International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer.

INTRODUCTION: miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC. MATERIAL AND METHODS: Two parallel studies on archived formalin-fixed paraffin-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors was investigated in FFPE tissue from invasive border and central tumor zones from 47 early metastatic CRCs matched with 47 non-metastatic CRCs. Intra-tumor expression gradients were analyzed on both cohorts. RESULTS: Mean intra-tumor coefficient of variation in the heterogeneity cohort was 38.5% (range: 33.1-49.0%) only slightly less than variation between patients (45.1%, range 37.0-49.5%). We demonstrated systematic expression gradients between tumor zones equal to a 3.23 (p=0.003) and 1.36 (p=0.014) fold lower expression in invasive areas for miR-200c, 1.52 (p<0.001) and 1.27 (p=0.021) fold lower expression in invasive areas for miR-92a. For miR-21 we found a 1.75 (p<0.001) and 1.21 (p=0.064) fold higher expression in invasive areas compared to luminal and central zones, respectively. No significant difference in expression levels between metastatic and non-metastatic tumors was demonstrated, nor a difference in intra-tumor gradients between metastatic and non-metastatic tumors. CONCLUSION: This study provides evidence for moderate intra-tumor and inter-patient heterogeneities of three well-described potential prognostic markers in CRC. We demonstrate intra-tumor expression gradients indicating a differentiated expression of the target miRNAs between functional tumor zones, but the potential role as markers of early metastatic disease is still not fully clarified.

Intra-tumor heterogeneity of microRNA-92a, microRNA-375 and microRNA-424 in colorectal cancer.

UNLABELLED: Various microRNAs (miRNAs) have been investigated in order to improve diagnostics and risk assessment in colorectal cancer (CRC). To clarify the potential of miRNA profiling in CRC, knowledge of intra-tumor heterogeneity in expression levels is crucial. The study aim was to estimate the intra-tumor variance of three selected miRNAs: miR-92a, miR-375 and miR-424 in CRC tissue. MATERIAL AND METHODS: A retrospective study on archived formalin-fixed paraffin embedded tissue from 9 patients with CRC. miRNA tissue expression levels were analyzed by qRT-PCR on tissue representing luminal, central and invasive border zones. Variance components were estimated based on ∆∆Cp values using mixed modeling and presented as coefficients of variation (CV). RESULTS: Intra-tumor variance was approximately half of the variance observed between patients with a mean intra-tumor CV of 56.4% (range 33.1-77.1%) and a mean inter-patient CV of 101.7% (range 48.8-152.7%). Furthermore we found a significant systematic difference in expression levels between tumor zones for miR-92a and miR-375 with a luminal-invasive difference equal to 0.60 Cp (95% CI: 0.30-0.89, p=0.0003) for miR-92a and a luminal-invasive difference equal to 0.78 Cp (95% CI: 0.10-1.46, p=0.027) for miR-375. Conclusion While the intra-tumor variance of miR-92a, miR-375 and miR-424 is substantial, it only constitutes approximately 30% of the total variation. Functional deregulation between tumor zones might contribute to variations in measured expression levels, and thus knowledge of specific intra-tumor expression patterns is crucial in tissue sampling for research as well as in future diagnostics.

A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells.

As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.

(19)F-heptuloses as tools for the non-invasive imaging of GLUT2-expressing cells.

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) µmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.

The value of next-generation sequencing in routine diagnostics and management of patients with cytopenia.

INTRODUCTION: We performed a single-center study of real-world health data to investigate the direct clinical consequence of targeted next-generation sequencing (NGS) results integrated in the clinicopathological evaluation of patients with cytopenia suspected of myelodysplastic syndrome (MDS). METHODS: The study included 87 newly referred patients, who had a bone marrow examination, which included targeted NGS analysis. NGS was requested at the discretion of either examining pathologist or hematologist. Data were collected retrospectively from patient files including pathology reports with integrated NGS results. RESULTS: The NGS results had a diagnostic impact in 67 cases (77%) when combining both histopathological and final clinical evaluation and provided prognostic value in 19 cases (22%). NGS supported a confident or tentative histopathological diagnosis in 52 cases (60%). Twenty cases (23%) had a final diagnosis of either Clonal Cytopenia of Undetermined Significance (CCUS) or Idiopathic Cytopenia of Undetermined Significance (ICUS). In 4 cases, NGS results affected the choice of principal treatment strategy, including considerations of allotransplantation. Twenty-one patients (24%) could be discharged to primary care physician. CONCLUSION: In a multidisciplinary clinicopathological real-world setting, NGS analysis of bone marrow samples from selected patients contributed substantially to the diagnostic evaluation and management of patients with cytopenia suspected of MDS. Consequently, we have now included NGS analysis in most routine bone marrow examinations from patients with MDS or unexplained cytopenia.

MicroRNA characteristics in epithelial ovarian cancer.

The purpose of the current study was to clarify differences in microRNA expression according to clinicopathological characteristics, and to investigate if miRNA profiles could predict cytoreductive outcome in patients with FIGO stage IIIC and IV ovarian cancer. Patients enrolled in the Pelvic Mass study between 2004 and 2010, diagnosed and surgically treated for epithelial ovarian cancer, were used for investigation. MicroRNA was profiled from tumour tissue with global microRNA microarray analysis. Differences in miRNA expression profiles were analysed according to histologic subtype, FIGO stage, tumour grade, type I or II tumours and result of primary cytoreductive surgery. One microRNA, miR-130a, which was found to be associated with serous histology and advanced FIGO stage, was also validated using data from external cohorts. Another seven microRNAs (miR-34a, miR-455-3p, miR-595, miR-1301, miR-146-5p, 193a-5p, miR-939) were found to be significantly associated with the clinicopathological characteristics (p ≤ 0.001), in our data, but mere not similarly significant when tested against external cohorts. Further validation in comparable cohorts, with microRNA profiled using newest and similar methods are warranted.

Improved Variant Detection in Clinical Myeloid NGS Testing by Supplementing a Commercial Myeloid NGS Assay with Custom or Extended Data Filtering and Accessory Fragment Analysis.

BACKGROUND: Commercial myeloid next-generation sequencing (NGS) panels may facilitate uniform generation of raw data between laboratories. However, different strategies for data filtering and variant annotation may contribute to differences in variant detection and reporting. Here, we present how custom data filtering or the use of Oncomine extended data filtering improve detection of clinically relevant mutations with the Oncomine Myeloid Research Assay. METHODS: The study included all patient samples (n = 264) analyzed during the first-year, single-site, clinical use of the Ion Torrent Oncomine Myeloid Research Assay. In data analysis, the default analysis filter was supplemented with our own data filtering algorithm in order to detect additional clinically relevant mutations. In addition, we developed a sensitive supplementary test for the ASXL1 c.1934dupG p.Gly646fs mutation by fragment analysis. RESULTS: Using our custom filter chain, we found 96 different reportable variants that were not detected by the default filter chain. Twenty-six of these were classified as variants of strong or potential clinical significance (tier I/tier II variants), and the custom filtering discovered otherwise undetected tier I/tier II variants in 25 of 132 patients with clinically relevant mutations (19%). The remaining 70 variants not detected by the default filter chain were classified as variants of unknown significance. Among these were several unique variants with possible pathogenic potential judged by bioinformatic predictions. The recently launched Oncomine 5.14 extended filter algorithm detects most but not all of the tier I/tier II variants that were not detected by the default filter. The supplementary fragment analysis for the ASXL1 c.1934dupG p.Gly646fs confidently detected a variant allele frequency of down to 4.8% (SD 0.83%). The assay also detected the ASXL1 c.1900_1922del23 mutation. CONCLUSION: Detection of clinically relevant variants with the Oncomine Myeloid Research NGS assay can be significantly improved by supplementing the default filter chain with custom data filtering or the recently launched Oncomine 5.14 extended filter algorithm. Our accessory fragment analysis facilitates easy testing for frequent ASXL1 mutations that are poorly or not covered by the NGS assay.

Therapeutic Cancer Vaccination With a Peptide Derived From the Calreticulin Exon 9 Mutations Induces Strong Cellular Immune Responses in Patients With CALR-Mutant Chronic Myeloproliferative Neoplasms.

BACKGROUND: The calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN. METHODS: The safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446). RESULTS: Patients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines. CONCLUSION: Therapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response.

An immunogenic first-in-human immune modulatory vaccine with PD-L1 and PD-L2 peptides is feasible and shows early signs of efficacy in follicular lymphoma.

Cells in the tumor microenvironment of Follicular lymphoma (FL) express checkpoint molecules such as programmed death ligands 1 and 2 (PD-L1 and PD-L2) and are suppressing anti-tumor immune activity. Stimulation of peripheral blood mononuclear cells (PBMC) with PD-L1 (IO103) or PD-L2 (IO120) peptides can activate specific T cells inducing anti-regulatory functions including cytotoxicity against PD-L1/PD-L2-expressing cells. In this study, we vaccinated eight FL patients with PD-L1 and PD-L2 peptides following treatment with standard chemotherapy. Patients experienced grade 1-2 injection site reaction (5/8) and mild flu-like symptoms (6/8). One patient experienced neutropenia and thrombocytopenia during pseudo-progression. Enzyme-linked immunospot detected vaccine-specific immune responses in PBMC from all patients, predominately toward PD-L1. The circulating immune composition was stable during treatment; however, we observed a reduction regulatory T cells, however, not significant. One patient achieved a complete remission during vaccination and two patients had pseudo-progression followed by long-term disease regression. Further examination of these early signs of clinical efficacy of the dual-epitope vaccine in a larger study is warranted.

Identification and validation of potential prognostic and predictive miRNAs of epithelial ovarian cancer.

BACKGROUND: Ovarian cancer is the leading cause of death by gynecologic cancers in the Western world. The aim of the study was to identify microRNAs (miRNAs) associated with prognosis and/or resistance to chemotherapy among patients with epithelial ovarian cancer. METHODS: Using information from the Pelvic Mass Study we identified a cohort of women with epithelial ovarian cancer. Tumor tissues were then collected and analyzed by global miRNA microarrays. MiRNA profiling was then linked to survival and time to progression using Cox proportional-hazards regression models. Logistic regression models were used for the analysis of resistance to chemotherapy. Our results were validated using external datasets retrieved from the NCBI Gene Expression Omnibus database. RESULTS: A total of 197 patients with epithelial ovarian cancer were included for miRNA microarray analysis. In multivariate analyses we identified a number of miRNAs significantly correlated with overall survival (miR-1183 (HR: 1.42, 95% CI:1.17-1.74, p = 0.0005), miR-126-3p (HR: 1.38, 95% CI:1.11-1.71, p = 0.0036), time to progression (miR-139-3p (HR: 1.48, 95% CI: 1.13-1.94, p = 0.0047), miR-802 (HR: 0.48, 95% CI: 0.29-0.78, p = 0.0035)), progression free survival (miR-23a-5p (HR:1.32, 95% CI:1.09-1.61, p = 0.004), miR-23a-3p (HR:1.70, 95% CI:1.15-2.51, p = 0.0074), miR-802 (HR: 0.48, 95% CI: 0.29-0.80, p = 0.0048)), and resistance to chemotherapy (miR-1234 (HR: 0.26, 95% CI: 0.11-0.64, p = 0.003)). A few miRNAs identified in our training cohort, were validated in external cohorts with similar results. CONCLUSION: Eight miRNAs were identified as significant predictors of overall survival, progression free survival, time to progression, and chemotherapy resistance. A number of these miRNAs were significantly validated using external datasets. Inter-platform and inter-laboratory variations may have influence on the ability to compare and reproduce miRNA results. The use of miRNAs as potential markers of relapse and survival in ovarian cancer warrants further investigation.

Digital image analysis of pan-cytokeratin stained tumor slides for evaluation of tumor budding in pT1/pT2 colorectal cancer: Results of a feasibility study.

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users. Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides. Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p < 0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p = 0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p = 0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3-4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.

Early metastatic colorectal cancers show increased tissue expression of miR-17/92 cluster members in the invasive tumor front.

Accurate prediction of regional lymph node metastases (LNM) in endoscopically resected pT1 colorectal cancer (CRC) is crucial in treatment stratification for subsequent radical surgery. Several miRNAs have been linked to CRC invasion and metastasis, including the oncogenic miR-17/92 cluster, and expression levels might have predictive value in the risk assessment of early metastatic progression in CRC. We performed global miRNA microarray using tissue samples from the invasive front of pT1 CRC and investigated associations of the miR-17/92 cluster and presence of LNM. In total, 56 matched pT1 CRCs were thoroughly clinicopathologically characterized, and miRNA microarrays were performed on invasive front tissue samples. Global miRNA intensities were screened using paired t-tests between pT1pN+ and pT1pN0. Associations between miR-17/92 and histopathological features were analyzed using general linear models and tumor cell adjusted expression intensities. miR-17-3p and miR-92a were significantly higher expressed in the invasive front of tumors with LNM compared to those without, corresponding to 1.53-fold higher expression of miR-17-3p (95%CI: 1.04-2.24, P = .030) and 1.28-fold higher expression of miR-92a (95%CI: 1.01-1.68, P = .042). An inverse association between miR-19a and presence of high-grade tumor budding was observed (1.55-fold, 95%CI: 1.13-2.12, P = .008). We provide evidence for associations between early regional LNM and high expression levels of the miR-17/92 cluster members: miR-17-3p and miR-92a, in the invasive front of CRC. Our results support a role for the miR-17/92 cluster in early metastatic progression of CRC and calls for further investigation.

Clinical validation of chemotherapy predictors developed on global microRNA expression in the NCI60 cell line panel tested in ovarian cancer.

OBJECTIVE: Ovarian cancer is the leading cause of death among gynecologic malignancies. This is partly due to a non-durable response to chemotherapy. Prediction of resistance to chemotherapy could be a key role in more personalized treatment. In the current study we aimed to examine if microRNA based predictors could predict resistance to chemotherapy in ovarian cancer, and to investigate if the predictors could be prognostic factors for progression free and overall survival. METHODS: Predictors of chemotherapy-resistance were developed based on correlation between miRNA expression and differences in measured growth inhibition in a variety of human cancer cell lines in the presence of Carboplatin, Paclitaxel and Docetaxel. These predictors were then, retrospectively, blindly validated in a cohort of 170 epithelial ovarian cancer patients treated with Carboplatin and Paclitaxel or Docetaxel as first line treatment. RESULTS: In a multivariate cox proportional analysis the predictors of chemotherapy-resistance were not able to predict time to progression after end of chemotherapy (hazard ratio: 0.64, 95% CI: 0.36-1.12, P = 0.117). However, in a multivariate logistic analysis, where time to progression was considered as either more or less than 6 months, the predictors match clinical observed chemotherapy-resistance (odds ratio: 0.19, 95% CI: 0.05-0.73, P = 0.015). Neither univariate nor multivariate, time-dependent, cox analysis for progression free survival (PFS) or overall survival (OS) in all 170 patients showed to match predicted resistance to chemotherapy (PFS: hazard ratio: 0.69, 95% CI: 0.40-1.19, P = 0.183, OS: hazard ratio: 0.76, 95% CI: 0.42-1.40, P = 0.386). CONCLUSION: In the current study, microRNA based predictors of chemotherapy-resistance did not demonstrate any convincing correlation to clinical observed chemotherapy-resistance, progression free survival, or overall survival, in patients with epithelial ovarian cancer. However the predictors did reflect relapse more or less than 6 months.

Current status on microRNAs as biomarkers for ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological malignancy in the Western world, and has a very poor prognosis, often due to late diagnosis and emergence of chemotherapy resistance. Therefore, there is an essential need for new diagnostic and prognostic markers that can improve and initiate more personalized treatment, eventually improving survival of the patients. MicroRNAs are small, non-coding RNA molecules, that post-transcriptionally regulate gene expression. Several studies have within the last decade shown that microRNAs are deregulated in OC and have potential as diagnostic and prognostic biomarkers for OC. Recently studies have also focused on microRNAs as predictors of chemotherapy responses and their potential as therapeutic targets. However, many of the published studies are difficult to interpret as a whole due to various methods of analysis. Future focus should be aimed at developing a general standardized analytical method, which can limit differences between studies thus allowing easier comparison across them. In addition, validation of studies in independent series that ideally should be histotype-specific is essential to determine the clinical role of microRNAs in different types of OC. In this review we summarize the current knowledge of microRNAs as potential biomarkers for OC, with focus on their clinical relevance.

Altering ß-cell number through stable alteration of miR-21 and miR-34a expression.

AIM: An insufficient functional ß-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore ß-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in ß-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced ß-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to ß cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net ß-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes.   MATERIALS AND METHODS: miRNA expression was manipulated using Lentiviral transduction of the ß-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. RESULTS: Overexpression of miR-21 decreased net ß-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net ß-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. CONCLUSION: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase ß-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce ß-cell death and dysfunction.

MicroRNA expression profiling of carcinoma in situ cells of the testis.

Testicular germ cell tumours, seminoma (SE) and non-seminoma (NS), of young adult men develop from a precursor cell, carcinoma in situ (CIS), which resembles foetal gonocytes and retains embryonic pluripotency. We used microarrays to analyse microRNA (miRNA) expression in 12 human testis samples with CIS cells and compared it with miRNA expression profiles of normal adult testis, testis with Sertoli-cell-only that lacks germ cells, testis tumours (SE and embryonal carcinoma (EC), an undifferentiated component of NS) and foetal male and female gonads. Principal components analysis revealed distinct miRNA expression profiles characteristic for each of the different tissue types. We identified several miRNAs that were unique to testis with CIS cells, foetal gonads and testis tumours. These included miRNAs from the hsa-miR-371-373 and -302-367 clusters that have previously been reported in germ cell tumours and three miRNAs (hsa-miR-96, -141 and -200c) that were also expressed in human epididymis. We found several miRNAs that were upregulated in testis tumours: hsa-miR-9, -105 and -182-183-96 clusters were highly expressed in SE, while the hsa-miR-515-526 cluster was high in EC. We conclude that the miRNA expression profile changes during testis development and that the miRNA profile of adult testis with CIS cells shares characteristic similarities with the expression in foetal gonocytes.