined progression events; crossover was mandatory following treatment unblinding in COMFORT-I. OS was a secondary endpoint in both studies and was evaluated in an intent-to-treat (ITT) analysis using a Cox proportional hazard model that estimated the treatment effect stratified by clinical trial and International Prognostic Scoring System (IPSS) risk. The crossover-corrected treatment effect was estimated using a rank-preserving structural failure time (RPSFT) method. Results: Overall, 528 patients were randomized: 301 to ruxolitinib (COMFORT-I, n=155; COMFORT-II, n=146) and 227 to placebo (n=154) or BAT (n=73). All ongoing patients in the control arms crossed over to ruxolitinib by the 3-year follow-up. Patient populations were similar between the two trials and their details were previously published. In the combined ruxolitinib group, 162 patients (53.8%) had high-risk MF and 139 (46.2%) had int-2 risk MF based on IPSS criteria. At the 5-year ITT analysis, 128 patients (42.5%) died in the ruxolitinib group compared with 117 (51.5%) in the control group. The risk of death was reduced by 30% with ruxolitinib compared with control (median OS: ruxolitinib, 63.5 mo; control, 45.9 mo; HR, 0.70; 95% CI, 0.54-0.91; P=0.0065; Figure A). After correcting for crossover using RPSFT, OS advantage was more pronounced for patients originally randomized to ruxolitinib (median OS: ruxolitinib, 63.5 mo; control, 27 mo; HR, 0.35; 95% CI, 0.23-0.59; Figure B). An analysis of OS censoring patients at the time of crossover also demonstrated that ruxolitinib prolonged survival compared with control (median OS: ruxolitinib, 63.5 mo; control, 28.3 mo; HR, 0.53; 95% CI, 0.36−0.78; P=0.0013; Figure C). Among all patients treated with ruxolitinib, those with lower-risk disease had longer survival compared with those with high-risk disease (median OS: int-2, not reached [estimated, 102 mo]; high-risk, 50 mo; HR, 2.86; 95% CI, 1.95-4.20; P65 or ≤65 y), sex, disease type (PMF, PPV-MF, PET-MF), risk status (int-2 or high), JAK2V617F mutation status, baseline spleen volume (>10 or ≤10 cm), anemia, white blood cell count (>25 or ≤25 × 109L), or platelet count (>200 or ≤200 × 109/L). Conclusion: Long-term treatment with ruxolitinib up to 5 years prolonged survival in patients with MF compared with BAT or placebo. Corrections for patients who crossed over to ruxolitinib suggested that the separation between ruxolitinib and control OS curves was primarily caused by a delay in ruxolitinib treatment. The results suggest that earlier treatment with ruxolitinib may provide a greater survival advantage for patients with MF. Disclosures Gupta: Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mesa:Incyte: Research Funding; Ariad: Consultancy; Novartis: Consultancy; Celgene: Research Funding; CTI: Research Funding; Promedior: Research Funding; Galena: Consultancy; Gilead: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kiladjian:AOP Orphan: Research Funding; Novartis: Research Funding. Cervantes:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sun:Incyte Corporation: Employment, Equity Ownership. Gao:Incyte Corporation: Employment, Equity Ownership. Dong:Novartis Pharmaceutical Corporation: Employment, Equity Ownership. Naim:Incyte Corporation: Employment, Equity Ownership. Gopalakrishna:Novartis Pharma AG: Employment, Equity Ownership. Harrison:Incyte Corporation: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau.

SummarySeveral members of a family from Scranton, Pennsylvania were identified to have normal levels of prothrombin antigen while their prothrombin clotting activity was approximately 50% of normal. There has been no previous history of bleeding or other clinical manifestations in this family. The genomic DNA from the proband was amplified for all exons in the prothrombin gene and analyzed by single strand conformation polymorphism (SSCP)/heteroduplex analysis followed by DNA sequence analysis and restriction enzyme digestion. A mutation at nucleotide 20040 in exon 14 was identified and confirmed by restriction enzyme digestion. This mutation results in the substitution of Thr for Lys at amino acid 556. Amino acid 556 has been reported as one of the key residues for the binding of Na+ in the thrombin portion of the protein.

Srdan Verstovsek,1 Jason Gotlib,2 Vikas Gupta,3 Ehab Atallah,4 John Mascarenhas,5 Alfonso Quintas-Cardama,1 William Sun,6 Nicholas J Sarlis,6 Victor Sandor,6 Richard S Levy,6 Hagop M Kantarjian,1 Ruben A Mesa71University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Stanford Cancer Institute, Stanford, CA, USA; 3Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada; 4Medical College of Wisconsin, Milwaukee, WI, USA; 5Mount Sinai School of Medicine, New York, NY, USA; 6Incyte Corporation, Wilmington, DE, USA; 7Mayo Clinic, Scottsdale, AZ, USAPurpose: Ruxolitinib is an oral Janus kinase (JAK) 1/JAK2 inhibitor approved in the US for the treatment of intermediate- or high-risk myelofibrosis (MF). Because thrombopoietin and erythropoietin signal through JAK2, dose-dependent cytopenias are expected with treatment. In the COMFORT-I (COntrolled Myelofibrosis study with Oral JAK inhibitor Treatment I) trial, these cytopenias were effectively managed with dose adjustments. These analyses were conducted to evaluate the relationship between ruxolitinib titrated doses and changes in platelet count and hemoglobin level as well as efficacy measures.Patients and methods: COMFORT-I was a randomized, placebo-controlled trial in 309 patients with intermediate-2 or high-risk MF and a platelet count ≥100 × 109/L. Ruxolitinib starting doses were 15 and 20 mg twice daily (bis in die [BID]) for patients with baseline platelet counts of 100–200 × 109/L and >200 × 109/L, respectively. Percentage changes from baseline to week 24 in spleen volume and MF-related symptoms were assessed in subgroups defined by final titrated dose (average daily dose during weeks 21 to 24).Results: The median final titrated doses for patients starting at doses of 15 and 20 mg BID were 10 and 20 mg BID, respectively, at week 24. Most dose reductions occurred in the first 8–12 weeks of treatment and coincided with decreases in platelet count and hemoglobin level. Subsequently, platelet counts stabilized and hemoglobin levels gradually returned to near baseline levels (red blood cell transfusion rates followed a similar trend). Final titrated doses of ≥10 mg BID were associated with clinically meaningful improvements in MF-related symptoms that were comparable across doses, while marginally greater reductions in spleen volume were observed at higher doses.Conclusion: This COMFORT-I analysis shows that dose-dependent cytopenias were effectively managed with ruxolitinib dose adjustments, and titrated doses of ≥10 mg BID were associated with clinically meaningful reductions in spleen volume and symptom improvement at week 24.Keywords: COMFORT-I, dose titration, JAK2 inhibitor, myelofibrosis, ruxolitinib, treatment-related cytopenias

Three members of a San Antonio, Texas, family were identified with prothrombin activity levels half the normal level but to have normal levels of antigen. All exons of the prothrombin gene from the proband were sequenced. A G-to-A mutation at nucleotide 7543 was found that resulted in the substitution of His for Arg at residue 320. The Arg320-Ile321 bond is 1 of 2 sites in prothrombin cleaved by Factor Xa in the prothrombinase complex to form thrombin. Substitution of His for Arg at this site resulted in the blockage of Factor Xa cleavage, forming a dysfunctional molecule. The proband, her mother, and her maternal aunt were found to be heterozygous for this mutation. This is the first known observation of an amino acid substitution at this site that resulted in dysprothrombinemia.

Prothrombin and thrombin are involved in diverse biological functions. The structure of prothrombin has been studied extensively and its cDNA has been cloned from several species. The tissue-specific expression of this protein has been studied, as well as the developmental expression pattern. The structure of the human gene coding for prothrombin has been determined, and gene regulation studies have been performed that indicate that HNF-1 might be responsible for the liver-specific expression of this protein. Other regulatory elements have been identified. In order to further study the biological properties of prothrombin, prothrombin-deficient mice have been generated using gene targeting technology. Prothrombin deficiency in mice results in partial embryonic lethality. The mice that survive to birth die from bleeding events. The embryonic lethality occurs between embryonic days 9.5 and 11.5 and appears to be due to the loss of integrity of the vasculature due to a failure in blood coagulation. These results indicate that prothrombin plays not only a key role in hemostasis but suggests that it may be important for mouse development.

Purpose There is a lack of definitive cell surface markers to differentiate cultured human corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used to identify and purify HCECs from stromal fibroblasts. Methods RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in HCECs and commercial antibodies against these overexpressed antigens were screened by immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by fluorescence-activated cell sorting (FACS). Results Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable. Conclusions Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.

Human embryonic stem cells (hESCs) are self-renewing and pluripotent cells that can differentiate into functional cardiomyocytes in vitro. Significant progress has been made over the past decade in improving the differentiation and enrichment of hESC-derived cardiomyocytes. These cells allow investigations into human cardiac biology, and have great potential for applications in cell therapy and drug discovery. However, several technical hurdles must be overcome before their routine use as therapeutic agents or as cellular models for drug screening. This is an exciting area of stem cell research that will have a major impact on cardiovascular medicine. Keywords: Blastocyst; Gastrulation; Pluripotent; Reprogramming; Myocardial infarction; QT interval; Teratoma; Torsades de Pointes; Xenogeneic

A B S T R AC T background Ruxolitinib, a selective inhibitor of Janus kinase (JAK) 1 and 2, has clinically significant activity in myelofibrosis. methodS In this double-blind trial, we randomly assigned patients with intermediate-2 or highrisk myelofibrosis to twice-daily oral ruxolitinib (155 patients) or placebo (154 patients). The primary end point was the proportion of patients with a reduction in spleen volume of 35% or more at 24 weeks, assessed by means of magnetic resonance imaging. Secondary end points included the durability of response, changes in symptom burden (assessed by the total symptom score), and overall survival. resulTS The primary end point was reached in 41.9% of patients in the ruxolitinib group as compared with 0.7% in the placebo group (P

Background: Age, prior thrombotic events, and an elevated hematocrit are established risk factors for increased risk of thrombosis in patients with polycythemia vera (PV). Evidence also suggests that elevated white blood cell (WBC) counts ≥ 11×109/L are a significant independent risk factor for thrombosis in patients with PV (P =0.02; Barbui T, et al. Blood. 2015; 126:560-561). Therefore, this analysis was conducted to evaluate the impact of ruxolitinib (Rux) and best available therapy (BAT) treatment on WBC counts among patients with Baseline WBC counts ≥ 11×109/L in the RESPONSE trial. RESPONSE is a global, multicenter, open-label phase 3 trial investigating Rux and BAT in patients with PV who are resistant to or intolerant of hydroxyurea (HU). In the RESPONSE trial, Rux was associated with durable improvements in hematocrit control without phlebotomy compared with BAT as well as reductions in WBC counts. Methods: Changes from Baseline in WBC counts in the Rux arm (n=110), BAT arm (n=112), and HU subgroup of the BAT arm (n=66) were evaluated as part of an exploratory analysis using the RESPONSE 80-Week analysis dataset. Patient subgroups with Baseline WBC counts ≥ 11×109/L and < 11×109/L were analyzed for changes in WBC counts at Weeks 12, 32, and 80. For patients with Baseline WBC counts ≥ 11×109/L, the proportion of patients who achieved a decrease in WBC counts to ≤ 10×109/L or a ≥50% reduction at Week 12 and Week 32, respectively, was summarized; time to achieve the decrease was analyzed by Kaplan-Meier method. Results: Baseline mean WBC counts were generally similar among patients in the Rux arm, BAT arm, and HU subgroup (17.6×109/L, 19.0×109/L, and 17.4×109/L, respectively). The proportion of patients with Baseline WBC counts ≥ 11×109/L was also similar among the Rux arm, BAT arm, and HU subgroup (75%, 71%, and 70%, respectively). In patients with Baseline WBC counts ≥ 11×109/L, patients treated with Rux had greater mean reductions in WBC counts compared with the BAT arm and HU subgroups, and these reductions were maintained over time; mean changes from Baseline in WBC values at Week 12/Week 32 (×109/L) were -7.7/-7.2 for Rux, -3.2/-4.2 for BAT, and -1.2/-2.2 for HU. In patients with lower Baseline WBC counts, mean values remained stable over time. The change from Baseline to Week 12 for individual patients with Baseline WBC counts ≥ 11×109/L is shown in Figure 1. In patients with Baseline WBC counts ≥ 11×109/L, worsening WBC counts were observed in 10.8% of patients in the Rux arm vs 35.4% in the BAT arm (P =0.0002) and 47.8% in the HU subgroup (P Conclusion: Rux treatment resulted in better control of WBC counts compared to BAT in patients with PV. These changes in the Rux arm occurred early after study initiation (within a median of 8 weeks) and were durable over time. Although RESPONSE was not powered to assess the impact of Rux on thromboembolic events, the lower rate of thromboembolic events observed in the Rux arm vs the BAT arm (1.8 vs 8.2 per 100 patient-years of exposure) is consistent with the observed effects of Rux on hematocrit, WBC counts, and C-reactive protein levels, which are all associated with thromboembolic risk. Disclosures Miller: Incyte Corporation: Honoraria, Research Funding. Kiladjian:Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot); Incyte Corporation: Consultancy; Novartis: Consultancy. Naim:Incyte Corporation: Employment, Equity Ownership. Sun:Incyte Corporation: Employment, Equity Ownership. Gadbaw:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Vannucchi:Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau.

Aims Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. Methods and results The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose–response (0.1–10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization. Conclusion Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.

OBJECTIVE 11-β-hydroxysteroid dehydrogenase type 1 (11βHSD1) converts inactive cortisone into active cortisol, thereby amplifying intracellular glucocorticoid action. The efficacy and safety of the 11βHSD1 inhibitor INCB13739 were assessed when added to ongoing metformin monotherapy in patients with type 2 diabetes exhibiting inadequate glycemic control (A1C 7–11%). RESEARCH DESIGN AND METHODS This double-blind placebo-controlled paralleled study randomized 302 patients with type 2 diabetes (mean A1C 8.3%) on metformin monotherapy (mean 1.5 g/day) to receive one of five INCB13739 doses or placebo once daily for 12 weeks. The primary end point was the change in A1C at study end. Other end points included changes in fasting glucose, lipids, weight, adverse events, and safety. RESULTS After 12 weeks, 200 mg of INCB13739 resulted in significant reductions in A1C (−0.6%), fasting plasma glucose (−24 mg/dl), and homeostasis model assessment–insulin resistance (HOMA-IR) (−24%) compared with placebo. Total cholesterol, LDL cholesterol, and triglycerides were all significantly decreased in hyperlipidemic patients. Body weight decreased relative to placebo after INCB13739 therapy. A reversible dose-dependent elevation in adrenocorticotrophic hormone, generally within the normal reference range, was observed. Basal cortisol homeostasis, testosterone in men, and free androgen index in women were unchanged by INCB13739. Adverse events were similar across all treatment groups. CONCLUSIONS INCB13739 added to ongoing metformin therapy was efficacious and well tolerated in patients with type 2 diabetes who had inadequate glycemic control with metformin alone. 11βHSD1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes.

Mice carrying a conditional prothrombin knockout allele (fIIlox) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fIIlox/lox mice or compound heterozygous mice carrying one fIIlox allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fIIlox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fIIlox allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short (∼5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.

Two members of a family from Carora, Venezuela, were found to have prothrombin activity levels at 4% of normal and undetectable antigen levels. All exons of the prothrombin gene from the proband were sequenced and a mutation at nucleotide 1305 was identified that would result in the substitution of Cys for Tyr at residue 44. Residue 44 is present in the aromatic stack region of the protein. Substitution of a Cys in this region would result in an abnormal folding of the protein which could be the cause for the observed lack of secretion of the abnormal prothrombin.

BACKGROUND: Combining statins with antihypertensive therapy has been demonstrated to provide an early reduction in cardiovascular events. This nested substudy of the AVALON trial assessed the effects of coadministered amlodipine and atorvastatin vs. either therapy alone or placebo on arterial compliance, to evaluate the vascular benefits of coadministered therapy. METHODS: During an initial 8-week, double-blind phase, patients with concomitant hypertension and dyslipidemia were randomized into four treatment groups (placebo, amlodipine 5 mg, atorvastatin 10 mg, or coadministered amlodipine 5 mg and atorvastatin 10 mg). The sustained effect of combined therapy was evaluated during subsequent 8-week, single-blind, and 12-week, open-label periods. In the single-blind phase, all patients were coadministered amlodipine 5 mg and atorvastatin 10 mg, which were then titrated to optimize blood pressure and low-density lipoprotein cholesterol control during the open-label phase. Arterial compliance was assessed every 4 weeks using the HDI/Pulsewave CR-2000. RESULTS: Overall, 668 patients (61% male, mean age 55 years) were randomized to treatment. A 19% improvement in small artery compliance (C2) was observed with coadministered amlodipine and atorvastatin from baseline to week 8, which was significantly greater than with either treatment alone or with placebo (P = 0.03 to 0.0001). After 28 weeks, C2 was increased from baseline in all groups, but the overall improvement was greatest in the group receiving coadministered drugs for the entire study period (P < 0.05). CONCLUSIONS: Early and sustained improvement in small artery compliance was observed following coadministration of amlodipine and atorvastatin, thus demonstrating a vascular benefit with simultaneous treatment of hypertension and dyslipidemia.

Current efforts to direct differentiation of human embryonic stem cells (hESC) into a particular cell lineage usually lead to a heterogeneous cell population with only a fraction of the desired cell type present. We show the generation of an essentially pure population of human cardiomyocytes from hESC using lineage selection.A construct comprising the murine alpha-myosin heavy chain (alpha-MHC) promoter driving the neomycin-resistance gene was introduced into hES3 cells to generate stable transgenic lines. Transgenic hESC lines were differentiated into cardiomyocytes and subjected to G418 selection. Both G418-selected and non-selected cardiomyocytes were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The teratoma-forming potential of differentiated cells was assessed by injection of about 2 million cells into the hind limb muscle of SCID mice. Results After cardiac differentiation and antibiotic selection in a suspension culture process, more than 99% of the transgenic cells showed immunoreactivity to alpha-MHC and alpha-actinin; this enrichment efficiency was observed for independent transgenic cell lines. Quantitative RT-PCR analysis revealed high levels of enrichment for cardiac-specific messages in the selected population. Importantly, injection of selected cells into six SCID mice resulted in no apparent teratoma formation, in contrast to differentiated but non-selected controls.Our results represent a significant step toward scalable production of pure human cardiomyocytes from stable, expandable hESC lines that will facilitate the development of cell therapies, safety pharmacology and drug discovery.

Human embryonic stem (hES) cells represent a potentially unlimited source of transplantable beta-cells for the treatment of diabetes. Here we describe a differentiation strategy that reproducibly directs HES3, an National Institutes of Health (NIH)-registered hES cell line, into cells of the pancreatic endocrine lineage. HES3 cells are removed from their feeder layer and cultured as embryoid bodies in a three-dimensional matrix in the presence of Activin A and Bmp4 to induce definitive endoderm. Next, growth factors known to promote the proliferation and differentiation of pancreatic ductal epithelial cells to glucose-sensing, insulin-secreting beta-cells are added. Pdx1 expression, which identifies pancreatic progenitors, is detected as early as day 12 of differentiation. By day 34, Pdx1+ cells comprise between 5% and 20% of the total cell population and Insulin gene expression is up-regulated, with release of C-peptide into the culture medium. Unlike another recent report of the induction of insulin+ cells in differentiated hES cell populations, we are unable to detect the expression of other pancreatic hormones in insulin+ cells. When transplanted into severe combined immunodeficiency (SCID) mice, differentiated cell populations retain their endocrine identity and synthesize insulin.

Objective To test the primary study hypothesis that a physician-delivered coronary heart disease risk evaluation and communication program can lower patients' predicted 10-year risk of myocardial infarction or death due to coronary heart disease by 10% within 6 months compared to usual care. Design Prospective, parallel group, open-label, controlled, cluster-randomized multinational trial; the study site is the unit of randomization. Setting Patients were recruited from 106 general practices located in nine European countries. Patients Men and women aged 45 to 64 (N = 1500) with a documented history of hypertension (treated or untreated), systolic blood pressure ≥ 140 mmHg (or ≥ 130 mmHg in the presence of renal or kidney disease), no history of cardiovascular disease, and a predicted 10-year risk of myocardial infarction or death due to coronary heart disease ≥ 10%. Intervention Sites were randomized to deliver a physician-directed coronary heart disease risk communication and education program or usual care. The intervention program included informing patients of their 10-year risk of myocardial infarction or death due to coronary heart disease, educating patients about modifiable risk factors and their control, and three follow-up phone calls by a physician or study nurse. Main outcome measure Predicted 10-year risk of myocardial infarction or death due to coronary heart disease at 6 months. Conclusions REACH OUT will evaluate a novel, patient-focused, physician-implemented application of coronary heart disease risk equations. Results of the study will be of practical relevance to physicians, health care organizations, and those who issue clinical guidelines for the reduction of cardiovascular risk.

The AVALON study was a randomized, multicenter trial to assess the efficacy and safety of coadministered amlodipine and atorvastatin in patients with hypertension and dyslipidemia. Phase one was an 8-week, double-blind, double-dummy, placebo-controlled period whereby patients received amlodipine 5 mg, atorvastatin 10 mg, amlodipine 5 mg and atorvastatin 10 mg, or placebo. Thereafter, all patients received single-blind amlodipine 5 mg and atorvastatin 10 mg for 8-weeks, followed by 12 weeks of open-label treatment where doses could be titrated to improve low-density lipoprotein cholesterol and blood pressure control. A total of 847 patients entered the double-blind phase. At Week 8, 45% of the patients receiving amlodipine 5 mg and atorvastatin 10 mg reached both their blood pressure and low-density lipoprotein cholesterol goals, compared with 8.3% with amlodipine (p

Background: The Janus kinase inhibitor ruxolitinib (RUX) has recently been shown to induce a meaningful reduction in bone marrow (BM) fibrosis in patients with intermediate-2 or high-risk myelofibrosis (MF) compared with best available therapy (BAT). The altered BM microenvironment in MF is generally associated with prominent pro-inflammatory changes and high levels of corresponding cytokines. We evaluated the complex BM stromal changes following up to 5 years of RUX therapy in a cohort of patients (pts) with primary, post-polycythemia vera, or post-essential thrombocythemia MF. Methods: BM biopsies were performed at protocol-defined intervals for RUX-treated pts at baseline (BL) and at 24 months (mo) (68 pts), 48 mo (38 pts), 54 mo (23 pts), 60 mo (10 pts), or 66 mo (4 pts). To compare the effects of long-term therapy on BM fibrosis we additionally included a historical cohort of pts with primary MF treated with BAT. Samples from the BAT cohort (192 pts) were matched with those from the RUX cohort according to BM morphology at BL. Analysis in the RUX cohort included immunohistochemical assessment of therapy-induced changes by applying specific antibodies (CD61 – megakaryocytes [MEG]; CD68 – macrophages [MAC]; MUM1 – plasma cells [PLC]). Changes in these parameters were determined by consensus after independent blinded review by 3 pathologists (HMK/JT/CBR). Results were categorized as improvement, stabilization, or worsening from BL. Changes in serum levels of 73 cytokines (BL vs 4 weeks) were available in 48 (of the 68; 71%) pts of the RUX cohort. Logarithmic transformation was applied to both BL and week 4 cytokine values, and a stepwise logistic regression method was implemented to identify significant predictors. A composite variable was constructed that reflects a linear combination of the most important predictors using the slope from the logistic analysis as the coefficients. ROC curves with the status of BM response as the dependent variable were generated and the area under the curve (AUC) was calculated as a measure of accuracy. Results: In line with previous results, the odds for improvement or stabilization of BM fibrosis were greater with RUX at all time points, whereas the odds of worsening were markedly reduced with RUX vs BAT. Improvement or stabilization of BM fibrosis with RUX was associated with concordant changes in both collagen deposition and osteosclerosis. As an indicator of therapy-induced reduction in the BM inflammatory stromal reaction, in more than 70% of pts a decrease in the amount of PLC was observed. In 48% of patients, RUX induced a reduction of MAC, while in 39% of cases their number remained unchanged. MEG frequency was reduced in 52% of cases post–RUX therapy, while an increase was observed in only 6.8% of samples. Furthermore, MEG atypia was reduced in 37% of cases, while in 55% of patients a stabilization effect of therapy was seen for this parameter. Multivariate regression identified 10 cytokines that strongly correlated with morphologic outcomes with different weighting factors for each of the predictive equations: CRP: C-reactive protein, IL-10: Interleukin 10, MDC: Macrophage- derived cytokine, SCF: Stem cell factor, TNFα: Tumor necrosis factor alpha, ApoA1: Apolipoprotein A1, EOT: Eotaxin, Hp: Haptoglobin, IgE: Immunoglobulin E, TIMP1: Tissue inhibitor of metalloproteinase-1. In all pts tested RUX induced a significant change of these identified cytokines by week 4. Individual changes in parameters of BM morphology were strongly associated with corresponding changes in cytokine levels. Decrease of BM fibrosis at 24 mo was strongly associated with reduced cytokine levels (AUC=0.85939, p=0.0134). Additionally, decreased frequencies of MEG, PLC, and MAC showed significant correlations with RUX-induced modulation of cytokines (MEG: AUC=0.942572, p=0.0176; PLC: AUC=0.838135, p=0.0331; MAC: AUC=0.965495, p=0.0033). Conclusions: These results demonstrate that long-term RUX therapy, unlike BAT, causes a marked modulation of the BM cellular microenvironment. The effect observed with RUX was seen across all aspects of the fibrotic process including the inflammatory BM stromal reaction associated with MF. The complex relationships between improvement of the BM microenvironment and RUX-dependent effects on inflammatory cytokines reinforce previous observations suggesting that sustained JAK inhibition may be disease-modifying in MF. Disclosures Kvasnicka: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria. Thiele:AOP Orphan Pharmaceuticals: Consultancy, Honoraria; Incyte Corporation: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria; AOP Orphan Pharmaceuticals: Consultancy, Honoraria. Sun:Incyte Corporation: Employment. Verstovsek:Incyte Corporation: Research Funding.