Circulating miR-26b-5p and miR-451a as diagnostic biomarkers in medullary thyroid carcinoma patients.

PURPOSE/METHODS: The determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated. RESULTS: The samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients. CONCLUSION: This study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine.

Chlamydia trachomatis elicits TLR3 expression but disrupts the inflammatory signaling down-modulating NFκB and IRF3 transcription factors in human Sertoli cells.

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases worldwide, can disseminate and localize to the upper genital tract impairing reproductive function. Specifically, ascending C. trachomatis genital infection has been demonstrated to cause epididymitis or epididymo-orchitis, well-known risk factors for male infertility. C. trachomatis possesses the ability to infect primary human Sertoli cells, key elements for the spermatogenetic process and the immune protection of germ cells. Therefore, herein, we investigated the innate immune response in Sertoli cells following C. trachomatis infection, as well as its indirect effects on human spermatozoa. Specifically, we evaluated C. trachomatis mediated induction of Toll-like Receptors (TLR) 2, 3 and 4 as well as of downstream intracellular signaling molecules (NFκB and IRF3) and the levels of the related inflammatory mediators (IL-1α, IL-6, IFN-α, IFN-ß and IFN-γ), in an in vitro infection model of primary human Sertoli cells. The main result of our study shows that C. trachomatis induced TLR3-mediated recognition in human Sertoli cells, accompanied by the down-modulation of NFκB and IRF3-dependent signaling pathways followed by no production of pro-inflammatory cytokines. In conclusion, our findings suggest that C. trachomatis can disrupt the innate immune response in Sertoli cells and evade intracellular killing, potentially giving rise to a long-term infection that may exert negative effects on the male reproductive system.

The miR-139-5p regulates proliferation of supratentorial paediatric low-grade gliomas by targeting the PI3K/AKT/mTORC1 signalling.

AIMS: Paediatric low-grade gliomas (pLGGs) are a heterogeneous group of brain tumours associated with a high overall survival: however, they are prone to recur and supratentorial lesions are difficult to resect, being associated with high percentage of disease recurrence. Our aim was to shed light on the biology of pLGGs. METHODS: We performed microRNA profiling on 45 fresh-frozen grade I tumour samples of various histological classes, resected from patients aged ≤16 years. We identified 93 microRNAs specifically dysregulated in tumours as compared to non-neoplastic brain tissue. Pathway analysis of the microRNAs signature revealed PI3K/AKT signalling as one of the centrally enriched oncogenic signalling. To date, activation of the PI3K/AKT pathway in pLGGs has been reported, although activation mechanisms have not been fully investigated yet. RESULTS: One of the most markedly down-regulated microRNAs in our supratentorial pLGGs cohort was miR-139-5p, whose targets include the gene encoding the PI3K's (phosphatidylinositol 3-kinase) catalytic unit, PIK3CA. We investigated the role of miR-139-5p in regulating PI3K/AKT signalling by the use of human cell cultures derived from supratentorial pLGGs. MiR-139-5p overexpression inhibited pLGG cell proliferation and decreased the phosphorylation of PI3K target AKT and phosphorylated-p70 S6 kinase (p-p70 S6K), a hallmark of PI3K/AKT/mTORC1 signalling activation. The effect of miR-139-5p was mediated by PI3K inhibition, as suggested by the decrease in proliferation and phosphorylation of AKT and p70 S6K after treatment with the direct PI3K inhibitor LY294002. CONCLUSIONS: These findings provide the first evidence that down-regulation of miR-139-5p in supratentorial pLGG drives cell proliferation by derepressing PI3K/AKT signalling.

Noncanonical GLI1 signaling promotes stemness features and in vivo growth in lung adenocarcinoma.

Aberrant Hedgehog/GLI signaling has been implicated in a diverse spectrum of human cancers, but its role in lung adenocarcinoma (LAC) is still under debate. We show that the downstream effector of the Hedgehog pathway, GLI1, is expressed in 76% of LACs, but in roughly half of these tumors, the canonical pathway activator, Smoothened, is expressed at low levels, possibly owing to epigenetic silencing. In LAC cells including the cancer stem cell compartment, we show that GLI1 is activated noncanonically by MAPK/ERK signaling. Different mechanisms can trigger the MAPK/ERK/GLI1 cascade including KRAS mutation and stimulation of NRP2 by VEGF produced by the cancer cells themselves in an autocrine loop or by stromal cells as paracrine cross talk. Suppression of GLI1, by silencing or drug-mediated, inhibits LAC cells proliferation, attenuates their stemness and increases their susceptibility to apoptosis in vitro and in vivo. These findings provide insight into the growth of LACs and point to GLI1 as a downstream effector for oncogenic pathways. Thus, strategies involving direct inhibition of GLI1 may be useful in the treatment of LACs.

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress.

The Hedgehog (Hh) signaling regulates tissue development, and its aberrant activation is a leading cause of malignancies, including medulloblastoma (Mb). Hh-dependent tumorigenesis often occurs in synergy with other mechanisms, such as loss of p53, the master regulator of the DNA damage response. To date, little is known about mechanisms connecting DNA-damaging events to morphogen-dependent processes. Here, we show that genotoxic stress triggers a cascade of signals, culminating with inhibition of the activity of Gli1, the final transcriptional effector of Hh signaling. This inhibition is dependent on the p53-mediated elevation of the acetyltransferase p300/CBP-associated factor (PCAF). Notably, we identify PCAF as a novel E3 ubiquitin ligase of Gli1. Indeed PCAF, but not a mutant with a deletion of its ubiquitination domain, represses Hh signaling in response to DNA damage by promoting Gli1 ubiquitination and its proteasome-dependent degradation. Restoring Gli1 levels rescues the growth arrest and apoptosis effect triggered by genotoxic drugs. Consistently, DNA-damaging agents fail to inhibit Gli1 activity in the absence of either p53 or PCAF. Finally, Mb samples from p53-null mice display low levels of PCAF and upregulation of Gli1 in vivo, suggesting PCAF as potential therapeutic target in Hh-dependent tumors. Together, our data define a mechanism of inactivation of a morphogenic signaling in response to genotoxic stress and unveil a p53/PCAF/Gli1 circuitry centered on PCAF that limits Gli1-enhanced mitogenic and prosurvival response.

No need for rescue medication (NNR) as an easily interpretable efficacy outcome measure in analgesic trials: validation in an individual-patient meta-analysis of dental pain placebo-controlled trials of naproxen.

WHAT IS KNOWN AND OBJECTIVE: In analgesic trials, pain relief is often assessed using a pain-relief score. We aimed to assess, through a meta-analysis, whether absence of need for rescue medication (NNR) is a reliable outcome measure in the evaluation of acute pain relief. METHODS: Individual-patient meta-analysis of placebo-controlled trials of single-dose naproxen sodium 220 or 440 mg in dental pain. Efficacy estimates were based on NNR and compared with the more commonly used 50% maximum total pain relief score (50% TOTPAR). The trials included were the full set of trials sponsored by one manufacturer. RESULTS AND DISCUSSION: Need for rescue medication and 50% TOTPAR gave comparable estimates of efficacy of naproxen sodium (220 and 440 mg) relative to placebo in dental pain at both 8 and 12 h after dosing. WHAT IS NEW AND CONCLUSION: No need for rescue medication is a reliable outcome measure for use in acute pain trials. As it is more readily understandable than 50% TOTPAR, it should be the preferred primary outcome measure in acute pain trials.

Barking up the wrong genome--we are not alone.

WHAT IS KNOWN AND OBJECTIVE: Pharmacogenetic studies, to help us understand variability in human drug response, have hitherto focussed largely on our own germline mutations, and in the context of anticancer and antimicrobial drugs, also on mutations of the tumour cell or invader microorganism. Here, we wish to draw attention to how our microbiome may contribute to variability in drug effects. COMMENT: Irinotecan, a prodrug which is activated to the topoisomerase I inhibitory metabolite (SN-38), is commonly used for the treatment of a range of cancers. SN-38 is subsequently detoxified by uridine diphosphate-glycosyltransferase 1, encoded by the UGT1A1 gene. It is well known that the variant allele UGT1A*18 is associated with the more common adverse effects of irinotecan. A recent study shows that the potentially dose-limiting irinotecan-induced diarrhoea is due to enterohepatic circulation of SN-38, and its reactivation in the gut by bacterial ß-glucuronidases. Importantly, the authors used specific inhibitors of the microbial enzymes to reduce the gastro-intestinal toxicity in mice. WHAT IS NEW AND CONCLUSION: We draw attention to the increasing range of diseases, including diabetes and obesity, associated with our microbiome. This pharmacogenetic example reminds us that in personalized medicine, there is more than our own genome to take account of.

Exploring Culturally Specific Drug Resistance Strategies of Hawaiian Youth in Rural Communities.

This qualitative study examined the drug resistance strategies of Hawaiian youth residing in rural communities in Hawai`i. Forty seven youth participated in 14 focus groups which focused on the social and environmental context of drug use for these youth. The findings indicated that there were 47 references to resistance strategies used in drug offer situations. These strategies fell within two different categories: (1) overt/confrontational drug resistance strategies, and (2) non-confrontational drug resistance strategies. These strategies occurred within the community context of relational networks of ascribed and biological family members, and differed in frequency of use by gender. Implications for culturally grounded drug prevention programs for rural Hawaiian youth are discussed.

When is a genetic test suitable for prime time? Predicting the risk of prostate cancer as a case-example.

Advances in genomics promise to deliver personalized medicine both for prevention and treatment of disease. Considerable effort is being directed towards translating observed associations between various genetic variants, such as single nucleotide polymorphisms (SNPs), and disease or drug response into clinically useful genetic tests. Unfortunately, because reported associations are usually weak or moderate, tests based on them are generally not accurate enough for use in routine clinical practice, and therefore, ensuring the appropriate use of genetic tests is important. In a recent report, a combination of 5 SNPs was claimed to improve the predictive value of the test for prostate cancer, compared with the individual SNPs. This led the authors to suggest that a 5-SNP-test could be used to predict the risk of prostate cancer. We evaluate the characteristics of the proposed test, comment on it, and summarize the views of others on its potential clinical utility. We hope that this may serve as a case-example for the evaluation of the many new genetic tests being suggested for adoption.

Community risk and resiliency factors related to drug use of rural Native Hawaiian youth: an exploratory study.

This exploratory, qualitative study examined the community-based risk and resiliency factors related to the drug use of rural Native Hawaiian youth. Forty-seven youth from five middle schools participated in focus groups that examined the ecological context of drug use for rural Hawaiian youth. Findings indicated that youth in the study were part of large extended networks of families and that these networks became a defining characteristic of the rural communities in the study. These familial networks functioned as sources of risk and protection related to drug use for youth participants. Implications for community-based practice are discussed.

'A'ole Drugs! Cultural Practices and Drug Resistance of Rural Hawaiian Youth.

This qualitative study examined how Native Hawaiian youth from rural communities utilized cultural practices to promote drug resistance and/or abstinence. Forty-seven students from 5 different middle schools participated in gender specific focus groups that focused on the cultural and environmental contexts of drug use for Native Hawaiian youth. The findings described culturally specific activities that participants used in drug related problem situations. The findings also suggested that those youth with higher levels of enculturation were able to resist drugs more effectively than those youth who were disconnected from their culture. The implications of these findings for social work practice are discussed.

Notch signaling is involved in expression of thyrocyte differentiation markers and is down-regulated in thyroid tumors.

CONTEXT: Notch genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch signaling in thyroid follicular cells has never been fully published. OBJECTIVE: The objective of the study was to characterize the expression of Notch pathway components in thyroid follicular cells and Notch signaling activities in normal and transformed thyrocytes. DESIGN/SETTING AND PATIENTS: Expression of Notch pathway components and key markers of thyrocyte differentiation was analyzed in murine and human thyroid tissues (normal and tumoral) by quantitative RT-PCR and immunohistochemistry. The effects of Notch overexpression in human thyroid cancer cells and FTRL-5 cells were explored with analysis of gene expression, proliferation assays, and experiments involving transfection of a luciferase reporter construct containing human NIS promoter regions. RESULTS: Notch receptors are expressed during the development of murine thyrocytes, and their expression levels parallel those of thyroid differentiation markers. Notch signaling characterized also normal adult thyrocytes and is regulated by TSH. Notch pathway components are variably expressed in human normal thyroid tissue and thyroid tumors, but expression levels are clearly reduced in undifferentiated tumors. Overexpression of Notch-1 in thyroid cancer cells restores differentiation, reduces cell growth rates, and stimulates NIS expression via a direct action on the NIS promoter. CONCLUSION: Notch signaling is involved in the determination of thyroid cell fate and is a direct regulator of thyroid-specific gene expression. Its deregulation may contribute to the loss of differentiation associated with thyroid tumorigenesis.

Participatory drug prevention research in rural Hawai'i with native Hawaiian middle school students.

BACKGROUND/OBJECTIVES: This paper describes a prevention study focused on the drug use scenarios encountered by Native Hawaiian youth. Priorities from communities on the Big Island of Hawai;i helped to shape the qualitative data collection and analysis of middle school students participating in the study. METHODS: Forty-seven youth from five different schools were interviewed in small, gender-specific focus groups during lunch hour or after school. RESULTS: The findings indicated that youth were exposed to drug offers that were direct-relational or indirect-contextual in nature. Direct-relational offers were didactic exchanges where drugs or alcohol were offered from one individual to another (e.g., "Do you want some beer?"). Indirect-contextual offers reflected complex exchanges among individuals, where drugs or alcohol were involved, but not offered directly (e.g., "Do you want to hang out with us?"). CONCLUSIONS: Implications are discussed regarding drug prevention research and programs that highlight indirect-contextual drug offers that are place based and culturally grounded.

Alternative splicing of the ErbB-4 cytoplasmic domain and its regulation by hedgehog signaling identify distinct medulloblastoma subsets.

Medulloblastoma (MB) results from aberrant development of cerebellar neurons in which altered hedgehog (Hh) signalling plays a major role. We investigated the possible influence of Hh signalling on ErbB-receptor expression in MB, in particular that of the ErbB-4 CYT-1 and CYT-2 isoforms generated by alternative splicing of the cytoplasmic domain. ErbB-4 expression was downregulated in Hh-induced MBs from Patched-1(+/-) mice. Hh signalling (reflected by enhanced expression of the Gli1 transcription factor) inhibited ErbB-4 expression in mouse cerebellar granule progenitors and human MB cells. Analysis of 26 human primary MBs revealed a subset of 11 tumors characterized by low Gli1 levels, upregulated ErbB-4 expression and increased CYT-1:CYT-2 ratios. Interestingly, CYT-1 and Gli1 levels were inversely correlated. ErbB-4 CYT-1 and CYT-2 had different phenotypic effects in cultured MB cells: in response to neuregulin treatment, CYT-2 overexpression inhibited proliferation whereas CYT-1, which includes a phosphatidylinositol 3-kinase (PI3K)-binding site that is missing in CYT-2, enhanced resistance to starvation- and etoposide-induced apoptosis by activating PI3K/Akt signalling. CYT-1:CYT-2 ratios displayed correlation with tumor histotype and ErbB-2 levels, which are established prognostic indices for MB. These findings demonstrate that low-level Hh signalling in human MB is associated with the selective maintenance of high ErbB-4 CYT-1 expression, an alteration that exerts tumor-promoting effects.

How high should total pain-relief score be to obviate the need for analgesic remedication in acute pain? Estimation using signal detection theory and individual-patient meta-analysis.

BACKGROUND: A pain relief score 50% of the maximum is often used as a clinically meaningful outcome in meta-analyses of analgesic trials. This arbitrary value requires validation. OBJECTIVE: To determine the optimum pain relief score for predicting pain relief sufficient to obviate the need for analgesic remedication in acute post-surgical pain. DESIGN: Individual-patient meta-analysis of randomized controlled trials and use of signal detection theory to identify the optimum cut-off point on the total pain relief score (TOTPAR). Analgesic remedication was used as the clinical outcome. DATA SOURCES: Seven parallel-group, active and placebo-controlled trials of minor analgesics. RESULTS: The predictive value of the TOTPAR score [expressed as a percentage of the maximum score (%maxTOTPAR)] for remedication was excellent for all the trials. The pooled estimate of the area under the receiver operating curve, an index of discriminative power, was outstanding 0.96 (95% CI 0.95-0.97). The pooled estimate of the optimal %maxTOTPAR for predictive purposes was 44.11 (95% CI 44.00-44.23). CONCLUSION: The analysis suggests that the arbitrary 50% cut-off point TOTPAR score often used in meta-analyses of analgesic trials in dental pain is reasonably acceptable. This is different to the 33% cut-off point reported for analgesic trials of acute breakthrough cancer pain and some chronic pain states such as diabetic neuropathy and postherpetic neuralgia. These differences deserve careful consideration when reading reports of analgesic trials and meta-analyses. Remedication itself should be considered as the preferred outcome measure for analgesic trials.

The clinical effectiveness and cost-effectiveness of newer drugs for children with epilepsy. A systematic review.

OBJECTIVES: To examine the clinical effectiveness and cost-effectiveness of newer antiepileptic drugs (AEDs) for epilepsy in children: gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate and vigabatrin. DATA SOURCES: Electronic databases. Drug company submissions. REVIEW METHODS: For the systematic review of clinical and cost-effectiveness, studies were assessed for inclusion according to predefined criteria. Data extraction and quality assessment were also undertaken. A decision-analytic model was constructed to estimate the cost-effectiveness of the newer agents in children with partial seizures, the only condition where there were sufficient trial data to inform a model. RESULTS: The quality of the randomised controlled trial (RCT) data was generally poor. For each of the epilepsy subtypes considered in RCTs identified for this review (partial epilepsy with or without secondary generalisation, Lennox-Gastaut syndrome, infantile spasms, absence epilepsy and benign epilepsy with centrotemporal spikes), there is some evidence from placebo-controlled trials that the newer agents tested are of some value in the treatment of these conditions. Where active controls have been used, the limited evidence available does not indicate a difference in effectiveness between newer and older drugs. The data are not sufficient to inform a prescribing strategy for any of the newer agents in any of these conditions. In particular, there is no clinical evidence to suggest that the newer agents should be considered as a first-choice treatment in any form of epilepsy in children. Annual drug costs of the newer agents ranges from around 400 pound to 1200 pound, depending on age and concomitant medications. An AED that is ineffective or has intolerable side-effects will only be used for a short period of time, and many patients achieving seizure freedom will successfully withdraw from drug treatment without relapsing. The results of the decision-analytic model do not suggest that the use of the newer agents in any of the scenarios considered is clearly cost-effective but, similarly, do not indicate that they are clearly not cost-effective. CONCLUSIONS: The prognosis for children diagnosed with epilepsy is generally good, with a large proportion responding well to the first treatment given. A substantial proportion, however, will not respond well to treatment, and for these patients the clinical goal is to find an optimal balance between the benefits and side-effects of any treatment given. For the newly, or recently, diagnosed population, the key question for the newer drugs is how soon they should be tried. The cost-effectiveness of using these agents early, in place of one of the older agents, will depend on the effectiveness and tolerability of these agents compared with the older agents; the evidence from the available trial data so far suggests that the newer agents are no more effective but may be somewhat better tolerated than the older agents, and so the cost-effectiveness for early use will depend on the trade-off between effectiveness and tolerability, both in terms of overall (long-term) treatment retention and overall utility associated with effects on seizure rate and side-effects. There are insufficient data available to estimate accurately the nature of this trade off either in terms of long-term treatment retention or utility. Better information is required from RCTs before any rational evidence-based prescribing strategy could be developed. Ideally, RCTs should be conducted from a 'public health' perspective, making relevant comparisons and incorporating outcomes of interest to clinicians and patients, with sufficiently long-term follow-up to determine reliably the clinical utility of different treatments, particularly with respect to treatment retention and the balance between effectiveness and tolerability. RCTs should mirror clinical practice with respect to diagnosis, focusing on defined syndromes or, where no syndrome is identified, on groups defined by specific seizure type(s) and aetiology. Epilepsy in children is a complex disease, with a variety of distinct syndromes and many alternative treatment options and outcomes. Diagnosis-specific decision-analytic models are required; further research may be required to inform parameter values adequately with respect to epidemiology and clinical practice.