Genome-wide DNA methylation analysis pre- and post-lenalidomide treatment in patients with myelodysplastic syndrome with isolated deletion (5q).

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets.

Observational, Retrospective Evaluation of a New Nurse-Initiated Emergency Department Pain Management Protocol.

BACKGROUND: Triage nurses are important in pain management and in early relief of pain among patients admitted to the emergency department (ED). AIMS: To assess a new nurse-initiated pain management protocol, without the requirement for medical prescription, wich was implemented in October 2016 for patients with moderate or severe pain in the ED. It allows the administration of oral acetaminophen and oral oxycodone chlorydrate during the first evaluation of the patient by a nurse and eliminates the use of codeine or tramadol. METHODS: We conducted a comparative, single-center, retrospective study that looked at the outcomes of a new nursing protocol for patients aged ≥16 years with moderate to severe pain. The primary outcome was the percentage of increase of analgesics delivered by the nurse. RESULTS: A total of 756 patients were included: 377 before and 379 after protocol implementation. Oral analgesic use on admission increased from 44.3% to 57.8% (p < .001), and from 50.2% to 76.6% among patients with severe pain (p < .001). Strong opioid analgesic administration increased from 2.1% to 41.2%. This increase was also observed among those with moderate pain (1.4% to 13.3%; p < .001) and those with severe pain (2.6% to 62.6%; p < .001). Analgesic prescriptions added by the clinician decreased from 28.6% to 21.4% (p = .028). CONCLUSIONS: We observed an increase in analgesic administration after the implementation of a new nurse-initiated pain treatment protocol, especially an increase in oral opioid analgesics, for patients with moderate to severe pain.

Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation.

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ acute lymphoblastic leukemia (ALL) treated with tyrosine kinase inhibitors (TKIs) and allogeneic stem cell transplantation (aSCT). Ninety-seven Ph+ ALL patients (median age 41 years; range 18-64 years) within the prospective multicenter German Multicenter ALL Study Group studies 06/99 (n = 8) and 07/2003 (n = 89) were analyzed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with single nucleotide polymorphism arrays and validated by multiplex ligation-dependent probe amplification. The frequencies of recurrently deleted genes were: IKZF1, 76%; CDKN2A/2B, 45%; PAX5, 43%; BTG1, 18%; EBF1, 13%; ETV6, 5%; RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (P = .023), disease-free survival (P = .012), and remission duration (P = .036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase 1 and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT.

Intranasal sufentanil versus intravenous morphine for acute severe trauma pain: A double-blind randomized non-inferiority study.

BACKGROUND: Intravenous morphine (IVM) is the most common strong analgesic used in trauma, but is associated with a clear time limitation related to the need to obtain an access route. The intranasal (IN) route provides easy administration with a fast peak action time due to high vascularization and the absence of first-pass metabolism. We aimed to determine whether IN sufentanil (INS) for patients presenting to an emergency department with acute severe traumatic pain results in a reduction in pain intensity non-inferior to IVM. METHODS AND FINDINGS: In a prospective, randomized, multicenter non-inferiority trial conducted in the emergency departments of 6 hospitals across France, patients were randomized 1:1 to INS titration (0.3 µg/kg and additional doses of 0.15 µg/kg at 10 minutes and 20 minutes if numerical pain rating scale [NRS] > 3) and intravenous placebo, or to IVM (0.1 mg/kg and additional doses of 0.05 mg/kg at 10 minutes and 20 minutes if NRS > 3) and IN placebo. Patients, clinical staff, and research staff were blinded to the treatment allocation. The primary endpoint was the total decrease on NRS at 30 minutes after first administration. The prespecified non-inferiority margin was -1.3 on the NRS. The primary outcome was analyzed per protocol. Adverse events were prospectively recorded during 4 hours. Among the 194 patients enrolled in the emergency department cohort between November 4, 2013, and April 10, 2016, 157 were randomized, and the protocol was correctly administered in 136 (69 IVM group, 67 INS group, per protocol population, 76% men, median age 40 [IQR 29 to 54] years). The mean difference between NRS at first administration and NRS at 30 minutes was -4.1 (97.5% CI -4.6 to -3.6) in the IVM group and -5.2 (97.5% CI -5.7 to -4.6) in the INS group. Non-inferiority was demonstrated (p < 0.001 with 1-sided mean-equivalence t test), as the lower 97.5% confidence interval of 0.29 (97.5% CI 0.29 to 1.93) was above the prespecified margin of -1.3. INS was superior to IVM (intention to treat analysis: p = 0.034), but without a clinically significant difference in mean NRS between groups. Six severe adverse events were observed in the INS group and 2 in the IVM group (number needed to harm: 17), including an apparent imbalance for hypoxemia (3 in the INS group versus 1 in the IVM group) and for bradypnea (2 in the INS group versus 0 in the IVM group). The main limitation of the study was that the choice of concomitant analgesics, when they were used, was left to the discretion of the physician in charge, and co-analgesia was more often used in the IVM group. Moreover, the size of the study did not allow us to conclude with certainty about the safety of INS in emergency settings. CONCLUSIONS: We confirm the non-inferiority of INS compared to IVM for pain reduction at 30 minutes after administration in patients with severe traumatic pain presenting to an emergency department. The IN route, with no need to obtain a venous route, may allow early and effective analgesia in emergency settings and in difficult situations. Confirmation of the safety profile of INS will require further larger studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT02095366. EudraCT 2013-001665-16.

Mutational hierarchies in myelodysplastic syndromes dynamically adapt and evolve upon therapy response and failure.

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.

Accurate quantification of chromosomal lesions via short tandem repeat analysis using minimal amounts of DNA.

BACKGROUND: Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings. METHODS: For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers. RESULTS: Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77-0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring. CONCLUSIONS: In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

Indication-Specific Effect of a Phytotherapeutic Remedy on Human Fetal Osteoblastic Cells: An in vitro Analysis.

BACKGROUND: Impaired fracture healing is a recurring interdisciplinary medical challenge. Alternative treatment concepts, apart from conventional medicine, are popular, but scientific evidence on their effects is still lacking. Plant-derived substances are widely assumed to support bone homeostasis. To clarify the effects on bone healing mechanisms, a commercially available, homeopathic-spagyric remedy, containing inter alia two herbal substances with assumed osteogenic potential, equisetum arvense and bellis perennis, was analyzed. METHODS: Human fetal osteoblastic (hFOB) 1.19 cells were incubated with the test substance in serial dilutions from 10 to 0.00001%. Cell viability has been evaluated through ATP level (CTG assay) and MTT tetrazolium reduction. Cell proliferation was analyzed by BrdU incorporation and cell migration by wound healing assay (WHA) via image analysis. Additionally, determination of the expression of key genes via real-time PCR and proteins via proteome array for inflammation, cell proliferation, and angiogenesis were performed. RESULTS: An incubation of hFOB 1.19 cells with the test substance for 24/72 h showed no reduction in cell number, viability, or proliferation. Cell migration was unimpaired. The test substance induced inflammatory genes and growth factors along with genes of osseous regeneration (ALP, Col1, IL-1α, IL-6, IL-8, IL-10, Osteocalcin, Osteonectin, RUMX2, TGF, VEGFA). Increased protein expression was found in multiple cytokines, chemokines, and acute phase proteins. CONCLUSION: The test substance did not impair cell vitality parameters (MTT, CTG, BrdU, and WHA). A tendency to activate growth factors, bone regeneration genes, and proteins was shown for osteoblasts, indicating a possible positive effect on osteogenic processes.Störungen des komplexen Prozesses der Knochenheilung stellen auch heutzutage noch eine interdisziplinäre Herausforderung dar. Es existieren zahlreiche alternative Therapiekonzepte, deren Evidenz jedoch häufig nicht belegt ist. Es wird davon ausgegangen, dass pflanzliche Substanzen die Knochenheilung unterstützen können. Wir analysierten die Wirkung eines kommerziellen, homeopathisch-spagyrischen Heilmittels, welches unter anderen zwei Pflanzenstoffe enthält, denen ein osteogenes Potential zugeschrieben wird (Equisetum arvense und Bellis perennis).Es erfolgte eine Inkubation humaner fetaler Osteoblastenzellen (hFOB 1.19) mit der Testsubstanz in absteigender Verdünnung von 10 bis 0.00001%. Die Zellvitalität wurde anhand der Zellzahlbestimmung durch ATP-abhängige metabolische Aktivität mittels CellTiter-Glo® (CTG) Test sowie durch Tetrazolium Reduktion (MTT) evaluiert. Die Zellproliferation wurde durch Inkorporation von Bromdesoxyuridin (BrdU) in die DNA aktiver Zellen analysiert. Der Wound Healing Assay (WHA) diente der Quantifizierung der Zellmigration. Zusätzlich wurde die Expression bestimmter Schlüsselgene mittels real-time PCR und die Proteinexpression via proteom array für Inflammation, Zellproliferation und Angiogenese erhoben.Die Inkubation von hFOB 1.19 mit der Testsubstanz für 24/72 Stunden führte zu keiner Reduktion von Zellzahl, -vitalität oder -proliferation. Auch die Zellmigration war unbeeinträchtigt. Es zeigte sich eine Induktion inflammatorischer Gene, Wachstumsfaktoren sowie Genen der knöchernen Regeneration (ALP, Col1, IL-1α, IL-6, IL-8, IL-10, Osteocalcin, Osteonectin, RUMX2, TGF, VEGFA). Verschiedene Zytokine, Chemokine und Akute Phase Proteine wurden vermehrt exprimiert.Die Testsubstanz hatte keine negativen Auswirkungen auf die gemessenen Zellvitalitätsparameter (MTT, CTG, BrdU and WHA). Es zeigte sich eine Aktivierungstendenz für Wachstumsfaktoren, Gene und Proteine der Knochenregeneration, die auf einen möglichen positiven Effekt der Substanz auf den Prozess des Knochenheilung hinweisen.

Temperature diagnostic to identify high risk areas and optimize Legionella pneumophila surveillance in hot water distribution systems.

Legionella pneumophila is frequently detected in hot water distribution systems and thermal control is a common measure implemented by health care facilities. A risk assessment based on water temperature profiling and temperature distribution within the network is proposed, to guide effective monitoring strategies and allow the identification of high risk areas. Temperature and heat loss at control points (water heater, recirculation, representative points-of-use) were monitored in various sections of five health care facilities hot water distribution systems and results used to develop a temperature-based risk assessment tool. Detailed investigations show that defective return valves in faucets can cause widespread temperature losses because of hot and cold water mixing. Systems in which water temperature coming out of the water heaters was kept consistently above 60 °C and maintained above 55 °C across the network were negative for Legionella by culture or qPCR. For systems not meeting these temperature criteria, risk areas for L. pneumophila were identified using temperature profiling and system's characterization; higher risk was confirmed by more frequent microbiological detection by culture and qPCR. Results confirmed that maintaining sufficiently high temperatures within hot water distribution systems suppressed L. pneumophila culturability. However, the risk remains as shown by the persistence of L. pneumophila by qPCR.

Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.