Peripheral arterial catheters in extremely preterm infants born at less than 28 weeks of gestation-a single-center experience.

The aim of this study was to perform a retrospective data analysis of established peripheral artery catheters (pAC) in extremely preterm infants. The primary outcome was the pAC life span and its correlation to gestational age, birth weight, localizations, and pAC removal. Retrospective data analysis of electronic patient records of all extremely preterm infants (born less than 28 weeks gestation) admitted to the neonatal intensive care unit in Graz (Austria) between January 2014 and December 2020. A total of 196 preterm infants with a median (IQR) gestational age of 25.7 (24.6-26.6) weeks and a birth weight of 730 (614-898) g were included. In 155 (79%) of these preterm infants, 286 pAC and six umbilical artery catheters were inserted successfully. The first pAC was inserted 2.5 (1.4-7.4) h after birth, and the median pAC life span was 57.5 (22.-107.2) h. Gestational age, birth weight, and catheter localization did not correlate with the pAC life span. The pAC localizations were the radial artery (63%), tibial posterior artery (21%), ulnar artery (6%), dorsal artery of the foot (6%), others (1%), and not documented (3%). Adverse reactions including temporarily impaired peripheral perfusion, local inflammation, extravasation, or bleeding were reported in 13% of all pAC, but none of these resulted in long-term sequelae. A median (IQR) of 9 (5-18) arterial blood samples were drawn via pAC, resulting in a notable reduction of pain stimuli.Conclusion: The use of pAC in extremely preterm infants is feasible and safe. Neither gestational age, birth weight nor localization did affect the life span of pAC. No long-term sequelae were observed, and pain events were reduced by using pAC for blood drawing. What is Known: • Peripheral artery catheters can be used for continuous blood pressure measurement and blood draw even in extremely preterm infants. • (Severe) adverse reactions such as bleeding, necrosis, or amputation occur between 1 and 4%. • What is New: • The median peripheral arty catheter life span is 58 h and is not affected by gestational age, birth weight, nor localization. • A median of nine blood samples can be taken per each single pAC and, therefore, prevent pain events in extremely preterm infants.

Rhabdomyosarcoma xenotransplants in zebrafish embryos.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. High-risk and metastatic disease continues to be associated with very poor prognosis. RMS model systems that faithfully recapitulate the human disease and provide rapid, cost-efficient estimates of antitumor efficacy of candidate drugs are needed to facilitate drug development and personalized medicine approaches. Here, we present a new zebrafish-based xenotransplant model allowing for rapid and easily accessible drug screening using low numbers of viable tumor cells and relatively small amounts of water-soluble chemicals. Under optimized temperature conditions, embryonal RMS xenografts were established in zebrafish embryos at 3 h postfertilization (hpf). In proof-of-principle experiments, chemotherapy drugs with established clinical anti-RMS efficacy (vincristine, dactinomycin) and the mitogen-activated protein kinase kinase inhibitor trametinib were shown to significantly reduce the cross-sectional area of the tumors by 120 hpf. RMS xenograft models in zebrafish embryos henceforth could serve as a valuable addition to cell culture and mammalian models of RMS and represent a rapid and cost-effective solution for preclinical candidate drug testing.

Combinatorial effects of azacitidine and trametinib on NRAS-mutated melanoma.

Congenital melanocytic nevus (CMN) syndrome represents a mosaic RASopathy, typically caused by postzygotic NRAS codon 61 mutations, which originate in ectodermal precursor cells and result in melanocyte deposits in the skin and central nervous system (CNS). Affected patients are prone to develop uniformly fatal melanomas in the skin and CNS. Here, we report the case of a 2.7-year-old male with CMN syndrome, diffuse leptomeningeal melanosis and CNS melanoma, who underwent experimental therapy with the DNA methyltransferase inhibitor azacitidine in combination with the mitogen-activated protein kinase (MEK) inhibitor trametinib with exceptional clinical and radiological response. Response to combination therapy appeared to be more durable than the treatment response observed in several other severely affected patients treated with trametinib for late-stage disease. Correspondingly, concomitant exposure to trametinib and azacitidine prevented development of trametinib resistance in NRAS-mutated human melanoma cells in vitro. Also, azacitidine was shown to inhibit growth and mitogen-activated protein kinase 1/2 (ERK1/2) phosphorylation of melanoma cells and act synergistically with trametinib to inhibit the growth of trametinib-resistant melanoma cells. These observations suggest that azacitidine enhances trametinib monotherapy and may represent a promising candidate drug for combination therapies to enhance the efficacy of MEK inhibitors in RAS-driven diseases.

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

BACKGROUND: Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. METHODS: This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. RESULTS: Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. CONCLUSIONS: Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.

Lack of Electron Acceptors Contributes to Redox Stress and Growth Arrest in Asparagine-Starved Sarcoma Cells.

Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD+ regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD+ regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.

Growth inhibition associated with disruption of the actin cytoskeleton by Latrunculin A in rhabdomyosarcoma cells.

Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multi-kinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).

Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma.

Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value.

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia.

CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.

Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR1 ribozymes.

Human cancers, including hepatocellular carcinoma (HCC), are characterized by a high degree of drug resistance. The multidrug resistance (MDR) transporters MDR1-P-glycoprotein and MRP2 (multidrug-associated protein 2) are expressed in almost 50% of human cancers, including HCCs. In this study, we analyzed the effect of anti-MDR1 ribozymes, especially AFP promoter-driven anti-MDR1 ribozymes, to specifically chemosensitize HCC cells. Epirubicin-selected HB8065/R cells were used as MDR1-P-glycoprotein-overexpressing cells. Adenoviral vectors were constructed to allow an efficient gene transfer of anti-MDR1 ribozyme constructs. AFP promoter-driven anti-MDR1 ribozymes reduced the IC(50) 30-fold for epirubicin in HCC cells, whereas human colorectal cancer cells were unaffected. Target sequences were either the translational start site or codon 196 of the human MDR1 gene. Adenoviral delivery of CMV promoter-driven anti-MDR1 ribozymes resulted in a reduced IC(50) for epirubicin and doxorubicin (60- and 20-fold, respectively). They completely restored chemosensitivity in stably transfected anti-MDR1 ribozyme-expressing HCC cells as well as in HCC cells transduced with adenoviruses expressing wild-type anti-MDR1 ribozymes. Adenoviral delivery of ribozymes was so efficient that chemosensitization of HCC cells could be demonstrated in cell cultures without further selection of transduced cells for single anti-MDR1 ribozyme-expressing HCC cell clones. Northern blots showed a decreased MDR1 mRNA expression, and fluorescence-activated cell sorting (FACS) analysis revealed a significantly reduced expression of MDR1-P-glycoprotein on the cell surface of HB8065/R cells after transduction with the anti-MDR1 ribozymes. In conclusion, our data demonstrate that adenoviral delivery of ribozymes can chemosensitize HCC cells and that chemosensitization can be specifically achieved by ribozymes driven by an AFP promoter directed against human MDR1.