TaqMan Low Density Array Cards Research Articles

Evaluation of circulating microRNA signature in patients with erosive hand osteoarthritis: The HOAmiR study

ObjectivesTo identify circulating micro-RNAs differentially expressed in patients with erosive hand osteoarthritis (HOA) compared to patients with non-erosive HOA and patients without HOA. MethodsIn the screening phase, 768 well-characterized micro-RNAs using Taqman Low Density Array cards were measured in 30 sera from 10 patients with erosive HOA, 10 patients with non-erosive HOA and 10 controls without HOA, matched for age and BMI. In a second step, we validated the micro-RNAs identified at the screening phase (adjusted p value < 0.05 after false discovery rate correction using Benjamini-Hochberg method and literature review) in larger samples (60 patients with erosive HOA and 60 patients without HOA matched for age and BMI). ResultsIn the screening phase, we identified 21 down-regulated and 4 up-regulated micro-RNAs of interest between erosive HOA and control groups. Among these, 9 micro-RNAs (miR-373-3p, miR-558, miR-607, miR-653-5p, miR-137 and miR448 were down-regulated and miR-142-3p, miR-144-3p and miR-34a-5p were up-regulated) were previously described in chondrocytes homeostasis or OA. We found only one significantly down-regulated micro-RNA between erosive and non-erosive HOA. In the validation phase, we showed replication of a single micro-RNA the significant downregulation of miR-196-5p, that had been previously identified in the screening phase among patients with erosive HOA compared to those without HOA.After reviewing the literature and the miRNA-gene interaction prediction model, we found that this microRNA could interact with bone homeostasis and HOXC8, which could explain its role in osteoarthritis. ConclusionsWe found that miR-196-5p was down regulated in patients with erosive HOA and some of its targets could explain a role in OA.

O-062 MicroRNAs in Blastocoel Fluid: a molecular signature for predicting human embryo implantation potential

Abstract Study question Can microRNA expression in blastocoel fluid (BF) predict embryo implantation potential? Summary answer Up-regulation of miR-106, miR-373, miR-301, miR-320 and miR-525-3p represents the molecular signature characterizing the embryos with high implantation capability. What is known already The discovery of DNA within the BF and in spent embryo culture media, has caused increased interest in the non-invasive preimplantation testing application for monogenic disorders and aneuploidies. Aneuploid embryos have a lower implantation potential, however, chromosomal abnormalities represent only a tiny percentage of the causes of implantation failure. MiRNAs can control all cellular pathways and are involved in pluripotency, self-renewal, and stemness, and their altered regulation affects different human diseases. Study design, size, duration From September 2018 to March 2022, 112 BFsampleswere collected from human embryos on the fifth day of development, before the blastocyst cryopreservation. The samples were classified according to blastocyst grade and the data on implantation outcome and term births were collected for the transferred embryos. We compared the expression profiles of 89 miRNAs, previously identified in BF, between 33 BF from implanted embryos and 30 from non-implanted ones, regardless of blastocyst grade. Participants/materials, setting, methods By custom-designed TaqMan Low-Density Array card (TLDA), we analyzed the expression of 89 miRNAs in 4 different BF samples simultaneously. Differentially expressed (DE) miRNAs were identified by Volcano plot and Significance Analysis of Microarrays (SAM) tests. Bioinformatic analysis was performed to identify the biological role of the DE miRNAs. To evaluate miRNA’s ability for predicting implantation, Pearson’s correlation analyses, classical univariate Receiver Operator Characteristic (ROC) curve analysis, and the optimal cut-off value determination were performed. Main results and the role of chance We found five miRNAs, miR-106, miR-373, miR-301, miR-320 and miR-525-3pup-regulated in BF from implanted blastocysts. The identified miRNAs perform an important role during the first phases of embryo development suggesting that their up-regulation may reflect embryo health. Moreover, four of the five miRNAs showed significant correlation coefficients in both implanted and non-implanted blastocysts, indicating that their expression changes in the same way in the single sample and reflects the potentiality of the embryo to implant. Finally, ROC curve analysis confirmed that our miRNAs could be considered potential biomarkers for implantation. Limitations, reasons for caution Successful implantation requires a close dialogue between the embryo and the endometrium, mediated by different proteins and miRNAs produced by both the embryo and maternal tissues. Determining the quality of the embryo and its implantation potential is not sufficient to predict successful pregnancy outcomes. Wider implications of the findings This study represents the first report correlating miRNA profiles in BFand implantation and suggests that miRNA signature could become an accurate tool to evaluate embryo quality. It could be associated or replaced with the PGT-A to choose the most competent embryo and improve the outcome of assisted reproduction cycles. Trial registration number The study has been approved by theEthical Committee of Azienda Ospedaliero Universitaria Policlinico &amp;quot;G.Rodolico -San Marco&amp;quot; Catania.

Comparison of multiple gene expression platforms for measuring a bladder cancer hypoxia signature.

Tumour hypoxia status provides prognostic information and predicts response to hypoxia‑modifying treatments. A previous study by our group derived a 24‑gene signature to assess hypoxia in bladder cancer. The objectives of the present study were to compare platforms for generating signature scores, identify cut‑off values for prospective studies, assess intra‑tumour heterogeneity and confirm hypoxia relevance. Briefly, RNA was extracted from prospectively collected diagnostic biopsies of muscle invasive bladder cancer (51patients), and gene expression was measured using customised Taqman Low Density Array (TLDA) cards, NanoString and Clariom S arrays. Cross‑platform transferability of the gene signature was assessed using regression and concordance analysis. The cut‑off values were the cohort median expression values. Intra‑ and inter‑tumour variability were determined in a retrospective patient cohort (n=51) with multiple blocks(2‑18) from the same tumour. To demonstrate relevance, bladder cancer cell lines were exposed to hypoxia (0.1% oxygen, 24h), and extracted RNA was run on custom TLDA cards. Hypoxia scores(HS) values showed good agreement between platforms: ClariomS vs. TLDA (r=0.72, P<0.0001; concordance73%); ClariomS vs. NanoString (r=0.84, P<0.0001; 78%); TLDA vs. NanoString (r=0.80, P<0.0001;78%). Cut‑off values were 0.047 (TLDA), 7.328 (NanoString) and 6.667 (ClariomS). Intra‑tumour heterogeneity in gene expression and HS (coefficient of variation3.9%) was less than inter‑tumour (7.9%) variability. HS values were higher in bladder cancer cells exposed to hypoxia compared with normoxia (P<0.02). In conclusion, the present study revealed that application of the 24‑gene bladder cancer hypoxia signature was platform agnostic, cut‑off values determined prospectively can be used in a clinical trial, intra‑tumour heterogeneity was low and the signature was sensitive to changes in oxygen levels invitro.

Reduced Levels of miR-342-5p in Plasma Are Associated With Worse Cognitive Evolution in Patients With Mild Alzheimer's Disease.

BackgroundProgressive cognitive decline is the most relevant clinical symptom of Alzheimer’s disease (AD). However, the rate of cognitive decline is highly variable between patients. Synaptic deficits are the neuropathological event most correlated with cognitive impairment in AD. Considering the important role of microRNAs (miRNAs) in regulating synaptic plasticity, our objective was to identify the plasma miRNAs associated with the rate of cognitive decline in patients with mild AD.MethodsWe analyzed 754 plasma miRNAs from 19 women diagnosed with mild AD using TaqMan low-density array cards. The patients were grouped based on the rate of decline in the MMSE score after 2 years [<4 points (N = 11) and ≥4 points (N = 8)]. The differentially expressed miRNAs between the two groups were validated in an independent cohort of men and women (N = 53) with mild AD using RT-qPCR.ResultsIn the discovery cohort, 17 miRNAs were differentially expressed according to the fold change between patients with faster declines in cognition and those with slower declines. miR-342-5p demonstrated differential expression between the groups and a good correlation with the rate of cognitive decline in the validation cohort (r = −0.28; p = 0.026). This miRNA had a lower expression level in patients who suffered from more severe decline than in those who were cognitively more stable after 2 years (p = 0.049).ConclusionLower levels of miR-342-5p in plasma were associated with faster cognitive decline in patients with mild AD after 2 years of follow-up.

GATA3 rs3824662A Allele Is Overrepresented in Adult Patients with Ph-like ALL, Especially in Patients with CRLF2 Abnormalities

Background: Ph-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL in both children and adults (Roberts, NEJM 2014; Roberts, JCO 2017; Jain, Blood 2017). Inherited GATA3 variants (rs3824662) have been described in higher frequency in children with Ph-like ALL (Perez-Andreu, Nat Genetics 2013). The frequency and clinical correlation of this genetic variant in adult patients with Ph-like ALL is unknown.Methods: Ph-like ALL status was defined by gene expression profiling using either U133 Plus 2.0 microarrays as previously described (Roberts, NEJM 2014), or a customized Taqman low-density array card. CRLF2 rearrangement (IGH - CRLF2 or P2RY8 - CRLF2) was confirmed using fluorescence-in-situ-hybridization (FISH). Genome-wide SNP genotyping was performed using Affymetrix SNP 6 or Illumina Omni2.5 array. Genetic ancestry was inferred by STRUCTURE using genotypes at 30,000 randomly selected SNPs. Association of GATA3 rs3824662 genotype with Ph-like ALL and/or CRLF2 rearrangement was evaluated by logistic regression, after adjusting for genetic ancestry as appropriate.Results: A total of 187 newly diagnosed B-ALL patients at MD Anderson Cancer Center (MDACC) were evaluated for Ph-like gene expression profile (Figure 1). Fifty-three patients had Ph-like ALL (30 CRLF2 rearranged; 23 non- CRLF2). We performed analyses for GATA3 rs3824662 variant in those with available samples (n=85). The rs3824662 AA genotype was identified in 20 of 42 (47.6%) patients with Ph-like ALL vs. 2 of 43 (4.6%) patients without Ph-like ALL (P= 4.215x10-6). The risk allele frequency (RAF) was 65.5% among the Ph-like ALL patients vs. 25.5% among the non-Ph-like ALL patients (P= 0.00056) (Figure 2). The rs3824662 AA genotype was particularly over-represented patients with CRLF2 rearranged Ph-like ALL (56%) vs. non- CRLF2 rearranged Ph-like ALL (35%). The RAF was 78% in the CRFL2 rearranged Ph-like ALL, compared to 47% in non- CRFL2 rearranged Ph-like ALL (Figure 2). Among the Ph-like ALL, GATA3 rs3824662 A allele was over-represented in Ph-like ALL with CRLF2, JAK2, or IKZF1 abnormalities (up to 80%). GATA3 rs3824662 A allele was significantly more common in US Hispanics than individuals of non-Hispanic background (P= 0.00055), as defined by Native American genetic ancestry. We hypothesize that GATA3 regulates CRFL2 transcription, and this may in turn leads to CRLF2 overexpression (Yang et al., ASH 2017 submitted).Conclusions: Similar to pediatric patients, GATA3 SNP rs3824662 is associated with susceptibility to Ph-like ALL in adults, with a preponderance of ALL patients with CRLF2 abnormalities. There is significant over-representation of this germline genetic variant in patients of Hispanic background, which may in part explain the high incidence of Ph-like ALL in this population. [Display omitted] DisclosuresJain:Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Genentech: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour:Bristol-Myers Squibb: Consultancy. Kantarjian:Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding. Mullighan:Amgen: Consultancy; Loxo Oncology: Research Funding.

Expression of cPLA2γ mRNA and protein differs the response of PBMC from severe and non-severe asthmatics to bacterial lipopolysaccharide and house dust mite allergen

Chronic inflammation in asthmatics is initiated/exacerbated by many environmental factors, such as bacterial lipopolysaccharide and allergens. Phospholipase A2 and histone acetyltransferase/deacetylases are enzymes involved in inflammatory process, particularly in lipid inflammatory mediators production and control of transcription of many inflammatory genes, respectively. The aim of the study was to identify differences in the inflammatory process in patients with severe and non-severe asthma, taking as a criterion expression of two groups of enzymes: phospholipases A2 and histone acetyltransferases/deacetylases. Thirty-two patients with severe, non-severe atopic to house dust mite asthmatics and 14 healthy volunteers were recruited. Peripheral blood mononuclear cells were stimulated with Dermatophagoides pteronyssinus allergen (nDer p1) and bacterial lipopolysaccharide (LPS). The expression of phospholipases A2 and histone acetyltransferases and deacetylases were assessed using TaqMan Low Density Array Cards. The protein expression was analyzed with immunoblot. Increased expression of phospholipase A2 Group IVC (PLA2G4C) and cytosolic phospholipase A2 gamma (cPLA2γ) protein was observed in peripheral blood mononuclear cells (PBMC) from severe asthmatics in response to LPS and nDer p1, compared to non-severe asthmatics. nDer p1-stimulated PBMC from severe asthmatics exhibit induced expression of HDAC1 and similar trend was observed in protein concentration. Decreased expression of EP300 occurred in PBMC of severe asthmatics. PBMC from non-severe asthmatics showed decreased expression of HDAC2 and PLA2G15 after LPS treatment. In conclusion, in response to LPS and dust mite allergen, PBMC from severe and non-severe asthmatics modulate expression of selected phospholipase A2, histone acetyltransferases and deacetylases, while increased expression of cPLA2γ characterizes PBMC response from severe asthmatics.

Metformin decreases miR-122, miR-223 and miR-29a in women with polycystic ovary syndrome.

Metformin is associated with increased insulin sensitivity, whereas oral contraceptive pills (OCP) could increase the risk for type 2 diabetes (T2D) in women with polycystic ovary syndrome (PCOS). Certain miRNAs might serve as biomarkers for the risk of T2D. The aim of this study was to investigate changes in circulating miRNA levels during treatment with metformin and OCP in women with PCOS. Sixty-five women with PCOS according to Rotterdam criteria were randomized to metformin (2 g/day), metformin + OCP (150 mg desogestrel + 30 µg ethinylestradiol) or OCP alone for 12 months. Serum miRNA analysis was performed with individual RT-qPCR or Taqman low density array cards of 22 selected miRNAs previously related to PCOS, glucose and/or lipid metabolism. miR-122 and miR-29a levels were decreased after treatment with metformin compared with metformin + OCP and OCP group: miR-122: log2 difference −0.7 (P = 0.01) and −0.7 (P = 0.02), miR-29a: log2 difference −0.5 (P = 0.01) and −0.4 (P = 0.04), while miR-223 levels were decreased in the metformin + OCP group after treatment: log2 difference −0.5 (P = 0.02). During the treatment period, a significant weight loss was observed in the metformin group compared with the OCP group. In the OCP group, miRNA levels were unchanged during the treatment period. Levels of circulating miRNAs associated with lipid and glucose metabolism decreased during metformin treatment. Changes in miRNA levels in the metformin group could be explained by the simultaneous weight loss in the same group. These results support the notion that metformin treatment alone may be superior for metabolic health compared with OCP.

Serum miRNAs Are Pharmacodynamic Biomarkers Associated With Therapeutic Response in Pediatric Inflammatory Bowel Disease.

BackgroundWe sought to identify microRNAs (miRNAs) associated with response to anti-TNF-α or glucocorticoids in children with inflammatory bowel disease (IBD) to generate candidate pharmacodynamic and monitoring biomarkers. MethodsClinical response was assessed by Pediatric Crohn’s Disease Activity Index and Pediatric Ulcerative Colitis Activity Index. Quantitative real-time polymerase chain reaction via Taqman Low-Density Array cards were used to identify miRNAs in a discovery cohort of responders (n = 11) and nonresponders (n = 8). Seven serum miRNAs associated with clinical response to treatment, along with 4 previously identified (miR-146a, miR-146b, miR-320a, miR-486), were selected for further study. Candidates were assessed in a validation cohort of serum samples from IBD patients pre- and post-treatment and from healthy controls. Expression of miRNA was also analyzed in inflamed mucosal biopsies from IBD patients and non-IBD controls. ResultsDiscovery cohort analysis identified 7 miRNAs associated with therapeutic response: 5 that decreased (miR-126, miR-454, miR-26b, miR-26a, let-7c) and 2 that increased (miR-636, miR-193b). In the validation cohort, 7 of 11 candidate miRNAs changed in the same direction with response to anti-TNF-α therapies, glucocorticoids, or both. In mucosal biopsies, 7 out of 11 miRNAs were significantly increased in IBD vs healthy controls.ConclusionsFive candidate miRNAs associated with clinical response and mucosal inflammation in pediatric IBD patients were identified (miR-126, let-7c, miR-146a, miR-146b, and miR-320a). These miRNAs may be further developed as pharmacodynamic and response monitoring biomarkers for use in clinical care and trials.

Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: searching for biomarkers of implantation

Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos? Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM. Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing. This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions. Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3hours from sample collection to data inspection. The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed. This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples. Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection. This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study. None.

Biomarkers of Overall Survival and Response to Glasdegib and Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia

Background: In a previously reported Phase 2 randomized study of patients with acute myeloid leukemia (AML), addition of the investigational agent glasdegib (PF-04449913) to low-dose cytarabine (LDAC) improved overall survival (OS) when compared with LDAC alone. In a non-randomized study arm, glasdegib together with 7+3 chemotherapy was well tolerated and associated with clinical activity. We used a comprehensive biomarker analysis, evaluating gene expression, circulating cytokine levels, and gene mutations, to identify molecular drivers that predict overall response (OR) and OS.Methods: In this Phase 2 multicenter study (NCT01546038), patients with AML who were suitable for non-intensive therapy were randomized (2:1) to LDAC + glasdegib 100 mg QD or LDAC alone, and patients suitable for intensive therapy were assigned 7+3 plus glasdegib 100 mg QD. Whole blood, serum, and bone marrow aspirate samples were collected at baseline, and used to assess 19 genes for expression analysis, 38 analytes for circulating cytokine levels, and 109 genes for mutation analysis. Gene expression was analyzed using TaqMan Low Density Array Cards (TLDCs), cytokine levels were analyzed using quantitative, multiplexed immunoassays (Myriad RBM), and mutation analysis was performed using the Illumina® MiSeq instrument (San Diego, CA). All correlations were performed either for OS or for OR. For gene expression and cytokine analysis, a cut-off value above or below the median expression level for each treatment arm was used to separate samples into two subgroups (< or ≥ the median value) to explore the relationship of expression levels with OS data. Criteria for significance in the non-intensive cohort required one subgroup to have a p-value of <0.05 in the between-treatment arms comparison and the HR difference between the two subgroups to be ≥2 fold. Responses were defined as patients with a complete remission (CR), CR with incomplete blood count recovery (CRi), morphologic leukemia-free state, partial remission (PR), or PRi. For response correlations, genes or cytokines were considered to be differentially expressed if they had a p-value <0.05 and were differentially expressed by ≥2-fold.Results: Within the non-intensive arm (LDAC + glasdegib, n=68; LDAC alone, n=30), expression levels of several genes correlated with improved OS with glasdegib plus LDAC. Lower levels of expression of FOXM1 and MSI2, and higher expression levels of BCL2 and CCND2 correlated with improved OS with the combination. Additionally, lower levels of the cytokines 6CKINE (CCL21), ICAM-1, MIP-1α, and MMP-3 correlated with improved OS. An analysis of correlations of gene expression and cytokine levels with OR could not be completed due to the low number of responders in the LDAC only group (n=2). In the intensive treatment arm (glasdegib and 7+3, n=59), higher PTCH1 expression correlated with improved OS (p=0.0219, median OS 10.8 versus 39.5 months). In this cohort, lower levels of IL-8 (p=0.0225) and MIP-3β (p=0.0403) correlated with lower OS. Expression levels of no genes or cytokines significantly correlated with OR in this arm. We also examined correlations between gene mutation status and OS in both study arms. In the non-intensive arm (LDAC + glasdegib, n=58; LDAC alone, n=25), no genes mutated in at least 5 patients correlated with OS. In the intensive treatment arm (n=47), mutations in FLT3, TP53, CEP170, NPM1, and ANKRD26 correlated with OS (all p<0.05). Patients in this arm with FLT3 mutations responded better than patients with wild type FLT3 (p=0.0336, median OS of 13.1 months versus unreached for FLT3 mutant).Conclusions: In this biomarker analysis, we found that expression levels of a select number of genes and circulating cytokines implicated in AML correlated with OS in the non-intensive and the intensive arms. The improved response for patients with FLT3 mutations and high PTCH1 expression levels in the intensive arm deserves further investigation. These findings need to be verified in larger controlled studies, which are ongoing. DisclosuresCortes:Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Astellas: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding. Chan:Pfizer: Employment, Equity Ownership. Wang:Pfizer: Employment, Equity Ownership. Ching:Pfizer Inc: Employment, Equity Ownership. Johnson:Pfizer Inc: Employment, Equity Ownership. O'Brien:Pfizer Inc: Employment, Equity Ownership.

Analytics of Cerebrospinal Fluid MicroRNA Quantitative PCR Studies

MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional gene expression. Recent studies have shown that human disease states correlate with measurable differences in the level of circulating miRNAs relative to healthy controls. Thus, there is great interest in developing clinical miRNA assays as diagnostic or prognostic biomarkers for diseases, and as surrogate measures for therapeutic outcomes. Our studies have focused on miRNAs in human cerebral spinal fluid (CSF) as biomarkers for central nervous system (CNS) diseases. Our objective here was to examine factors that may affect the outcome of quantitative PCR (qPCR) studies on CSF miRNAs, in order to guide planning and interpretation of future CSF miRNA TaqMan® low-density array (TLDA) studies. We obtained CSF from neurologically normal (control) donors and used TLDAs to measure miRNA expression. We examined sources of error in the TLDA outcomes due to (1) nonspecific amplification of products in total RNA, (2) variations in RNA isolations performed on different days, (3) miRNA primer probe efficiency, and (4) variations in individual TLDA cards. We also examined the utility of card-to-card TLDA corrections and use of an unchanged "reference standard" to remove batch processing effects in large-scale studies.

Circulating miRNAs in diabetic kidney disease: case-control study and in silico analyses.

The aim of this study was to investigate a miRNA expression profile in type 1 diabetes mellitus (T1DM) patients with DKD (cases) or without this complication (controls). Expression of 48 miRNAs was screened in plasma of 58 T1DM patients (23 controls, 18 with moderate DKD, and 17 with severe DKD) using TaqMan Low Density Array cards (Thermo Fisher Scientific). Then, five of the dysregulated miRNAs were selected for validation in an independent sample of 10 T1DM controls and 19 patients with DKD (10 with moderate DKD and 9 with severe DKD), using RT-qPCR. Bioinformatic analyses were performed to explore the putative target genes and biological pathways regulated by the validated miRNAs. Among the 48 miRNAs investigated in the screening analysis, 9 miRNAs were differentially expressed between DKD cases and T1DM controls. Among them, the five most dysregulated miRNAs were chosen for validation in an independent sample. In the validation sample, miR-21-3p and miR-378-3p were confirmed to be upregulated in patients with severe DKD, while miR-16-5p and miR-29a-3p were downregulated in this group compared to T1DM controls and patients with moderate DKD. MiR-503-3p expression was not validated. Bioinformatic analyses indicate that the four validated miRNAs regulate genes from PI3K/Akt, fluid shear stress and atherosclerosis, AGE-RAGE, TGF-β1, and relaxin signaling pathways. Our study found four miRNAs differentially expressed in patients with severe DKD, providing significant information about the biological pathways in which they are involved.

MP87-03 TARGETED GENE EXPRESSION PROFILING WITHIN CIRCULATING TUMOR CELLS IN MCRPC

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV1 Apr 2018MP87-03 TARGETED GENE EXPRESSION PROFILING WITHIN CIRCULATING TUMOR CELLS IN MCRPC Udit Singhal, Yugang Wang, Zachary Reichert, Scott Tomlins, and Todd Morgan Udit SinghalUdit Singhal More articles by this author , Yugang WangYugang Wang More articles by this author , Zachary ReichertZachary Reichert More articles by this author , Scott TomlinsScott Tomlins More articles by this author , and Todd MorganTodd Morgan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2903AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Moving from simple enumeration of circulating tumor cells (CTCs) to molecular characterization continues to be a critical need in order for CTC-based liquid biopsy approaches to move into the clinic. CTCs provide a unique avenue to assess tumor-specific RNA expression, and methods that replicate tissue-based approaches may help address key technical challenges. Tissue-based molecular risk classifiers, such as Prolaris and OncotypeDx, were previously developed by targeted expression profiling using the TaqMan Low Density Array (TLDA) platform. Here we describe the development of an approach to perform CTC-based, targeted gene expression profiling using the TLDA platform in patients with metastatic castrate resistant prostate cancer (mCRPC). METHODS CTCs were isolated from 5 mL whole blood of mCRPC patients using an anti-EpCAM-microbead based protocol. Following cell lysis, mRNA from CTCs was captured using Oligo(dT)25 mRNA Dynabeads followed by reverse transcription. TLDA cards (Applied Biosystems) were used to perform simultaneous, singleplex qPCR detection of a custom panel of 48 prostate cancer related genes and controls, including AR, AR-V7, PCA3, PSMA, SChLAP1, TMPRSS2:ERG, and WNT5B. Blood processed from 22 healthy controls was used as a baseline referent to account for leukocyte contamination. Gene expression data from prostate cancer cells (VCaP, PC3, and LNCaP) spiked into normal control blood was used for CTC isolation assay development, and CTCs from 68 patients with mCRPC were subsequently evaluated. RESULTS CTC expression was assessed in 68 patients with mCRPC. We identified 46 (68%) that showed increased androgen signaling, including 27 (40%) with increased expression of AR and 19 (28%) with increased AR-V7, 27 (40%) with increased expression of KLK2, and 14 (21%) with increased TMPRSS2:ERG. Wnt signaling was upregulated in 29 (43%) patients, with increased expression of WNT5B in 21 (31%) and BMP7 in 14 (21%) patients. The prostate cancer specific long non-coding RNAs SChLAP1 and PCA3 were upregulated in 5 (7%) and 29 (43%) samples, respectively. 38 (56%) samples showed expression of epithelial-mesenchymal transition genes SOX2, SOX9, and TWIST1 and cell proliferation was upregulated in 51 (75%) as indicated by expression of GAS6, MDK, MET, and TSPAN8. CONCLUSIONS CTC-based targeted gene expression profiling using TLDA cards allows RNA assessment on a platform that has shown analytic validity in the tissue-based setting. Additional work will be required to assess the analytic and clinical validity of this approach when applied to CTCs. Further correlation of long-term, objective outcomes with CTC-based gene expression is ongoing and may inform prognosis in patients with advanced prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1188 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Udit Singhal More articles by this author Yugang Wang More articles by this author Zachary Reichert More articles by this author Scott Tomlins More articles by this author Todd Morgan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mineralocortidoid Receptor Mediates Uremic Serum‐Induced Increase in Endothelial Cell Dysfunction

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD) and mortality compared with the age‐matched general population. The high concentration of circulating uremic toxins in CKD patients leads to vascular inflammatory, thereby inducing endothelial dysfunction, which is associated with CVD development and progression. Plasma aldosterone levels are increased in CKD, and aldosterone has been found to increase vascular inflammation and fibrosis. The aim of our study was to analyze the influence of CKD in the expression of endothelial ion channels and its potential modification by mineralocorticoid receptor (MR) inhibition, which would consequently affect endothelial cell function. To that end we used primary human endothelial cells cultured in medium supplemented with pooled sera from either healthy or uremic patients undergoing dialysis. To obtain a complete profile of ion channel subunit expression in both cellular types, we performed high‐throughput qPCR of 92 ion channel genes using a custom‐designed Taqman low‐density array card. In addition, we characterized the influence of uremic serum on nitric oxide (NO) production and measured cortical stiffness using atomic force microscopy. Our data show that uremic serum potently increased MR expression. In addition, it induced remodeling of ion channel expression patterns, including increased expression of αENaC, a known transcriptional target of MR that promotes cortical stiffness and endothelial dysfunction. Exposure of endothelial cells to uremic serum significantly decreased NO production and produced stiffening of the endothelial cell cortex, which was prevented by inhibition of MR using spironolactone. Our data identifies MR as a possible mediator of uremia‐induced endothelial dysfunction and supports the proposed beneficial effects of MR inhibition during CKD.Support or Funding InformationThis work was supported by a grant from Ministerio de Economía e Innovación (MINECO, grant BFU2016‐78374‐R) to D.A.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic CD36 downregulation parallels steatosis improvement in morbidly obese undergoing bariatric surgery.

The notion that hepatic expression of genes involved in lipid metabolism is altered in obese patients is relatively new and its relationship with hepatic steatosis and cardiometabolic alterations remains unclear. We assessed the impact of Roux-en-Y gastric bypass surgery (RYGB) on the expression profile of genes related to metabolic syndrome in liver biopsies from morbidly obese individuals using a custom-made, focused cDNA microarray, and assessed the relationship between the expression profile and hepatic steatosis regression. Plasma and liver samples were obtained from patients at baseline and 12 months after surgery. Samples were assayed for chemical and gene expression analyses, as appropriate. Gene expression profiles were assessed using custom-made, focused TaqMan low-density array cards. RYGB-induced weight loss produced a favorable reduction in fat deposits, insulin resistance (estimated by homeostasis model assessment of insulin resistance (HOMA-IR)), and plasma and hepatic lipid levels. Compared with the baseline values, the gene expression levels of key targets of lipid metabolism were significantly altered: CD36 was significantly downregulated (-40%; P=0.001), whereas APOB (+27%; P=0.032) and SCARB1 (+37%; P=0.040) were upregulated in response to surgery-induced weight reduction. We also observed a favorable reduction in the expression of the PAI1 gene (-80%; P=0.007) and a significant increase in the expression of the PPARA (+60%; P=0.014) and PPARGC1 genes (+36%; P=0.015). Notably, the relative fold decrease in the expression of the CD36 gene was directly associated with a concomitant reduction in the cholesterol (Spearman's r=0.92; P=0.001) and phospholipid (Spearman's r=0.76; P=0.04) contents in this tissue. For the first time, RYGB-induced weight loss was shown to promote a favorable downregulation of CD36 expression, which was proportional to a favorable reduction in the hepatic cholesterol and phospholipid contents in our morbidly obese subjects following surgery.

The role of Mineralocorticoid Receptor on Human Aortic Endothelial Ion Channel Expression Profile in Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular events and cardiovascular disease (CVD) mortality compared with the age-matched general population. The high concentration of circulating uremic toxins in CDK patients may trigger vascular inflammatory responses, thereby inducing endothelial dysfunction, which is associated with CVD development and progression. In addition, plasma aldosterone levels are increased in CKD, and aldosterone has been found to increase vascular inflammation and fibrosis. The aim of our study was to analyze the influence of CKD in the expression of endothelial ion channels and its potential modification by mineralocorticoid receptor (MR) antagonism, which would consequently affect endothelial dysfunction. To that end we used human aortic endothelial cells (HAEC) cultured in medium supplemented with pooled sera from either healthy or uremic patients undergoing dialysis, as well as both groups in the presence of spironolactone. HAEC were found to express MR under the culture conditions used. To obtain a global portrait of ion channel expression in HAEC in the four groups tested, we performed high-throughput real-time polymerase chain reaction of 92 ion channel genes using a custom-designed Taqman low-density array card. We have obtained a profile of ion channel gene expression altered by uremic serum and the effect of spironolactone both on basal and altered expression of ion channel subunits.

Exacerbating Factors Induce Different Gene Expression Profiles in Peripheral Blood Mononuclear Cells from Asthmatics, Patients with Chronic Obstructive Pulmonary Disease and Healthy Subjects

Background: Despite several common phenotypic features, chronic obstructive pulmonary disease (COPD) and severe asthma differ with regard to their causative factors and pathophysiology. Both diseases may be exacerbated by environmental factors, however, the molecular profiles of disease episodes have not been comprehensively studied. We identified differences in gene and protein expression profiles expressed by peripheral blood mononuclear cells (PBMC) of COPD patients, patients with atopic asthma and healthy subjects when challenged with exacerbating factors in vitro: lipopolysaccharide (LPS), house dust mite (HDM) and cat allergen. Methods: PBMC isolated from patients with severe atopic asthma and COPD, as well as healthy subjects were stimulated with rDer p 1 DG, rFel d 1 DG and LPS. The changes in the expression of 47 genes belonging to five groups (phospholipase A₂, eicosanoids, transcription factors, cytokines and airway remodeling) were studied using TaqMan low density array cards. Immunoblotting was used to study relative protein expression. Results: rDer p 1 significantly up-regulated the expression of PLA2G4A, PLA2G6, PLA2G15, CYSLTR1, LB4R2, PTGS1, PTGS2, FOXP1, GATA3, HDAC2, IREB2, PPARG, STAT4, TSLP and CHI3L1 genes in asthmatics in comparison to healthy subjects. LPS induced significant expression of ANXA1 and LTA4H in asthmatics when compared to COPD patients and healthy subjects. SOX6,STAT4 and IL1RL1 were induced in COPD after LPS stimulation. Analysis of protein expression revealed a pattern similar to mRNA expression. Conclusions: LPS-induced exacerbation of asthma and COPD is characterized by differential expression of selected genes in PBMC. HDM allergen changed the expression profile of inflammatory genes between patients with asthma of atopic origin and healthy controls.

Gene Expression of Desaturase (FADS1 and FADS2) and Elongase (ELOVL5) Enzymes in Peripheral Blood: Association with Polyunsaturated Fatty Acid Levels and Atopic Eczema in 4-Year-Old Children

BackgroundIt is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children.MethodsAE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson’s rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE.ResultsChildren with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1.Conclusions and SignificanceChanges in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children.

Adaptation of Impression Cytology to Enable Conjunctival Surface Cell Transcriptome Analysis

Purpose: This study investigates the extent of the human transcriptome that can be quantified from conjunctival impression cytology extracts. The aim is to determine if sufficient RNA can be isolated from a patient’s conjunctival surface to identify differences in gene expression between dry eye and normal patients of (a) an array of 96 inflammatory biomarkers and associated receptors, and (b) if this comparison can be expanded to the entire transcriptome.Materials and Methods: CIC was used to collect conjunctival surface cells from 53 qualifying normal and dry eye patients. Based on prior optimization of all assay steps, RNA was isolated from the samples using a Qiagen RNeasy Plus Mini Kit and qRT-PCR was used to determine gene expression of 96 genes using TaqMan Low Density Array cards. Samples from six normal and six dry eye patients were then assayed on an Illumina Human HT-12 BeadChip.Results: Optimization steps yielded an RNA processing procedure that improved yield from an initial 12 genes through 96, then to the entire human transcriptome. For the HT-12 BeadChip, more than 30 genes differed by a factor of >1.5 between the dry eye and normal groups and seven genes were down-regulated by a factor of >2.0 in the dry eye group: HLA-DRB5, PSCA, FOS, lysozyme, TSC22D1, CAPN13 and CXCL6.Conclusions: Conjunctival impression cytology can be used to collect sufficient RNA from conjunctival surface cells that, when processed optimally, allows successful transcriptome-wide expression analysis. While the current transcriptome analysis used a limited patient group, larger studies of patients with various types and severities of dry eye should reveal significant gene expression trends that can then be targeted to improve dry eye treatment options.

Evaluating gene expression profiling by quantitative polymerase chain reaction to develop a clinically feasible test for outcome prediction in multiple myeloma

The gene expression profiles (GEPs) of 96 selected genes were analysed by real-time quantitative polymerase chain reaction (qPCR) with a TaqMan low-density array card in isolated tumour plasma cells (PCs) from 157 newly diagnosed multiple myeloma (MM) patients. This qPCR-based GEP correctly classified cases following the Translocation-cyclin D classification. Classic prognostic parameters and qPCR-based GEP predicted MM patient outcome and, although multivariate analyses revealed that cytogenetic risk (standard vs. high risk) was the variable that most strongly predicted prognosis, GEP added significant information for risk stratification. Considering only the standard risk cytogenetic patients, multivariate analyses revealed that high β2-microglobulin, low CDKN1A and high SLC19A1 gene expression levels independently predicted a short time-to-progression (TTP), while high International Staging System stage, low CDKN2B and high TBRG4 gene expression predicted poor overall survival (OS). A gene expression risk score enabled the division of standard risk patients into two groups with different TTPs (83% vs. 38% at 3years, P<0·0001) and OS rates (88% vs. 61% at 5years; P=0·003). This study demonstrates that quantitative PCR is a robust, accurate and feasible technique for implementing in the daily routine as a surrogate for GEP-arrays.