Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is associated with thrombotic complications in adults, but the incidence of COVID-19 related thrombosis in children and adolescents is unclear. Most children with acute COVID-19 have mild disease, but coagulopathy has been associated with multisystem inflammatory syndrome in children (MIS-C), a post-infectious complication. We conducted a multicenter retrospective cohort study to determine the incidence of thrombosis in children hospitalized with COVID-19 or MIS-C and to evaluate associated risk factors. We classified patients into one of three groups for analysis: COVID-19, MIS-C, or asymptomatic SARS-CoV-2. Among a total of 853 admissions (426 COVID-19, 138 MIS-C, and 289 asymptomatic SARS-CoV-2) in 814 patients, there were 20 patients with thrombotic events (TE) (including 1 stroke). Patients with MIS-C had the highest incidence (6.5%, 9/138) versus COVID-19 (2.1%, 9/426) or asymptomatic SARS-CoV-2 (0.7%, 2/289). In patients with COVID-19 or MIS-C, the majority of thrombotic events (89%) occurred in patients ≥12 years. Patients > 12 years with MIS-C had the highest rate of thrombosis at 19% (9/48). Notably, 71% of TE that were not present on admission occurred despite thromboprophylaxis. Multivariable analysis identified the following as significantly associated with thrombosis: age ≥12 years, cancer, presence of a central venous catheter, and MIS-C. In patients with COVID-19 or MIS-C, hospital mortality was 2.3% (13/564), but was 28% (5/18) in patients with thrombotic events. Our findings may help inform pediatric thromboprophylaxis strategies.

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer.

The cause of hemophilia A carrier bleeding is not well established. Desmopressin (DDAVP), used clinically to treat or prevent bleeding, can also be used as a medical stress surrogate. This study’s objective was to compare the response to DDAVP in hemophilia A carriers with that in normal control patients. Bleeding was assessed by the International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT). DDAVP (0.3 μg/kg) was administered either IV or subcutaneously (SC), and blood was drawn at baseline and 1, 2, and 4 hours postadministration. Blood was assessed for factor VIII (FVIII) level, von Willebrand factor (VWF) antigen (VWF:Ag), VWF activity (VWF:RCo or VWF:GPIbM), thromboelastography (TEG), and thrombin generation assay (TGA) at all points, and for VWF propeptide (VWFpp):Ag ratio and ABO blood type at baseline. Carriers were older than control patients (median age, 34 and 21 years, respectively; P = .003) and had higher ISTH-BAT bleeding scores (median bleeding score, 8 and 0, respectively; P = .001). Carriers had a significantly reduced FVIII response to DDAVP compared with control patients (P ≤ .0001). When only carriers with normal baseline FVIII levels (n = 10) were included, carriers maintained a reduced FVIII response (P ≤ .0001). Furthermore, participants with abnormal bleeding scores had a significantly lower FVIII response to DDAVP compared with those with normal bleeding scores (P = .036). Hemophilia A carriers have a lower and less sustained FVIII response to DDAVP, suggesting an impaired ability to respond to hemostatic stress, which contributes to bleeding.

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Background Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is caused by quantitative and qualitative defects in von Willebrand factor (VWF). The laboratory diagnosis of VWD in pediatric patients is complicated by VWF interassay and intra‐assay variability, stress‐induced elevations in VWF levels, and a lack of significant bleeding history with which to correlate test results. Objective Guidelines recommend repeat testing in patients with a high suspicion of VWD and unclear laboratory assay results; however, no studies have evaluated the utility of repeat VWF testing in pediatric patients. Methods This retrospective single‐center cohort study aimed to determine clinical variables associated with requiring more than one test to diagnose VWD and to establish a cutoff VWF value above which further testing is not informative. Results Of 811 patients evaluated for a suspected bleeding disorder, 22.2% were diagnosed with VWD, with ~70% diagnosed on the first test. Patients with VWD were younger (5.8 vs. 8.5 years, P = .002) and more likely to have a family history of VWD (38% vs. 22%, P < .001) than those without VWD. Univariate analysis failed to identify any clinical variables that correlated with needing multiple tests for a VWD diagnosis. A cutoff of 100 IU/dL for VWF antigen or activity on the first test yielded negative predictive values >95%. Conclusions We demonstrate that the majority of pediatric patients had diagnostic VWF values on the first set of testing. Pediatric patients without a family history of VWD and VWF levels >100 IU/dL may not need further testing to rule out the diagnosis of VWD.

Background Virtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA. Methods Rapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3’UTR of the lymphocyte-specific protein tyrosine kinase ( LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth. Results In this study, a previously unannotated lncRNA lying within exon six and 3’UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA “HULLK” for Hormone-Upregulated lncRNA within LCK . Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation. Conclusions HULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa. Electronic supplementary material The online version of this article (10.1186/s12943-019-1039-6) contains supplementary material, which is available to authorized users.

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Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is diagnosed via 3 cardinal features: 1) personal history of bleeding, 2) laboratory assays and 3) family history of VWD. The diagnosis of VWD in pediatric patients is complicated by von Willebrand factor (VWF) inter- and intra-assay variability, phlebotomy-induced physiologic stress increasing VWF levels from baseline, and a lack of prior hemostatic challenges. Given these challenges, NHLBI guidelines recommend repeated testing in patients with mildly low or normal levels and a high suspicion of VWD. However, no studies to date have evaluated the utility of repeat VWF testing in pediatric patients. Currently, our center's standard of care is to complete 3 separate sets of VWF testing to rule out VWD. The primary objective of this study was to determine the clinical variables associated with requiring more than 1 test to diagnose VWD and to establish a cutoff value for the first set of VWF assays above which further testing would not be informative. This single center retrospective cohort study included patients ≤ 18 years of age who either had a diagnosis of or evaluation for VWD between January 2012 and July 2017. Patients were excluded if the VWD laboratory evaluation was completed at another institution or due to the presence of another bleeding disorder. Medical charts were abstracted for demographic information, medications, reason for testing, family history of VWD, results of VWF assays, and other illness at time of evaluation. All patients had a retrospective ISTH BAT score completed. Data were analyzed using SAS and are reported as median (IQR). Statistical analysis was done with non-parametric tests (Mann-Whitney or Wilcoxon sign-rank) for two groups comparisons. Odds ratios were calculated using Fisher's Exact test for clinical factors associated with a VWD diagnosis. Univariate logistic regression was performed, modeling the odds of requiring more than 1 diagnostic test to diagnose VWD. One thousand unique patients were evaluated and 189 excluded, yielding a final cohort of 811 patients. Of these, 631 (77.8%) did not have VWD and 180 (22.2%) were diagnosed with VWD. Patients diagnosed with VWD were younger than those without (median age 5.8 vs 8.5 years, p=0.0019) and were more likely to have a family history of VWD (38% vs 22%, p < 0.0001) but there was no difference in race or sex between cohorts. As expected, patients in the VWD cohort had lower VWF activity (34 vs 78 IU/dl, p < 0.0001), VWF antigen (45 vs 89 IU/dL, p < 0.0001) and FVIII (57 vs 100 IU/dL, p < 0.0001) than those without VWD. ISTH BAT scores were higher in the VWD cohort (2.47 vs. 2.07, p = 0.027). As shown in Table 1 and Figure 1, increased odds of VWD diagnosis were noted in those tested due to a family history of VWD (OR 1.75, 95% CI 1.21-2.51) or abnormal coagulation studies (OR 1.61, 95% CI 1.07-2.24). Subjects with VWD required fewer tests than those without VWD (median 1 vs 2, p < 0.001). Univariate analysis failed to identify any significant associations with needing > 1 test for the diagnosis of VWD (Table 1), so a multivariable model was not performed. In 69.4% (125/180) of subjects with VWD, the first test was diagnostic. In receiver-operating curve analysis, the first VWF antigen and activity have a high power for diagnosis of VWD with AUC of 0.88 and 0.92, respectively (Fig 1). A cutoff of 100 IU/dL for VWF antigen or activity on first test yielded a sensitivity of 95%, specificity of 38% and negative predictive value of 96.6% for VWF antigen compared to 98%, 38% and 98.6% for VWF activity, respectively. Here we demonstrate that the majority of pediatric subjects had diagnostic VWF values on the first set of testing. Unfortunately, no clinical variables were identified for patients with VWD who required > 1 test for diagnosis. However increased odds of VWD diagnosis were noted in those with a family history of VWD and abnormal coagulation studies. A cutoff of 100 IU/dL for VWF activity or antigen on the first test resulted in > 95% negative predictive value to rule out the diagnosis. Pediatric patients without a family history of VWD and VWF levels > 100 IU/dL on initial test may not need further testing to rule out the diagnosis of VWD. Disclosures Doshi: Bayer Hemophilia Awards Program: Research Funding. Butler:Pfizer: Consultancy; Genentech: Consultancy; HemaBiologics: Consultancy.