Low Density Array Research Articles

Circulating miR-145 as a marker of therapeutic response to anti-TNF therapy in patients with ankylosing spondylitis

Circulating miRNAs appear promising therapeutic and prognostic biomarkers. We aimed to investigate the predictive value of circulating miRNAs on the disease outcome following anti-TNF therapy in patients with ankylosing spondylitis (AS). Our study included 19 AS patients assessed at baseline (M0), after three (M3) and twelve months (M12) of therapy. Total RNA was isolated from plasma. A comprehensive analysis of 380 miRNAs using TaqMan Low Density Array (TLDA) was followed by a single assay validation of selected miRNAs. All AS patients had high baseline disease activity and an excellent response to anti-TNF therapy at M3 and M12. TLDA analysis revealed the dysregulation of 17 circulating miRNAs, including miR-145. Single assay validation confirmed that miR-145 is significantly downregulated at M3 compared to baseline. The decrease in the levels of miR-145 from M0 to M3 negatively correlated with the change in BASDAI from M0 to M3; and positively correlated with disease activity improvement from M3 to M12 as per BASDAI and ASDAS. The predictive value of the early change in miR-145 and levels of miR-145 at M3 were further validated by Receiver operating curves analysis. We show thatthe early change in circulating miR-145 may be a predictor for the future outcome ofAS patients treated with TNF inhibitors. Patients with a more significant decrease in miR-145 levels may show further significant improvement of disease activity after 12 months. Monitoring the expression of miR-145 in plasma in AS patients may, therefore, influence our therapeutic decision-making.

Analyzing the impact of 900\xa0MHz EMF short-term exposure to the expression of 667 miRNAs in human peripheral blood cells

More than ever before, people around the world are frequently exposed to different sections of the electromagnetic spectrum, mainly emitted from wireless modern communication technologies. Especially, the level of knowledge on non-thermal biological EMF effects remains controversial. New technologies allow for a more detailed detection of non-coding RNAs which affect the post-transcriptional control. Such method shall be applied in this work to investigate the response of human blood cells to electromagnetic irradiation. In this ex vivo in vitro study, we exposed peripheral blood cells from 5 male donors to a continuous wave of 900 MHz EMF for 0, 30, 60 and 90 min. Significant micro RNA (miRNA) expression changes (p ≤ 0.05) above or below the SHAM exposed samples were evaluated using a quantitative real time PCR platform for simultaneous detection of 667 miRNAs called low density array. Only significant miRNA expression changes which were detectable in at least 60% of the samples per exposure group were analyzed. The results were compared with data from room temperature + 2 °C (RT + 2 °C) samples (here referred to as hyperthermia) to exclude miRNA expression altered by hyperthermia. The validation study by using the same donors and study design was performed after an interval of 2 years. When analyzing a total of 667 miRNAs during the screening study, 2 promising candidate miRNAs were identified, which were down regulated almost twice and showed a complete separation from the unexposed control group (miR-194 at 30 min and miR-939 at 60 min). The p-values even survived the Bonferroni correction for multiple comparisons (p = 0.0007 and p = 0.004, respectively). None of these miRNAs were expressed at a second time point after EMF exposure. Following an alternative analysis approach, we examined for miRNAs revealing an expected significant association of differential miRNA expression with the dose-time EMF exposure product, separately for each donor. Donors 2 and 3 revealed 11 and 10 miRNA species being significantly associated with EMF exposure which differed significantly from the other donors showing a minor number of differentially expressed miRNAs and could identify donors 2 and 3 as particularly EMF-responsive. The measurements were repeated after 2 years. The number of expressed/non-expressed miRNAs was almost similar (97.4%), but neither the number nor the previously differentially expressed miRNAs could be reproduced. Our data neither support evidence of early changes at miRNA expression level in human whole blood cells after 900 MHz EMF exposure nor the identification of EMF-responsive individuals.

Performance of genome prediction for morphological and growth-related traits in Yellow River carp

Common Carp (Cyprinus carpio) is the earliest truly domesticated fish, and genetic improvement of its economic traits is important for satisfying human protein demand.However, very little is known about the performance of Genomic selection (GS) in common carp by used SNP array data. Here we estimated the genetic parameters and evaluated the performance of GS for 24 morphological and growth-related traits in Yellow River carp. The result showed that the heritability of these traits ranged from medium to high. The predictive ability of most traits based on family cross-validation was higher than that of random cross-validation. Compared with best linear unbiased prediction (BLUP) method, genomic BLUP (GBLUP) method has more advantage on predictive ability of low heritability traits, with a maximum increase ratio of 37.5%. Gene annotation information is not helpful to improving predictive ability, but GBLUP based on haplotype information obtained highest accuracy on 6 of 24 traits, and compared with GBLUP, the average ratio of prediction abilities increased by 2%. This study shows that it is necessary to assess the effect of family structure on reference group construction before calculated the breeding value. In additon, biological information has a certain potential for improving prediction ability. Low density array genotyping, as a kind of method can decrease the cost of genotyping and facilitate the implementation of genomic selection in aquatic breeding.

Plasma miR-193a-3p can be a potential biomarker for the diagnosis of diabetic nephropathy

Diabetic nephropathy is one of the most common microvascular complications in patients with diabetes. MicroRNA (miRNA, miR) is closely related to the formation, development and pathophysiology of diabetic nephropathy. We aimed to investigate whether miR-193a-3p could be used as a potential biomarker for the diagnosis of diabetic nephropathy. Plasma samples were collected from all the participants. TaqMan Low Density Array analysis was employed to obtain the miRNA profiles of plasma samples, and qRT-PCR was used to confirm the result. Receiver operating characteristic curves were employed to evaluate the specificity and sensitivity of miR-193a-3p for predicting diabetic nephropathy. The expression of miR-193a-3p and miR-320c was elevated and miR-27a-3p was decreased in diabetic nephropathy patients compared to patients with type 2 diabetes and healthy controls. We found that, in diabetic nephropathy patients, the elevated miR-193a-3p expression had a negative correlation with the level of evaluate glomerular filtration rate, while a positive correlation with the level of proteinuria. We further demonstrated that miR-193a-3p could be employed to distinguish patients with diabetic nephropathy. The Kaplan-Meier analysis showed that the high expression of miR-193a-3p significantly shortened the dialysis-free survival of diabetic nephropathy patients. In conclusion, miR-193a-3p is involved in diabetic nephropathy pathogenesis and may serve as a potentially novel diagnostic biomarker for diabetic nephropathy.

Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer.

miRNAs in circulating extracellular vesicles (EVs) are promising biomarkers for cancer. However, their diagnostic ability for early‐stage non‐small‐cell lung cancer (NSCLC) is not well known. In this study, the circulating EV miRNAs profiling was initially performed in 36 untreated NSCLC patients and 36 healthy controls by TaqMan Low Density Array (TLDA). Subsequently, we performed quantitative reverse‐transcription PCR assay (RT‐qPCR) validation in several independent cohorts that included 159 NSCLC patients, 120 age/sex‐matched healthy controls and 31 benign nodule patients enrolled from three different clinical centres. In addition, 38 cases of NSCLC were analysed before and after surgery. We demonstrated that miR‐520c‐3p and miR‐1274b were significantly and steadily increased in NSCLC patients in comparison with healthy controls and benign nodule patients (P < 0.001) and decreased markedly after tumour resection (P < 0.001). The areas under the curve (AUCs) of the ROC curve of the two‐miRNA panel were 0.857 (95% CI, 0813–0.901; P < 0.0001) and 0.845 (95% CI, 0.793–0.896; P < 0.0001) for NSCLC and NSCLC stage I, respectively. Furthermore, the panel was able to differentiate NSCLC from benign nodules with an AUC of 0.823 (95% CI, 0.730–0.915; P < 0.0001). Furthermore, logistic regression analysis revealed the two‐miRNA panel as an independent risk factor for NSCLC (OR = 16.128, P < 0.0001). In conclusion, miR‐520c‐3p and miR‐1274b have biomarker potential for early diagnosis of NSCLC in multiple centres.

A novel microfluidic-based assay to determine high-risk pancreatic cysts for surgery utilizing pancreatic cyst fluid: an international multicenter study

Presenter: Ajay Maker MD | University of California San Francisco Background: Pancreatic cysts are increasingly diagnosed in the general population. We previously reported that the majority of cysts resected in the country are low risk lesions on final pathology, despite multiple established clinical guidelines. Thus, selecting which patients harbor a cyst at high-risk of occult malignancy or malignant degeneration for operative resection remains a clinical dilemma. The aim of the current study is to develop a high-throughout molecular diagnostic platform to identify pancreatic cysts at high risk of malignancy or high-grade dysplasia. Methods: Pancreatic cyst fluid from an international multi-center patient cohort were analyzed on a newly constructed microfluidic TaqMan low density array that was customized with our gene target panel of five miRNAs and seven mRNAs. Signatures were validated utilizing a machine learning random forest model for the optimal accuracy to determine high-risk cysts (high-grade dysplasia or invasive cancer) from low risk cysts for operative intervention. Results: Of 358 pancreatic cyst fluids analyzed, 157 cysts were intraductal papillary mucinous neoplasms (IPMNs), 62 were mucinous cystic neoplasms (MCNs), and the remainder were of various non-mucinous histologies. Of the 219 mucinous cysts, 60 (27%) were found to harbor high-grade dysplasia (HGD) or invasive cancer on final pathological examination. The models were established and then internally validated revealing a maximal accuracy of 87% to determine benign from potentially malignant cysts with an out-of-bag error of 8-12%. Further, the assay performed with an accuracy of 81% to identify high-risk from low-risk mucinous lesions (IPMN+MCN) for operative intervention. Conclusion: A high-throughput, cost-effective microfluidics-based molecular diagnostic assay has been developed that identifies high-risk cystic lesions of the pancreas for surgical evaluation. In this large, international, multi-center study, robust validation sets were performed. Performance of the biosignatures are currently being enhanced with clinical and radiographic features.

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.

MiRNA Expression in Patients with Gaucher Disease Treated with Enzyme Replacement Therapy

Aims: The aim of the work was to establish potential biomarkers or drug targets by analysing changes in miRNA concentration among patients with Gaucher disease (GD) compared to in healthy subjects. Methods: This study was an observational, cross-sectional analysis of 30 adult participants: 10 controls and 20 adults with GD type 1. Patients with GD type 1 were treated with enzyme replacement therapy (ERT) for at least two years. The control group was composed of healthy volunteers, unrelated to the patients, adjusted with age, sex and body mass index (BMI). The miRNA alteration between these groups was examined. After obtaining preliminary results on a group of six GD patients by the high-output method (TaqMan low-density array (TLDA)), potential miRNAs were selected for confirming the results by using the qRT-PCR method. With Diane Tools, we analysed miRNAs of which differential expression is most significant and their potential role in GD pathophysiology. We also determined the essential pathways these miRNAs are involved in. Results: 266 dysregulated miRNAs were found among 753 tested. Seventy-eight miRNAs were downregulated, and 188 were upregulated. Thirty miRNAs were significantly altered; all of them were upregulated. The analysis of pathways regulated by the selected miRNAs showed an effect on bone development, inflammation or regulation of axonal transmission in association with Parkinson’s disease. Conclusions: We revealed few miRNAs, like miR-26-5p, which are highly altered and fit the GD pathophysiological model, might be considered as novel biomarkers of disease progression but need further evaluation.

The O6-methyguanine-DNA methyltransferase inhibitor O6-benzylguanine enhanced activity of temozolomide + irinotecan against models of high-risk neuroblastoma.

DNA-damaging chemotherapy is a major component of therapy for high-risk neuroblastoma, and patients often relapse with treatment-refractory disease. We hypothesized that DNA repair genes with increased expression in alkylating agent resistant models would provide therapeutic targets for enhancing chemotherapy. In-vitro cytotoxicity of alkylating agents for 12 patient-derived neuroblastoma cell lines was assayed using DIMSCAN, and mRNA expression of 57 DNA repair, three transporter, and two glutathione synthesis genes was assessed by TaqMan low-density array (TLDA) with further validation by qRT-PCR in 26 cell lines. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was upregulated in cell lines with greater melphalan and temozolomide (TMZ) resistance. MGMT expression also correlated significantly with resistance to TMZ+irinotecan (IRN) (in-vitro as the SN38 active metabolite). Forced overexpression of MGMT (lentiviral transduction) in MGMT non-expressing cell lines significantly increased TMZ+SN38 resistance. The MGMT inhibitor O6-benzylguanine (O6BG) enhanced TMZ+SN38 in-vitro cytotoxicity, H2AX phosphorylation, caspase-3 cleavage, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling. TMZ+IRN+O6BG delayed tumor growth and increased survival relative to TMZ+IRN in two of seven patient-derived xenografts established at time of death from progressive neuroblastoma. We demonstrated that high MGMT expression was associated with resistance to alkylating agents and TMZ+IRN in preclinical neuroblastoma models. The MGMT inhibitor O6BG enhanced the anticancer effect of TMZ+IRN in vitro and in vivo. These results support further preclinical studies exploring MGMT as a therapeutic target and biomarker of TMZ+IRN resistance in high-risk neuroblastoma.

Design and evaluation of Taqman low density array for monitoring post-transplant viral infections.

The majority of transplant recipients undergo immunosuppressive treatment to prevent organ or tissue rejection. Consequently, they are more susceptible to infection agents including a number of viruses causing a significant morbidity and mortality. Only a limited number of viruses are currently tested for in transplant donors and recipients due to the cost and complexity. Taqman low density array (TLDA) may provide a suitable format to address more systematic testing approach. One hundred and one liver transplant recipient samples were retrospectively tested for 48 viral targets including two controls (bovine viral diarrhea virus and MS2) and two common viruses (TTV and HPgV), using a custom designed TLDA. Eight samples were analysed simultaneously on 384-well TLDA. Samples giving a signal considered positive/indeterminant were re-tested by different individual confirmatory assays. Infections with six previously untested for viruses-EBV, HPIV3, HuPuV9, KIV, HMPV and HPV-were detected in fourteen patients. Previously detected HCV infections were also confirmed. These infections did not seem have an effect on 5 year post-transplant outcome. 55 of 79 and 17 of 87 samples available for confirmatory assays were positive for TTV and HPgV, included for the evaluation of the TLDA performance. The custom viral TLDA can be successfully used for simultaneous detection of a range of post-transplant viral infections. To fully exploit its potential for monitoring and intervention, a whole blood testing should be applied in a prospective setting.

Identification and validation of endogenous control miRNAs in plasma samples for normalization of qPCR data for Alzheimer\u2019s disease

BackgroundMicroRNAs (miRNAs) are noncoding RNAs that are highly relevant as disease biomarkers. Several studies that explored the role of miRNAs in Alzheimer’s disease (AD) demonstrated their usefulness in clinical identification. Nevertheless, miRNAs that may act as endogenous controls (ECs) have not yet been established. The identification of ECs would contribute to the standardization of these biomarkers in AD. The objective of the study was to identify miRNAs that can be used as ECs in AD.MethodsWe evaluated 145 patients divided into two different cohorts. One was a discovery cohort of 19 women diagnosed with mild to moderate AD (Mini-Mental State Examination (MMSE) score ≥ 20) and with confirmed pathologic levels of Aβ42 in CSF. The stability assessment cohort consisted of 126 individuals: 24 subjects without AD or any kind of dementia and negative for all core CSF biomarkers of AD, 25 subjects with MCI and negative for CSF biomarkers (MCI −), 22 subjects with MCI and positive for CSF biomarkers (MCI +), and 55 subjects with AD and positive for CSF biomarkers. In the discovery cohort, a profile of 384 miRNAs was determined in the plasma by TaqMan low-density array. The best EC candidates were identified by mean-centering and concordance correlation restricted normalization methods. The stability of the EC candidates was assessed using the GeNorm, BestKeeper, and NormFinder algorithms.ResultsNine miRNAs (hsa-miR-324-5p, hsa-miR-22-5p, hsa-miR-103a-2-5p, hsa-miR-362-5p, hsa-miR-425-3p, hsa-miR-423-5p, hsa-let-7i-3p, hsa-miR-532-5p, and hsa-miR-1301-3p) were identified as EC candidates in the discovery cohort. The validation results indicated that hsa-miR-103a-2-5p was the best EC, followed by hsa-miR-22-5p, hsa-miR-1301-3p, and hsa-miR-425-3p, which had similar stability values in all three algorithms.ConclusionsWe identified a profile of four miRNAs as potential plasma ECs to be used for normalization of miRNA expression data in studies of subjects with cognitive impairment.

Evaluation of circulating microRNA expression in patients with trigeminal neuralgia: An observational study.

Trigeminal neuralgia (TN) is a chronic neuropathic pain that seriously affects the daily life of patients. There is increasing evidence that microRNAs (miRNAs) play an important role in the development of neuropathic pain.In this study, the TaqMan Low Density Array (TLDA) was used to analyze the serum miRNA levels of 28 TN patients, and 31 healthy people without any neuropathic pain were used as controls.The results showed that the expression profile of serum miRNA in TN patients was different from that in healthy controls. Compared with the control group, 13 miRNAs in the serum of TN patients were up-regulated and 115 miRNAs were down-regulated by >2 times. Quantitative reverse transcription PCR (RT-qPCR) analysis and receiver operating characteristic (ROC) curve were performed. The analysis further confirmed that the expression levels of 4 miRNAs, including miR-132-3p, miR-146b-5p, miR-155-5p, and miR-384, were significantly higher than those of healthy controls, and the difference was statistically significant.This study preliminarily confirmed the changes of serum miRNA expression profile in TN patients. Among them, 4 kinds of serum miRNA are likely to be related to the occurrence and development of TN.

MicroRNA Profile of Cardiovascular Risk in Patients with Obstructive Sleep Apnea

Background: Obstructive sleep apnea (OSA) is a common disease caused by repeated episodes of collapse of the upper airway during sleep and is associated with the development of cardiovascular disease (CVD). However, there is high heterogeneity in the impact of OSA on patients. Until now, the profile of OSA patients at risk of developing CVD has not been defined, including the measurable variables that could be used to predict the CVD risk of a patient with OSA. Objective: The aim of this study was to identify the microRNA (mi­RNA) profile associated with CVD in patients with OSA. Method: This is an observational, cross-sectional study that included 132 male patients. Three groups were defined as OSA patients, OSA patients with hypertension, and OSA patients who developed a major cardiovascular event. Polysomnography and ambulatory blood pressure measurements were performed. The expression profiling of 188 miRNAs in plasma was performed in 21 subjects (matched by BMI and age) by the TaqMan low density array (TLDA). miRNAs differentially expressed in the different subgroups of patients and miRNAs that correlated with the cardiovascular risk SCORE were selected for validation by RT-qPCR in the 111 remaining patients. Results: From the TLDA analysis, 7 miRNAs were selected for validation. Differential expression was not confirmed in any of the miRNAs. miR-143 was associated with nocturnal systolic blood pressure. miR-107 correlated with 24-h blood pressure parameters and with nocturnal hypertension. miR-486 was associated with the cardiovascular risk SCORE. Conclusions: The circulating profile of miRNAs does not seem to be different in any of the subgroups of patients with OSA and different cardiovascular risk factors. Nevertheless, miR-107 and miR-143 are associated with specific blood pressure parameters in patients with OSA and miR-486 is associated with the cardiovascular risk SCORE.

“Detection of BCR-ABL1-like Subtype in Adult Acute Lymphoblastic Leukemia Using Digital Ncounter Nanostring Technology”

Introduction A new provisional entity of "B-lymphoblastic leukaemia/lymphoma, BCR-ABL1-like" has been introduced in the 2017 revised edition of WHO classification of tumours of haematopoietic and lymphoid tissues. BCR-ABL1-like cases are negative for Ph chromosome or t(9:22)(q34;q11.2) translocation, do not express the fusion BCR-ABL1 RNA transcripts and proteins resulting from the Ph chromosome,and are characterized by similar gene expression profiles as that of BCR-ABL1-positive acute lymphoblastic leukemia (BCR-ABL1-positive ALL).There is no 'short-cut approach' for making an accurate diagnosis of BCR-ABL1-like ALL. Two approaches namely Gene expression profiling (GEP) or Next-generation sequencing (NGS) and TLDA (TaqMan low-density array) are used for the detection of BCR-ABL1-like ALL cases. NGS is very costly and data interpretation requires a lot of bioinformatics skills and TLDA is not commercially available in India. Aims We planned to study the whole transcriptome of BCR-ABL1-positive ALL cases using microarray GEP, followed by customizing targeted gene panel using nCounter NanoString technology, for the detection of BCR-ABL1-like cases. METHODS Flow cytometric immunophenotying (FCM-IP) and multiplex RT-PCR were performed on 200 B-ALL cases to detect BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (Gene Chip Human Genome U133 Plus 2.0 Array). The results were analyzed using TAC 4.0 software. Finally, a targeted panel of 50 differentially expressed genes [including 5 Housekeeping genes (HKGs)] was constructed according to our microarray findings and previously published data (Harvey RC et al.ASH 2013). A total of 96 B-ALL cases (16 BCR-ABL1-positive cases &amp; 80 BCR-ABL1-negative cases) were subjected to GEP using nCounter Platform. The results were analyzed using nSolver4.0 software. RESULTS In the study cohort of 200 adult B-ALL cases, BCR-ABL1 chimeric fusion transcripts were detected in 34% (b2a2 and b3a2=18.05% &amp; e1a2=15.5%), as revealed by multiplex assay. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 as DE genes (460 genes in e1a2, 515 genes in b2a2 and 599 genes in b3a2). DE genes were further filtered through hierarchical clustering analysis and a total of 45 DE genes with 10- to -86-fold change were identified. These genes were further analyzed using nCounter NanoString. To further identify the best classifier genes, log2 normalized expression values were analyzed using penalized logistic regression. Based on previous literature and regression coefficient values, 15 genes were finally selected whose performance was individually analyzed using receiver operating characteristic curve (ROC) and area under the curve (AUC). Optimal thresholds for these genes were estimated as the values with maximum sensitivity and specificity. Out of 78 examined BCR-ABL1-negative cases, 33(42.30%) BCR-ABL1-negative cases were clustered together with 15 BCR-ABL1-positive cases and were attributed as BCR-ABL1-like ALL cases in principal component analysis. Further, we categorized CRLF2 in two categories; high CRLF2 cases 25/33 (75.75%) &amp; low 8/33 (24.24%) in BCR-ABL1-like ALL cases. JAK2p.R683G mutation was screened in CRLF2 high cases and showed positivity in 19/24 (79.16%) by the Amplification Refractory Mutation (ARMS) PCR. In 25 cases, the average log fold change of -0.80 &amp;-5.83 was seen in P2YR8 &amp; CSF2RA respectively by qPCR. In CRLF2 low expressing cases, the average log fold change of 11 kinase genes showed -0.75 in CENPC, -0.66 FOXP1, -0.16 NUP153, 1.04 RCSD1, 1.50 PAX5, 1.12 FLT3, -5.65 EPOR, -4.03 ILR2B, -3.46 PDGRFB, -7.49 NTRK3 &amp;-2.83 ZNF274 respectively. The average log fold change of IKZF1 in 80 BCR-ABL1-negative cases was found to be 1.07. DISCUSSION &amp; CONCLUSION We have devised a method that includes 15 genes according to AUC/ROC for the detection of BCR-ABL1-like ALL cases, using nCounter NanoString technology for the first time in Indian patients. Furthermore, we are planning to validate this model in future, on 50 BCR-ABL1-positive and 150 BCR-ABL1-negative cases and devise a simple, efficient, cost-effective qPCR method. It is very important to detect BCR-ABL1-like ALL cases to start the desirable TKI therapy &amp; aid in treatment stratification, prognostication, and improve the overall survival of these patients. Figure Disclosures No relevant conflicts of interest to declare.

MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment.

To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.

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.

Integration of Next-Generation Sequencing in Diagnosing and Minimal Residual Disease Detection in Patients With Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia.

Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype of B cell ALL. It accounts for 20% of all B cell ALL cases and is similar to BCR-ABL1 in gene expression profile but lacks BCR-ABL fusion. It is highly heterogeneous and is characterized by genetic alterations that activate kinase and cytokine receptor signaling. Most of these alterations are amenable to tyrosine kinase inhibitors. Ph-like ALL is prevalent in pediatric and young adults, more common in males, and frequently seen in patients with Hispanic ancestry. It is associated with inadequate response to induction therapy, high minimal residual disease (MRD) levels, and increased risk of relapse. Overall survival and event-free survival are also inferior in these patients as compared to non-Ph-like ALL. In the clinical practice, low-density array, real-time quantitative polymerase chain reaction (RQ-PCR), flow cytometry, fluorescence in situ hybridization are used to identify genetic alteration in these patients. With the advent of next-generation sequencing (NGS), our understanding of disease pathogenesis and precision medicine has been improved. In this review, we analyzed data from several studies that used NGS as one of the diagnostic methods to identify genomic lesions in this high-risk subtype of B cell ALL. Studies have shown that NGS is a vital technique to identify various genomic lesions at diagnosis and throughout the treatment that can be missed by the widely used current methods. NGS has improved our understanding of various genomic lesions associated with Ph-like ALL and has helped define disease pathogenesis, MRD evaluation, and stratify therapy to prevent over or under treatment. We are in the era of precision medicine. Therefore unbiased, comprehensive genomic characterization of Ph-like ALL is important to implicate treatment directed against these genomic lesions and improve outcomes in these patients. We also analyzed data from studies that compared NGS with multi-flow cytometry and RQ-PCR for the evaluation of MRD. In the future, more extensive prospective studies are required to confirm the prognostic usefulness of NGS.

Expression differences of miR-142-5p between treatment-na\xefve chronic myeloid leukemia patients responding and non-responding to imatinib therapy suggest a link to oncogenic ABL2, SRI, cKIT and MCL1 signaling pathways critical for development of therapy resistance

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients’ prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy.MethodsTaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes.ResultsSignificant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples.ConclusionMiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.

Identification of Acute Myeloid Leukemia Bone Marrow Circulating MicroRNAs.

Background: In addition to their roles in different biological processes, microRNAs in the tumor microenvironment appear to be potential diagnostic and prognostic biomarkers for various malignant diseases, including acute myeloid leukemia (AML). To date, no screening of circulating miRNAs has been carried out in the bone marrow compartment of AML. Accordingly, we investigated the circulating miRNA profile in AML bone marrow at diagnosis (AMLD) and first complete remission post treatment (AMLPT) in comparison to healthy donors (HD). Methods: Circulating miRNAs were isolated from AML bone marrow aspirations, and a low-density TaqMan miRNA array was performed to identify deregulated miRNAs followed by quantitative RT-PCR to validate the results. Bioinformatic analysis was conducted to evaluate the diagnostic and prognostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). Results: We found several deregulated miRNAs between the AMLD vs. HD vs. AMLPT groups, which were involved in tumor progression and immune suppression pathways. We also identified significant diagnostic and prognostic signatures with the ability to predict AML patient treatment response. Conclusions: This study provides a possible role of enriched circulating bone marrow miRNAs in the initiation and progression of AML and highlights new markers for prognosis and treatment monitoring.

Digging Deeper into Breast Cancer Epigenetics: Insights from Chemical Inhibition of Histone Acetyltransferase TIP60 In Vitro.

Breast cancer is often sporadic due to several factors. Among them, the deregulation of epigenetic proteins may be involved. TIP60 or KAT5 is an acetyltransferase that regulates gene transcription through the chromatin structure. This pleiotropic protein acts in several cellular pathways by acetylating proteins. RNA and protein expressions of TIP60 were shown to decrease in some breast cancer subtypes, particularly in triple-negative breast cancer (TNBC), where a low expression of TIP60 was exhibited compared with luminal subtypes. In this study, the inhibition of the residual activity of TIP60 in breast cancer cell lines was investigated by using two chemical inhibitors, TH1834 and NU9056, first on the acetylation of the specific target, lysine 4 of histone 3 (H3K4) by immunoblotting, and second, by chromatin immunoprecipitation (ChIP)-qPCR (-quantitative Polymerase Chain Reaction). Subsequently, significant decreases or a trend toward decrease of H3K4ac in the different chromatin compartments were observed. In addition, the expression of 48 human nuclear receptors was studied with TaqMan Low-Density Array in these breast cancer cell lines treated with TIP60 inhibitors. The statistical analysis allowed us to comprehensively characterize the androgen receptor and NR3C2 receptors in TNBC cell lines after TH1834 or NU9056 treatment. The understanding of the residual activity of TIP60 in the evolution of breast cancer might be a major asset in the fight against this disease, and could allow TIP60 to be used as a biomarker or therapeutic target for breast cancer progression in the future.