Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Left ventricular pumping during the transition-adaptation sequence in preterm infants: impact of the patent ductus arteriosus.

BackgroundPostnatally, the immature left ventricle (LV) is subjected to high systemic afterload. Hypothesizing that LV pumping would change during transition-adaptation, we analyzed the LV in preterm infants (GA≤32+6), clinically stable or with a hemodynamically significant patent ductus arteriosus (hPDA) by applying a pump model.MethodsPumping was characterized by EA (effective arterial elastance, reflecting afterload), EES (end-systolic LV elastance, reflecting contractility), EA/EES coupling ratios, descriptive EA:EES relations, and EA/EES graphs. Data calculated from echocardiography and blood pressure were analyzed by diagnosis (S group: clinically stable, no hPDA, n=122; hPDA group, n=53) and by periods (early transition: days of life 1-3; late transition: 4-7; and adaptation: 8-30).ResultsS group: LV pumping was characterized by an increased EA/EES coupling ratio of 0.65 secondary to low EES in early transition, a tandem rise of both EA and EES in late transition, and an EA/EES coupling ratio of 0.45 secondary to high EES in adaptation; hPDA group: time-trend analyses showed significantly lower EA (P<0.0001) and EES (P=0.006). Therefore, LV pumping was characterized by a lower EA/EES coupling ratio (P=0.088) throughout transition-adaptation.ConclusionsIn stable infants, facing high afterload, the immature LV, enhanced by the physiological PDA, increases its contractility. In hPDA, facing low afterload, the overloaded immature LV exhibits a consistently lower contractility.

Preterm infants who later require duct ligation show different vital signs and pH in early postnatal life.

AIM: The study investigated early postnatal vital signs in very low birthweight (VLBW) infants who later developed patent ductus arteriosus (PDA). We hypothesised that the early postnatal course of vital signs and blood gas variables might differ between infants whose PDA closed spontaneously, those who responded to ibuprofen and those who later required PDA ligation. METHODS: We analysed computerised records of VLBW infants born <28 weeks of gestational age, including vital signs, arterial pH values and echocardiographic data from the first postnatal days. RESULTS: In total, 104 infants were included in the study. In the group of infants born <26 weeks of gestational age and requiring ibuprofen for PDA (n = 34), 12 infants ultimately required surgical ligation. Infants requiring ligation showed significantly lower oxygen saturation (p = 0.019), mean blood pressure (p = 0.034) and higher heart rate fluctuation ranges (p = 0.040) in the first five postnatal days than those who responded to ibuprofen. In multivariable logistic regression analysis, lower pH values in the first 48 h predicted the subsequent requirement for ligation independent of gestational age (p = 0.004). CONCLUSION: Patients <26 weeks of gestational age requiring PDA ligation showed significant differences in the course of vital signs and pH during the first days of life.

Delineation of target expression profiles in CD34+/CD38- and CD34+/CD38+ stem and progenitor cells in AML and CML.

In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38- and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38- and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38- LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38- cells variably expressed "aberrant" membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication-mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38- LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Cerebral regional tissue Oxygen Saturation to Guide Oxygen Delivery in preterm neonates during immediate transition after birth (COSGOD III): an investigator-initiated, randomized, multi-center, multi-national, clinical trial on additional cerebral tissue oxygen saturation monitoring combined with defined treatment guidelines versus standard monitoring and treatment as usual in premature infants during immediate transition: study protocol for a randomized controlled trial.

BACKGROUND: Transition immediately after birth is a complex physiological process. The neonate has to establish sufficient ventilation to ensure significant changes from intra-uterine to extra-uterine circulation. If hypoxia or bradycardia or both occur, as commonly happens during immediate transition in preterm neonates, cerebral hypoxia-ischemia may cause perinatal brain injury. The primary objective of the COSGOD phase III trial is to investigate whether it is possible to increase survival without cerebral injury in preterm neonates of less than 32 weeks of gestation by targeting cerebral tissue oxygen saturation (crSO2) using specified clinical treatment guidelines during the immediate transition period after birth (the first 15 min) in addition to the routine monitoring of arterial oxygen saturation (SpO2) and heart rate (HR). METHODS/DESIGN: COSGOD III is an investigator-initiated, randomized, multi-center, multi-national, phase III clinical trial. Inclusion criteria are neonates of less than 32 weeks of gestation, decision to provide full life support, and parental informed consent. Exclusion criteria are severe congenital malformations of brain, heart, lung, or prenatal cerebral injury or a combination of these. The premature infants will be randomly assigned to study or control groups. Both groups will have a near-infrared spectroscopy (NIRS) device (left frontal), pulse oximeter (right palm/wrist), and electrocardiogram placed immediately after birth. In the study group, the crSO2, SpO2, and HR readings are visible, and the infant will receive treatment in accordance with defined treatment guidelines. In the control group, only SpO2 and HR will be visible, and the infant will receive routine treatment. The intervention period will last for the first 15 min after birth during the immediate transition period and resuscitation. Thereafter, each neonate will be followed up for primary outcome to term date or discharge. The primary outcome is mortality or cerebral injury (or both) defined as any intra-ventricular bleeding or cystic periventricular leukomalacia (or both). Secondary outcomes are neonatal morbidities. DISCUSSION: crSO2 monitoring during immediate transition has been proven to be feasible and improve cerebral oxygenation during immediate transition. The additional monitoring of crSO2 with dedicated interventions may improve outcome of preterm neonates as evidenced by increased survival without cerebral injury. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03166722 . Registered March 5, 2017.

Discrepancy between invasive and non-invasive blood pressure readings in extremely preterm infants in the first four weeks of life.

BACKGROUND: The agreement between invasive and non-invasive blood pressure (BP) readings in the first days of life of preterm infants is contentiously debated. OBJECTIVE: To compare mean, systolic and diastolic invasive (IBP) and non-invasive BP (NBP) readings obtained during routine care in the first four weeks of life of extremely preterm infants. METHODS: We extracted pairs of IBP and NBP readings obtained from preterm infants born below 28 weeks of gestation from the local database. After exclusion of erroneous measurements, we investigated the repeated measures correlation and analyzed the agreement (bias) and precision adjusted for multiple measurements per individual. RESULTS: Among 335 pairs of IBP and NBP readings obtained from 128 patients, we found correlation coefficients >0.65 for mean, systolic and diastolic BP values. The bias for mean BP readings was -0.4 mmHg (SD 6.1), for systolic BP readings 6.2 mmHg (SD 8.1), and for diastolic BP readings -4.3 mmHg (SD 6.5). Overestimation of systolic IBP and underestimation of diastolic IBP by the non-invasive measurement were found both in the group with gestational age from 23 to 25.9 weeks and in the group with gestational age from 26 to 27.9 weeks. Systolic NBP readings tended to exceed invasive readings in the range <50 mmHg (bias 9.9 mmHg) whereas diastolic NBP readings were lower than invasive values particularly in the range >30 mmHg (bias -5.5 mmHg). CONCLUSION: The disagreement between invasive and non-invasive BP readings in infants extends to the first four weeks of life. Biases differ for mean, systolic and diastolic BP values. Our observation implies that they may depend on the range of the blood pressure. Awareness of these biases and preemptive concomitant use of IBP and NPB readings may contribute to reducing over- or under-treatment.

Long-term treatment with imatinib results in profound mast cell deficiency in Ph+ chronic myeloid leukemia.

Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0 ± 11.1 ng/ml; post-therapy: 3.4 ± 1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.

Ventriculo-arterial coupling in children with Still's murmur.

Still's murmur is the most common innocent heart murmur in children and considered flow related; however, so far the cause of the murmur has not yet been fully explained. Assessment of the hemodynamic ventriculo-arterial interaction and the proportional anatomical dimensions of the left ventricle and the aortic root were the objective for this study. This case-control study was conducted at the Division of Pediatric Cardiology, Vienna Medical University, including healthy children with and without Still's murmur. To assess ventriculo-arterial interaction, the model of ventriculo-arterial coupling (VAC) was applied. The model describes the interaction between the left ventricle (left ventricular contractility, ELV) and the arterial system (effective arterial elastance, EA) by the VAC ratio EA/ELV. The parameters EA and ELV can be derived from M-mode echocardiography thereby allowing a noninvasive pressure-volume analysis. Outcomes comprised VAC ratio and diameters of both the aortic root (AOD) and the left ventricle in end diastole (LVED) and end systole (LVES) as well as their relative proportions, ejection fraction (EF), stroke volume (SV), blood pressure (BP), and heart rate (HR). Forty-three healthy children with Still's murmur (mean age 5.2 years) and 42 healthy children without murmur (mean age 5.8 years) participated in this study. Children with Still's murmur had a significantly lower VAC ratio EA/ELV (0.5 ± 0.13 vs. 0.59 ± 0.15; P < 0.005), a significantly higher EF% (67.1 ± 5.8 vs. 63.3 ± 5.6; P < 0.005, P < 0.01), and a larger SV per kg bodyweight (1.84 ± 0.33 vs. 1.68 ± 0.38; P < 0.05) than controls. BP, HR, and diameters of AOD, LVED, and LVES as well as their relative anatomic proportions did not differ between children with Still's murmur and controls. Still's murmur seems to be generated by a subtle alteration in ventriculo-arterial coupling in healthy children. This result can be translated to parents, as they may be informed that their child's innocent murmur is caused by a more "lively interplay between the heart and the aorta."

The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells.

Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC50: 0.057 µM), in HMC-1.2 cells expressing KIT D816V (IC50: 0.72 µM), and in HMC-1.1 cells lacking KIT D816V (IC50: 0.09 µM), as well as in C2 cells (IC50: 0.74 µM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.