Correction: Ordoñez, et al.; DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters? Cancers 2019, 11, 1424

The authors would like to make a correction to their published pape

DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters?

Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms

TET2 mutations are associated with specific 5-methylcytosine and 5-hydroxymethylcytosine profiles in patients with chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Comprehensive DNA methylation profiling of chronic myeloproliferative neoplasms and discovery of ZFP36L1, a novel tumour suppressor gene epigenetically regulated by enhancer DNA methylation in myelofibrosis

La Policitemia vera (PV), trombocitemia esencial (TE) y la mielofibrosis primaria (MFP) forman parte de las neoplasias mieloproliferativas crónicas (NMPC). Éstas neoplasias presentan un curso clínico variable, supervivencia prolongada, riesgo de progresión a mielofibrosis secundaria (PV y TE) y un riesgo variable de transformación a leucemia mielode aguda (LMA). Las mutaciones genéticas no consiguen explicar la heterogeneidad fenotípica y clínica de las NMPC, no obstante su prevalencia y diversidad. La epigenética y en concreto la metilación del DNA podrían contribuir a explicar la heterogeneidad observada en las NMPC. Se realizó un estudio de la metilación del DNA en NPMC mediante microarrays de metilación. En la primera parte, se estudió la metilación del DNA mediante el array Illumina Infinium® 27k en muestras de PV, ET, MFP y transformaciones a LMA; en la segunda parte, se estudió la mielofibrosis (MF) tanto primaria como secundaria, utilizando el array de Illumina Infinium® 450K (segunda generación). El análisis bioinformático se realizó identificando regiones diferencialmente metiladas y posteriormente utilizando herramientas bioinformáticas para contextualizar regiones/genes diferencialmente metilados. En la primera parte se analizaron 71 muestras (24 PV, 23 TE, 24 MFP), 13 muestras de transformación a LMA y 8 controles sanos. Se encontraron 176 regiones diferencialmente metiladas, que permitieron segregar las muestras de donantes sanos y las de NMPC mediante clusterización supervisada. El estudio de los genes diferencialmente metilados reveló que la vía NF-kB se encuentra alterada por perdida de metilación en las NMPC (Ingenuity Pathway analysis) y que los genes identificados están involucrados en procesos celulares relevantes (Gene Ontology) para el fenotipo neoplásico. El perfil de metilación de las NMPC transformadas permitió segregar las muestras de transformación a LMA de las NMPC en fase crónica y las muestras de donantes sanos, sugiriendo que la metilación del DNA contribuye al proceso de transformación a LMA. En la segunda parte se analizaron 22 muestras de MFP, 17 muestras de MF secundaria y 8 donantes sanos. Los perfiles de metilación del DNA de la MF primaria y secundaria fueron comparables, permitiendo agrupar la MF en una única cohorte. Se observó un enriquecimiento significativo de metilación aberrante en las regiones enhancer, nunca antes descrito en la MF. Utilizando los enhancers diferencialmente metilados, se obtuvo un perfil de metilación de DNA que permitió segregar la MF de los donantes sanos, con clara predominancia de la pérdida de metilación del DNA. Los genes adyacentes a los 9000 enhancers mostraron enriquecimiento en procesos celulares relevantes (Gene Ontology) y una correlación inversa entre la hipermetilación y la expresión, sugiriendo que la metilación de los enhancers es relevante para el control de la expresión génica en MF. Se obtuvo una lista de 27 genes candidatos con hipermetilación de su enhancer y se escogió ZFP36L1 para estudios funcionales. Se demostró que el enhancer de ZFP36L1 tiene ganancia de metilación y pérdida de expresión en muestras independientes de MF y en líneas celulares mieloides. Experimentos con el vector p-CPGL, demostraron la funcionalidad de la región enhancer de ZFP36L1, sugiriendo que ésta región es suficiente para la regulación de la expresión del gen. La proteína ZFP36L1 participa en el control de la degradación exosómica del mRNA. La re-expresión del ZFP36L1 en la línea celular SET-2 (infección lentiviral o PMA) consiguió alterar el fenotipo neoplásico, asociándose a una reducción de la proliferación celular, aumento de apoptosis y a una disminución de la expresión de CDK6. La pérdida epigenética de ZFP36L1 podría asociarse a una menor degradación y aumento de la disponibilidad de mRNA CDK6, suscitando progresión descontrolada del ciclo celular y constituyendo ZFP36L1 como un potencial supresor tumoral en MF

Correction: Ordoñez, et al.; DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters? Cancers 2019, 11, 1424

The authors would like to make a correction to their published pape

DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters?

Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms

Seguridad y eficacia de un concentrado de complejo protrombínico en pacientes con coagulopatía y hemorragia

Fundamento. Los concentrados de factores del complejo protrombínico (CCP) están indicados para reversión del efecto de antagonistas de vitamina K (AVK). Recientemente se han utilizado en el manejo de la coagulopatía de la hemorragia masiva. El objetivo del presente trabajo es evaluar la seguridad y eficacia del CCP en dos situaciones clínicas, para reversión de AVK y manejo integral de la hemorragia masiva. Material y métodos. Revisión retrospectiva de los casos tratados con CCP entre enero de 2010 y febrero de 2013 en un único centro universitario. El objetivo primario fue la seguridad de administración del CCP en cuanto a reacciones inmediatas y episodios trombóticos. El objetivo secundario fue la eficacia, en 2 grupos: 1) Reversión de AVK y 2) Corrección de coagulopatía en hemorragia masiva. Resultados. El análisis de seguridad incluyó 31 pacientes (22 varones), edad mediana 61 años (rango 30-86). No se registraron reacciones adversas durante la infusión y solo se observó un evento trombótico. La eficacia en la reversión de AVK fue del 100% (6/6 pacientes), alcanzando normalización del INR en todos los pacientes. En hemorragia masiva, la supervivencia a las 24 horas fue 64% (16/25). Se requirieron procedimientos invasivos adicionales en 28% de los pacientes (7/25). El uso de CCP se asoció a cese de hemorragia en 44% de los pacientes (11/25), que correlacionó positivamente con la supervivencia (p=0,01). Conclusión. El uso de CCP es una alternativa segura y eficaz, para la reversión urgente del efecto de AVK, así como para el control de sangrado en situación de hemorragia masivaBackground. Prothrombin complex concentrates (PCC) are approved for urgent reversal of vitamin K antagonists (VKA). Recently, PCC have been used in the management of massive bleeding-associated coagulopathy. The present work evaluates safety and efficacy of PCC in a case series of both VKA reversal and massive bleeding. Methods. Retrospective review of cases treated with CCP (January 2010 to February 2013). Safety endpoints were infusion reactions and incidence of thromboembolic events. Efficacy endpoints were: 1) VKA reversal efficacy and 2) Massive bleeding coagulopathy reversal and 24h mortality. Results. Thirty-one patients were included (22 male), median age 61 years (range 30-86). No infusion reactions were detected, and only 1 thrombotic episode was observed. VKA reversal was effective in 100% of patients (6/6), all of them with complete reversal of INR value. In massive bleeding, 24-hour survival was 64% (16/25). Invasive hemostatic procedures were required in 28% of patients (7/25). CCP use was correlated with bleeding control in 44% of cases (11/25), and also significantly associated with survival (p=0.01). Conclusion. CCP are safe and effective for the novel indication of adjuvant treatment in massive bleeding patients, as well as for traditional urgent reversal of VKA

Seguridad y eficacia de un concentrado de complejo protrombínico en pacientes con coagulopatía y hemorragia

Fundamento. Los concentrados de factores del complejo protrombínico (CCP) están indicados para reversión del efecto de antagonistas de vitamina K (AVK). Recientemente se han utilizado en el manejo de la coagulopatía de la hemorragia masiva. El objetivo del presente trabajo es evaluar la seguridad y eficacia del CCP en dos situaciones clínicas, para reversión de AVK y manejo integral de la hemorragia masiva. Material y métodos. Revisión retrospectiva de los casos tratados con CCP entre enero de 2010 y febrero de 2013 en un único centro universitario. El objetivo primario fue la seguridad de administración del CCP en cuanto a reacciones inmediatas y episodios trombóticos. El objetivo secundario fue la eficacia, en 2 grupos: 1) Reversión de AVK y 2) Corrección de coagulopatía en hemorragia masiva. Resultados. El análisis de seguridad incluyó 31 pacientes (22 varones), edad mediana 61 años (rango 30-86). No se registraron reacciones adversas durante la infusión y solo se observó un evento trombótico. La eficacia en la reversión de AVK fue del 100% (6/6 pacientes), alcanzando normalización del INR en todos los pacientes. En hemorragia masiva, la supervivencia a las 24 horas fue 64% (16/25). Se requirieron procedimientos invasivos adicionales en 28% de los pacientes (7/25). El uso de CCP se asoció a cese de hemorragia en 44% de los pacientes (11/25), que correlacionó positivamente con la supervivencia (p=0,01). Conclusión. El uso de CCP es una alternativa segura y eficaz, para la reversión urgente del efecto de AVK, así como para el control de sangrado en situación de hemorragia masivaBackground. Prothrombin complex concentrates (PCC) are approved for urgent reversal of vitamin K antagonists (VKA). Recently, PCC have been used in the management of massive bleeding-associated coagulopathy. The present work evaluates safety and efficacy of PCC in a case series of both VKA reversal and massive bleeding. Methods. Retrospective review of cases treated with CCP (January 2010 to February 2013). Safety endpoints were infusion reactions and incidence of thromboembolic events. Efficacy endpoints were: 1) VKA reversal efficacy and 2) Massive bleeding coagulopathy reversal and 24h mortality. Results. Thirty-one patients were included (22 male), median age 61 years (range 30-86). No infusion reactions were detected, and only 1 thrombotic episode was observed. VKA reversal was effective in 100% of patients (6/6), all of them with complete reversal of INR value. In massive bleeding, 24-hour survival was 64% (16/25). Invasive hemostatic procedures were required in 28% of patients (7/25). CCP use was correlated with bleeding control in 44% of cases (11/25), and also significantly associated with survival (p=0.01). Conclusion. CCP are safe and effective for the novel indication of adjuvant treatment in massive bleeding patients, as well as for traditional urgent reversal of VKA

Anti-PD1 associated fulminant myocarditis after a single pembrolizumab dose: the role of occult pre-existing autoimmunity

Multiple myeloma is a promising candidate for anti-PD1 checkpoint inhibitor therapy.1–3 Results of phase I trials of pembrolizumab, in combination with lenalidomide or pomalidomide in relapsed/refractory patients have shown encouraging results. These trials showed a 35% and 65% response rate in patients already refractory to IMIDs with a median PFS of 7.2 and 14 months for the lenalidomide and pomalidomide combinations, respectively.4,5 These positive results prompted the activation of phase III trials, which are currently underway in relapsed (clinicaltrials.gov identifier 02576977) and first-line setting (clinicaltrials.gov identifier 02579863). Immune-related adverse events (irAE) as a result of uncontrolled activation of autoreactive T-cells,6 are the most important emerging safety issues of checkpoint inhibitors. Myocarditis is rare among the irAE; however, several cases of lethal immune-related myocarditis have recently been published.7–9 The Nivolumab patient database has revealed an incidence of myocarditis of 0.09% in over 20,000 patients already treated;7 however, this figure may be an underestimation since only symptomatic cases were recorded. Myocarditis seems to be frequent with the nivolumab-ipilimumab combination (0.27%), with two reports of a lethal outcome.8 To the best of our knowledge, no fatal cases have been reported with pembrolizumab or nivolumab as single checkpoint inhibitor agents. Here, we report a newly diagnosed multiple myeloma patient who developed a lethal immune-related myocarditis after a single dose of pembrolizumab, which was combined with lenalidomide and dexamethasone, not with other checkpoint inhibitors