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.
•Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases.•Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.
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.
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.
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.
ClinicalTrials.gov NCT02095366. EudraCT 2013-001665-16.
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.
•Genomic deletions of CDKN2A/2B are a new independent prognostic risk factor in adult Ph+ ALL.
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.
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•Temperature profiles were generated for hot water distribution systems points-of-use.•Risk assessment based on temperature profile results at control points was developed.•L. pneumophila positive areas were predicted using the risk assessment tool.•A temperature diagnostic flowchart is proposed to identify L. pneumophila risk areas.
Triage nurses are important in pain management and in early relief of pain among patients admitted to the emergency department (ED).
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.
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.
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).
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.
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.
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•Disease-initiating cells in lower-risk MDS are restricted to Lin−CD34+CD38− subset•Myelodysplastic cells in patients reprogram mesenchymal stromal cells•Reprogrammed MSCs are essential for propagation of myelodysplastic syndromes•MDS and niche cells interact via bidirectional signaling crosstalk
Myelodysplastic syndrome (MDS) stem and progenitor cells induce mesenchymal niche cells to adopt an altered expression pattern, which in turn supports MDS stem cell propagation in vivo.
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.
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.
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.
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.
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.
Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of ...relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG.
Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors.
Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells.
Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells.
No relevant conflicts of interest to declare.
Introduction
The acquisition of large-scale chromosomal lesions is a frequent event in malignant disorders. One example is the recurrent deletion of chromosome 5q (del(5q)) in myelodysplastic ...syndromes (MDS). The detection and monitoring of such deletions are important elements in routine clinical diagnostics and cancer genomic studies. Currently established methods for their assessment are metaphase cytogenetics (MC), fluorescence in situ hybridization (FISH) and micro-array based techniques. However, each of these methods harbours specific disadvantages as they depend on (viable) cells, are expensive, labour-intensive or only semi-quantitative. One possible approach to interrogate chromosomal deletions constitutes the assessment of allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Therefore, we aimed to establish a robust, quick and inexpensive PCR assay that measures allelic imbalance at such STR loci in order to reliably estimate frequencies of cells carrying del(5q) from only minute amounts of DNA.
Methods
Genomic DNA (gDNA) was isolated from bone marrow (BM) or blood cells of MDS and acute myeloid leukemia (AML) patients with cytogenetically confirmed del(5q). Based on NCBI UniSTS database, we designed 12 fluorochrome-labelled PCR amplicons with size ranges of 100-400 bp that surround STR loci between chromosomal bands 5q21 and 5q31. Using only 10 ng gDNA, all 12 PCR amplicons were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis on an ABI 3130 Genetic Analyzer. Allele size quantification of informative heterozygous loci was performed using ABI Genemapper software. Furthermore, size calculations of individual alleles were corrected for PCR-stutter, which was estimated from corresponding loci in homozygous samples. Finally, using mesenchymal stromal cells as a germline control, the degree of skewing in the allelic ratios of all informative STR markers in the tumor sample relative to the corresponding allelic ratios in the control was averaged and subsequently translated into fractions of cells carrying the del(5q) lesion.
Results
Application of our novel assay for quantification of del(5q) burden in n=559 samples from n=67 patients revealed a high frequency of informative markers with an average of 7 heterozygous STR loci per patient. The data shows a strong inter-marker concordance with a standard deviation (SD) of 2.3% for del(5q) cell frequencies. Moreover, duplicate analysis of 328 samples revealed an average SD of 0.86%. Most importantly, paired analysis of the proportion of del(5q) cells estimated using interphase-FISH and our PCR assay was carried out for n=9 samples and resulted in strong correlation with r²=0.93. A serial dilution series with deleted and non-deleted gDNA also revealed highly concordant results with r²=0.96. When comparing matched germline and tumor STR profiles, no case of microsatellite instability was detectable in our MDS/AML cohort thus highlighting the suitability of STR based lesion quantification in this disease entity. Furthermore, we could use our large dataset to calculate amplification efficiencies for each locus in order to predict “surrogate” germline profiles eventually allowing us to calculate del(5q) frequencies in tumor samples that lack germline controls. Finally, our assay reliably confirmed molecular remission in BM from del(5q) MDS patients after Lenalidomide treatment, in agreement with concomitant MC analyses.
Conclusion
Using a multiplexed PCR assay for measurement of STRs in deleted chromosomal regions we present a highly adaptable tool for precise quantification of large scale lesions. We show a very good correlation with established methods exemplarily for del(5q) lesions. Even without available germline control our assay provides robust results. Requiring only minute amounts of gDNA, this assay is ideally suitable for copy number quantification in samples for which only residual archival gDNA and no cells for FISH analysis are available. Due to the small amplicon sizes it should also be useful for investigation of fragmented gDNA from formalin-fixed archival specimen. In summary, our newly developed assay offers a mean to obtain quantitative data for basically any large scale chromosomal deletion which contains STRs and should be easily applicable to other clonal diseases.
Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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