Evaluation of microRNA expression in patient bone marrow aspirate slides

<div><p>Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.</p> </div

Hypermethylation and down-regulation of DLEU2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-1

BACKGROUND: Acute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML. METHODS: We interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p 0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons. RESULTS: We identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis. CONCLUSIONS: The DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology

The stability of miRNA species expression in hematopoietic samples.

<p>The stability of miRNA species expression using 41 samples of leukaemic and non-leukaemic cell lines, paediatric archived bone marrow aspirate slides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042951#pone.0042951.s002" target="_blank">Table S1</a>), and 14 potential miRNA candidates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042951#pone.0042951.s003" target="_blank">Table S2</a>). geNorm and NormFinder (NF) were used to calculate the expression stability values for each miRNA tested. The most ‘stably’ expressed gene in a sample group is that with an average expression stability value (<i>M</i>) closest to zero. The most stably expressed miRNA were; NF Ungrouped: hsa-miR-16 <i>M</i> = 1.143; NF Grouped by Cancer Status: hsa-miR-16 <i>M</i> = 0.457; NF Grouped by Leukaemia subtype: hsa-miR-16 <i>M</i> = 0.573; geNorm: hsa-miR-16 and hsa-miR-26b are equal at <i>M</i> = 1.817.</p

A comparison of RNA extraction methods for the evaluation of RNA concentration (ng/µl) and miRNA expression from archived bone marrow aspirate slides.

<p>The comparison of miRNA extraction methods from matched unstained archived bone marrow smears (n = 6 or n = 8 patients; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042951#pone.0042951.s004" target="_blank">Table S3</a>). Six different methods were used based on traditional TRIzol or using the Roche High Pure miRNA Column extraction kit (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042951#pone-0042951-t001" target="_blank">Table 1</a>). (<b>A</b>) <b>Comparison of Total RNA yield from experimental extraction protocols</b>. The average RNA concentration was measured by NanoDrop (ng/µl ±s.e.m.). Kit Total RNA extraction methods with a Proteinase K digestion (‘FFPE protocol’) are higher in RNA yield compared to kit methods without a digestion step (‘Tissue protocol’), or when extracting small RNA only. These methods are also significantly better than TRIzol extractions. The archived slide extraction method with the highest RNA yield was the Roche kit ‘isolation from FFPE Tissue’ Total RNA extraction with an overnight Proteinase K digestion. * Indicates RNA concentration is significantly different to the Kit Overnight FFPE: Total RNA protocol. (<b>B</b>) <b>Comparison of hsa-miR-16 expression from experimental extraction protocols</b>. The average expression of our reverence gene (hsa-miR-16) measured by qRT-PCR (raw C_t ±s.e.m.). Raw C_t value tracks with total RNA yield, whereby as the concentration of RNA increases the C_t value decreases. This analysis shows extractions with a Proteinase K digestion return a lower C_t value compared to extractions without a digestion step. TRIzol extractions also return a higher C_t value than the Proteinase K digestion protocols.</p

Modified protocols used for the extraction of RNA from archived bone marrow aspirate slides.

<p>The modified extraction protocols used in this study for the extraction of total RNA and small RNA from unstained archived bone marrow aspirate slides. Two main methods were used, the Roche High Pure miRNA Isolation Kit and TRIzol. We followed two provided extraction protocols for the kit; ‘miRNA isolation from tissue’, and ‘miRNA isolation from FFPE tissue’. The methods provided by the manufacturers were modified to include a slide scraping step, and a digestion/homogenization step to break down the slide material. The kit allows for the extraction of total RNA as well as small RNA only; both these methods were trialled here; samples listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042951#pone.0042951.s004" target="_blank">Table S3</a>.</p

RESEARCH Open Access

in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-

Epigenetic deregulation in pediatric acute lymphoblastic leukemia

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (&gt; 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes