Oncolytic virotherapy augments self-maintaining natural killer cell line cytotoxicity against neuroblastoma.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death. METHODS: We utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo. RESULTS: NK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression. CONCLUSIONS: To the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma.

Non-randomized studies should be considered for assessing surgical techniques in rectal prolapse: prospective cohort study.

AIM: Randomized trials comparing surgical techniques for rectal prolapse are not always feasible. We assessed whether non-randomized comparisons of those who have had surgery with those still waiting would be confounding baseline health status. METHOD: This was a prospective cohort study in seven UK hospitals. Participants were ≥ 18 years and listed for surgical interventions of equivalent intensity for rectal prolapse. They were defined as short or long waiters (≤ 18 or > 18 weeks, respectively). Time on the waiting list was compared with baseline comorbidity (Charlson comorbidity index) and change from baseline in health status (EQ-5D-5L) at the time of surgery. RESULTS: In all, 203 patients were analysed. Median (interquartile range) waiting time was 13.7 weeks (8.1, 20.4) varying across sites. Baseline comorbidity was not an important predictor of waiting time. Median Charlson comorbidity index was 2 (0, 3) for short and 1 (0, 3) for long waiters. A change in waiting time by a week was associated with negligible improvement in the EQ-5D-5L index of 0.001 (95% CI -0.000 to 0.003, P = 0.106). CONCLUSION: Negligible change in patient reported health status while on the waiting list and lack of effect of comorbidities in influencing waiting time support the use of non-randomized pre-/post-studies to compare the effects of surgical interventions for rectal prolapse.

Prediction of pre-eclampsia: review of reviews.

OBJECTIVE: Primary studies and systematic reviews provide estimates of varying accuracy for different factors in the prediction of pre-eclampsia. The aim of this study was to review published systematic reviews to collate evidence on the ability of available tests to predict pre-eclampsia, to identify high-value avenues for future research and to minimize future research waste in this field. METHODS: MEDLINE, EMBASE and The Cochrane Library including DARE (Database of Abstracts of Reviews of Effects) databases, from database inception to March 2017, and bibliographies of relevant articles were searched, without language restrictions, for systematic reviews and meta-analyses on the prediction of pre-eclampsia. The quality of the included reviews was assessed using the AMSTAR tool and a modified version of the QUIPS tool. We evaluated the comprehensiveness of search, sample size, tests and outcomes evaluated, data synthesis methods, predictive ability estimates, risk of bias related to the population studied, measurement of predictors and outcomes, study attrition and adjustment for confounding. RESULTS: From 2444 citations identified, 126 reviews were included, reporting on over 90 predictors and 52 prediction models for pre-eclampsia. Around a third (n = 37 (29.4%)) of all reviews investigated solely biochemical markers for predicting pre-eclampsia, 31 (24.6%) investigated genetic associations with pre-eclampsia, 46 (36.5%) reported on clinical characteristics, four (3.2%) evaluated only ultrasound markers and six (4.8%) studied a combination of tests; two (1.6%) additional reviews evaluated primary studies investigating any screening test for pre-eclampsia. Reviews included between two and 265 primary studies, including up to 25 356 688 women in the largest review. Only approximately half (n = 67 (53.2%)) of the reviews assessed the quality of the included studies. There was a high risk of bias in many of the included reviews, particularly in relation to population representativeness and study attrition. Over 80% (n = 106 (84.1%)) summarized the findings using meta-analysis. Thirty-two (25.4%) studies lacked a formal statement on funding. The predictors with the best test performance were body mass index (BMI) > 35 kg/m2 , with a specificity of 92% (95% CI, 89-95%) and a sensitivity of 21% (95% CI, 12-31%); BMI > 25 kg/m2 , with a specificity of 73% (95% CI, 64-83%) and a sensitivity of 47% (95% CI, 33-61%); first-trimester uterine artery pulsatility index or resistance index > 90th centile (specificity 93% (95% CI, 90-96%) and sensitivity 26% (95% CI, 23-31%)); placental growth factor (specificity 89% (95% CI, 89-89%) and sensitivity 65% (95% CI, 63-67%)); and placental protein 13 (specificity 88% (95% CI, 87-89%) and sensitivity 37% (95% CI, 33-41%)). No single marker had a test performance suitable for routine clinical use. Models combining markers showed promise, but none had undergone external validation. CONCLUSIONS: This review of reviews calls into question the need for further aggregate meta-analysis in this area given the large number of published reviews subject to the common limitations of primary predictive studies. Prospective, well-designed studies of predictive markers, preferably randomized intervention studies, and combined through individual-patient data meta-analysis are needed to develop and validate new prediction models to facilitate the prediction of pre-eclampsia and minimize further research waste in this field. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Predicción de la preeclampsia: revisión de revisiones OBJETIVO: Los estudios primarios y las revisiones sistemáticas proporcionan estimaciones de precisión variable para diferentes factores en la predicción de la preeclampsia. El objetivo de este estudio fue revisar las revisiones sistemáticas publicadas para recopilar evidencia sobre la capacidad de las pruebas disponibles para predecir la preeclampsia, identificar avenidas de investigación futura valiosas y minimizar el desperdicio futuro de investigación en este campo. MÉTODOS: Se realizaron búsquedas de artículos relevantes en bibliografías sobre el tema y en las bases de datos MEDLINE, EMBASE y The Cochrane Library, incluida DARE (Database of Abstracts of Reviews of Effects), desde el inicio de cada base de datos hasta marzo de 2017, sin restricciones de idioma, para obtener revisiones sistemáticas y metaanálisis sobre la predicción de la preeclampsia. La calidad de las revisiones incluidas se evaluó utilizando la herramienta AMSTAR y una versión modificada de la herramienta QUIPS. Se evaluó la amplitud de la búsqueda, el tamaño de la muestra, las pruebas y los resultados evaluados, los métodos de síntesis de datos, las estimaciones de la capacidad de predicción, el riesgo de sesgo relacionado con la población estudiada, la medición de los predictores y los resultados, la deserción del estudio y el ajuste por confusión. RESULTADOS: De las 2444 citas identificadas, se incluyeron 126 revisiones, que informaron sobre más de 90 predictores y 52 modelos de predicción para la preeclampsia. Alrededor de un tercio (n=37 (29,4%)) de todas las revisiones investigaron únicamente marcadores bioquímicos para predecir la preeclampsia, 31 (24,6%) investigaron asociaciones genéticas con la preeclampsia, 46 (36,5%) informaron sobre las características clínicas, cuatro (3,2%) evaluaron sólo marcadores ecográficos y seis (4,8%) estudiaron una combinación de pruebas; dos (1,6%) revisiones adicionales evaluaron los estudios primarios que investigaron cualquier prueba de diagnóstico de la preeclampsia. Las revisiones incluyeron entre dos y 265 estudios primarios, que incluyeron hasta 25 356 688 mujeres en la revisión más grande. Sólo aproximadamente la mitad (n=67 (53,2%)) de las revisiones evaluaron la calidad de los estudios incluidos. En muchas de las revisiones incluidas hubo un alto riesgo de sesgo, particularmente en relación con la representatividad de la población y la deserción de los estudios. Más del 80% (n=106 (84,1%)) resumió los hallazgos utilizando el metaanálisis. Treinta y dos (25,4%) estudios carecían de una declaración formal sobre la financiación. Los predictores con el mejor rendimiento de la prueba fueron el índice de masa corporal (IMC) >35 kg.m-2 , con una especificidad del 92% (IC 95%, 89-95%) y una sensibilidad del 21% (IC 95%, 12-31%); IMC >25 kg.m-2 , con una especificidad del 73% (IC 95%: 64-83%) y una sensibilidad del 47% (IC 95%: 33-61%); índice de pulsatilidad de la arteria uterina en el primer trimestre o índice de resistencia >90° percentil (especificidad del 93% (IC 95%: 90-96%) y sensibilidad del 26% (IC 95%: 23-31%)); factor de crecimiento placentario (especificidad 89% (IC 95%, 89-89%) y sensibilidad 65% (IC 95%, 63-67%)); y proteína placentaria 13 (especificidad 88% (IC 95%, 87-89%) y sensibilidad 37% (IC 95%, 33-41%)). Ningún marcador por sí solo tuvo un rendimiento de la prueba adecuado para el uso clínico rutinario. Los modelos que combinan marcadores son prometedores, pero ninguno fue sometido a una validación externa. CONCLUSIONES: Esta revisión de revisiones ha puesto en duda la necesidad de un metaanálisis agregado adicional en esta área, dado el gran número de revisiones publicadas sujetas a las limitaciones comunes de los estudios predictivos primarios. Se necesitan estudios prospectivos bien diseñados de marcadores predictivos, preferiblemente en estudios de intervención aleatorios, y combinados mediante el metaanálisis de datos de pacientes individuales, para desarrollar y validar nuevos modelos predictivos que faciliten la predicción de la preeclampsia y minimicen el desperdicio de investigación adicional en este campo.

Surgery for constipation: systematic review and practice recommendations: Graded practice and future research recommendations.

AIM: This manuscript forms the final of seven that address the surgical management of chronic constipation (CC) in adults. The content coalesces results from the five systematic reviews that precede it and of the European Consensus process to derive graded practice recommendations (GPR). METHODS: Summary of review data, development of GPR and future research recommendations as outlined in detail in the 'introduction and methods' paper. RESULTS: The overall quality of data in the five reviews was poor with 113/156(72.4%) of included studies providing only level IV evidence and only four included level I RCTs. Coalescence of data from the five procedural classes revealed that few firm conclusions could be drawn regarding procedural choice or patient selection: no single procedure dominated in addressing dynamic structural abnormalities of the anorectum and pelvic floor with each having similar overall efficacy. Of one hundred 'prototype' GPRs developed by the clinical guideline group, 85/100 were deemed 'appropriate' based on the independent scoring of a panel of 18 European experts and use of RAND-UCLA consensus methodology. The remaining 15 were all deemed uncertain. Future research recommendations included some potential RCTs but also a strong emphasis on delivery of large multinational high-quality prospective cohort studies. CONCLUSION: While the evidence base for surgery in CC is poor, the widespread European consensus for GPRs is encouraging. Professional bodies have the opportunity to build on this work by supporting the efforts of their membership to help convert the documented recommendations into clinical guidelines.

Surgery for constipation: systematic review and clinical guidance: Paper 1: Introduction & Methods.

AIM: This manuscript provides the introduction and detailed methodology used in subsequent reviews to assess the outcomes of surgical interventions with the primary intent of treating chronic constipation in adults and to develop recommendations for practice. METHOD: PRISMA guidance was adhered to throughout. A literature search was performed in public databases between January 1960 and February 2016. Studies that fulfilled strictly-defined PICOS (patients, interventions, controls, outcome, and study design) criteria were included. The process involved two groups of participants: (i): 'a clinical guidance group' of 18 UK experts (including junior support) who performed the systematic reviews and produced summary evidence statements (SES) based strictly on data synthesis in each review. The same group then produced prototype graded practice recommendations (GPRs) based on coalescence of SES and expert opinion; (ii): a European Consensus group of 18 ESCP (European Society of Coloproctology) nominated experts from nine European countries evaluated the appropriateness of each prototype GPR based on published RAND/UCLA methodology. RESULTS: An overview of the search results is provided in this manuscript. A total of 156 studies from 307 full text articles (from 2551 initially screened records) were included, providing data on procedures characterized by: (i) colonic resection (n = 40); (ii) rectal suspension (n = 18); (iii) rectal wall excision (n = 44); (iv) rectovaginal septum reinforcement (n = 47); (v) sacral nerve stimulation (n = 7). The overall quality of evidence was poor with 113/156 (72.4%) studies providing only Oxford level IV evidence. The best evidence was extracted for rectal excisional procedures, where the majority of studies were Oxford level I or II. The five subsequent reviews provide a total of 99 SES (reflecting perioperative variables, efficacy, harms and prognostic variables) that contributed to 100 prototype GPRs covering patient selection, procedural considerations and patient counselling. The final manuscript details the 85/100 GPRs that were deemed appropriate by European Consensus (remaining 15 were all uncertain) and future research recommendations. CONCLUSION: This manuscript and the following 6 papers suggest that the evidence base for surgical management of chronic constipation is currently poor although some expert consensus exists on best practice. Further studies are required to inform future commissioning of treatments and of research funding.

A brief mindfulness meditation can ameliorate the effects of exposure to idealised social media images on self-esteem, mood, and body appreciation in young women: An online randomised controlled experiment.

The study examined whether a 10-minute mindfulness meditation intervention could ameliorate effects of exposure to social media thinspiration and fitspiration images on women's self-esteem, mood, and body appreciation. A total of 162 women aged 18-42 years (M = 25.94, SD = 4.38) completed an online experiment which involved viewing thinspiration and fitspiration images before random allocation to either a brief, ten-minute mindfulness meditation audio intervention or a ten-minute control audio about jujitsu. Participants completed self-reported measures of self-esteem, positive and negative mood, and body appreciation at baseline (Time 1), post-exposure to idealised social media images (Time 2), and immediately post-intervention (Time 3). Mixed, repeated-measures ANOVAs showed that scores were lower for body appreciation, self-esteem, and positive mood, and higher for negative mood, in both groups after exposure to idealised imagery. However, a brief mindfulness intervention ameliorated the negative effects of social media exposure. Specifically, self-esteem, body appreciation, and mood were higher in the mindfulness meditation group at Time 3, compared to the control group. Future interventions should explore the utility of mindfulness practices to provide long term buffering effects against such social media content, as well as targeting the idealisation of female physiques portrayed in thinspiration and fitspiration content.

Preclinical evidence for employing MEK inhibition in NRAS mutated pediatric gastroenteropancreatic neuroendocrine-like tumors.

BACKGROUND: Pediatric gastroenteropancreatic neuroendocrine tumors are exceedingly rare, resulting in most pediatric treatment recommendations being based on data derived from adults. Trametinib is a kinase inhibitor that targets MEK1/2 and has been employed in the treatment of cancers harboring mutations in the Ras pathway. METHODS: We utilized an established human pediatric gastroenteropancreatic neuroendocrine-like tumor patient-derived xenograft (PDX) with a known NRAS mutation to study the effects of MEK inhibition. We evaluated the effects of trametinib on proliferation, motility, and tumor growth in vivo. We created an intraperitoneal metastatic model of this PDX, characterized both the phenotype and the genotype of the metastatic PDX and again, investigated the effects of MEK inhibition. RESULTS: We found target engagement with decreased ERK1/2 phosphorylation with trametinib treatment. Trametinib led to decreased in vitro cell growth and motility, and decreased tumor growth and increased animal survival in a murine flank tumor model. Finally, we demonstrated that trametinib was able to significantly decrease gastroenteropancreatic neuroendocrine intraperitoneal tumor metastasis. CONCLUSIONS: The results of these studies support the further investigation of MEK inhibition in pediatric NRAS mutated solid tumors.

Endocrine therapy in prostate cancer: time for reappraisal of risks, benefits and cost-effectiveness?

In the 70 years following the first description of the benefits of surgical castration, despite advances in medical therapy e.g. cabazitaxel, enzalutamide, abiraterone, androgen deprivation therapy (ADT) remains the cornerstone of treatment for advanced prostate cancer. However, with increasing numbers of men undergoing PSA testing, the disease is being diagnosed earlier and the costs of ADT, with uncertain survival benefits and associated risks, have risen dramatically. Clinical studies of potent novel agents have shown survival benefits in advanced disease, but timing, risks and cost-effectiveness of treatment remain controversial. As new agents enter clinical practice, a comprehensive research strategy is essential to optimise benefits whilst minimising harm.

Comparing diagnostic tests: trials in people with discordant test results.

Diagnostic tests are traditionally compared for accuracy against a gold standard but can also be compared prospectively in a trial. A conventional trial comparing two tests would randomize each participant to a testing strategy, but a more efficient alternative is to give both tests to all participants and follow up those with discordant results. Participants could be randomized before or after testing. The statistical analysis of such a trial has not previously been described. We investigated two estimates of the risk difference for a binary outcome: one based on analysing outcomes as if from a conventional trial and one combining estimates of different parameters in the manner of a decision analysis. We show that the trial estimate and decision analysis estimate are both unbiased and derive approximate formulae for their standard errors. By using the decision analysis estimate (but not the trial estimate), the same precision can be achieved by randomizing before testing as by randomizing after. To avoid destroying equipoise, and to allow consenting and randomizing to be carried out at the same visit, we recommend randomizing before testing. Giving both tests to all participants means fewer need to be recruited: in one example from the literature, the proposed design was nearly four times more efficient in this sense than a conventional trial design.

Long-term density-dependent changes in habitat selection in red deer (Cervus elaphus).

Understanding how habitat selection changes with population density is a key concept in population regulation, community composition and managing impacts on biodiversity and ecosystem services. At low density, it is expected that individuals select habitats in terms of their preference, but as population density increases, the availability of resources per individual declines on preferred habitats, leading to competition which forces some individuals to exploit less preferred habitats. Using spatial information of Scottish red deer (Cervus elaphus) winter counts, carried out in 110 areas across Scotland between 1961 and 2004 (a total of 1,206,495 deer observations), we showed how winter habitat niche breadth in red deer has widened with increasing population density. Heather moorland and montane habitats were most and least preferred for deer, respectively. Increasing density favoured the selection of grassland, to the detriment of the selection of heather moorland. The selection of heather and grassland decreased when temperature increased, while the selection of montane and peatland habitats increased. These findings are important for understanding how habitat use, density and population are likely to be affected by weather, and allow us to predict habitat impacts by large mammal herbivory and climate.

The Effects of Protein Phosphatase 2A Activation with Novel Tricyclic Sulfonamides on Hepatoblastoma.

BACKGROUND: The tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. We aimed to examine the effects of two novel compounds of the tricyclic sulfonamide class, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression, on human hepatoblastoma. METHODS: An established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were treated with increasing doses of 3364 or 8385, and viability, proliferation, cell cycle and motility were investigated. Cancer cell stemness was evaluated by real-time PCR and tumorsphere forming ability. Effects on tumor growth were examined using a murine model. RESULTS: Treatment with 3364 or 8385 significantly decreased viability, proliferation, cell cycle progression and motility in HuH6 and COA67 cells. Both compounds significantly decreased stemness as demonstrated by decreased abundance of OCT4, NANOG, and SOX2 mRNA. The ability of COA67 to form tumorspheres, another sign of cancer cell stemness, was significantly diminished by 3364 and 8385. Treatment with 3364 resulted in decreased tumor growth in vivo. CONCLUSION: Novel PP2A activators, 3364 and 8385, decreased hepatoblastoma proliferation, viability, and cancer cell stemness in vitro. Animals treated with 3364 had decreased tumor growth. These data provide evidence for further investigation of PP2A activating compounds as hepatoblastoma therapeutics.

Using 3D-bioprinted models to study pediatric neural crest-derived tumors.

The use of three-dimensional (3D) bioprinting has remained at the forefront of tissue engineering and has recently been employed for generating bioprinted solid tumors to be used as cancer models to test therapeutics. In pediatrics, neural crest-derived tumors are the most common type of extracranial solid tumors. There are only a few tumor-specific therapies that directly target these tumors, and the lack of new therapies remains detrimental to improving the outcomes for these patients. The absence of more efficacious therapies for pediatric solid tumors, in general, may be due to the inability of the currently employed preclinical models to recapitulate the solid tumor phenotype. In this study, we utilized 3D bioprinting to generate neural crest-derived solid tumors. The bioprinted tumors consisted of cells from established cell lines and patient-derived xenograft tumors mixed with a 6% gelatin/1% sodium alginate bioink. The viability and morphology of the bioprints were analyzed via bioluminescence and immunohisto chemistry, respectively. We compared the bioprints to traditional twodimensional (2D) cell culture under conditions such as hypoxia and therapeutics. We successfully produced viable neural crest-derived tumors that retained the histology and immunostaining characteristics of the original parent tumors. The bioprinted tumors propagated in culture and grew in orthotopic murine models. Furthermore, compared to cells grown in traditional 2D culture, the bioprinted tumors were resistant to hypoxia and chemotherapeutics, suggesting that the bioprints exhibited a phenotype that is consistent with that seen clinically in solid tumors, thus potentially making this model superior to traditional 2D culture for preclinical investigations. Future applications of this technology entail the potential to rapidly print pediatric solid tumors for use in high-throughput drug studies, expediting the identification of novel, individualized therapies.

Neurodevelopmental delay in small babies at term: a systematic review.

OBJECTIVE: Being small for gestational age (SGA) or having fetal growth restriction (FGR) may be associated with poorer neurodevelopmental outcomes compared to being appropriate for gestational age (AGA). The aim of this paper was to evaluate the existence and magnitude of decrease in neurodevelopmental scores in SGA and FGR infants born at term from a systematic review of the existing literature. METHODS: Studies of neurodevelopment in SGA/FGR babies were identified from a search of the internet scientific databases. Studies that included preterm births and those that did not define absolute indices of standardized cognitive outcome were excluded. SGA was defined as birth weight below the 10(th) centile for gestation and FGR as the same birth-weight standard with abnormal umbilical artery Doppler ultrasound or evidence of abnormal placentation on pathology specimen studies. Effect size was calculated as the standardized mean difference between neurodevelopment scores of controls and SGA/FGR children. RESULTS: There were 28 studies of SGA, with a total of 7861 SGA and 91 619 control AGA babies, and three studies of FGR, with a total of 119 FGR and 49 control AGA babies. Data synthesis showed that standardized neurodevelopmental scores in SGA babies were 0.32 SD (95% CI, 0.25-0.38) below those for normal controls, though with heterogeneity between studies (I(2) = 68.3%). Insufficient data were available for FGR babies. CONCLUSION: The findings of the study demonstrate that among babies born at term, being SGA is associated with lower scores on neurodevelopmental outcomes compared to AGA controls. A trial designed to evaluate the effects of intervention in small fetuses born at term in order to improve the neurodevelopmental outcome is urgently needed.

Assessment of the 10-year risk of coronary heart disease events for Qatar Petroleum's firefighters and non-firefighter staff in Qatar.

Coronary heart disease is a major public health problem worldwide and firefighters may be at particular occupational risk. In a cross-sectional study in Ras Laffan Industrial City, Qatar, we assessed the 10-year risk of coronary heart disease events for 369 Qatar Petroleum staff at their periodic medical examination. The subjects of the study (all males) were divided into firefighters and non-firefighters groups. Based on the Framingham risk score calculations, 69.9% of the subjects were categorized as low risk, 27.1% as intermediate risk and 2.9% as high risk. None of the firefighters was categorized as high risk, 15.5% were intermediate and the rest were low risk. In the whole group, low high-density lipoprotein cholesterol was the most prevalent risk factor (68.8%), followed by hypertension (32.0%) and smoking (15.4%). The mean risk of developing coronary heart disease in firefighters [6.5% (SD 3.7%)] was significantly lower than in non-firefighters 19.5% (SD 6.5%)].

CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.

Metastatic human hepatoblastoma cells exhibit enhanced tumorigenicity, invasiveness and a stem cell-like phenotype.

BACKGROUND/PURPOSE: Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. MATERIALS/METHODS: Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. RESULTS: The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. CONCLUSION: This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

Rapid Characterization of Solid Tumors Using Resonant Sensors.

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors.

PIM3 kinase promotes tumor metastasis in hepatoblastoma by upregulating cell surface expression of chemokine receptor cxcr4.

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

Targeting High-Risk Neuroblastoma Patient-Derived Xenografts with Oncolytic Virotherapy.

Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Forced vital capacity, airway obstruction and survival in a general population sample from the USA.

BACKGROUND: Many studies show a link between forced expiratory volume in 1 s (FEV(1)) and survival in the general population and this has been interpreted as a link between airway obstruction and survival. However, the observation that vital capacity is also associated with survival weakens this interpretation. METHODS: Data on spirometry and survival were taken from the Atherosclerosis Risk in Communities (ARIC) limited access dataset. Survival among 7489 participants with usable spirometry and complete data was regressed against measures of ventilatory function after controlling for many other factors likely to be associated with survival. RESULTS: Survival was strongly associated with forced vital capacity (FVC) after adjustment for FEV(1), but not the other way round. The fully adjusted hazard ratio (HR) associated with high FVC was 0.90 in men (95% CI 0.80 to 1.00; p=0.049) and 0.82 in women (95% CI 0.70 to 0.95; p=0.01). This compares with 0.98 for FEV(1) in men (95% CI 0.90 to 1.07; p.0.72) and 1.01 in women (95% CI 0.89 to 1.15; p=0.84). There was no association between survival and airway obstruction as measured by the FEV(1)/FVC ratio. CONCLUSIONS: FVC but not airway obstruction predicts survival in asymptomatic adults without chronic respiratory diagnoses or persistent respiratory symptoms. The association is not explained by age, anthropometry, smoking, income occupation or blood pressure. As FVC later in life, cardiovascular risk, type II diabetes mellitus and low-grade systemic inflammation are all associated with poor fetal growth, these other conditions may be partly responsible for the poor survival in those with low FVC.