Investigating the targets of MIR-15a and MIR-16-1 in patients with chronic lymphocytic leukemia (CLL).

BACKGROUND: MicroRNAs (miRNAs) are short, noncoding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. The miRNAs, MIR-15a/16-1, at chromosome band 13q14 are down-regulated in the majority of patients with chronic lymphocytic leukaemia (CLL). METHODOLOGY/PRINCIPAL FINDINGS: We have measured the expression of MIR-15a/16-1, and 92 computationally-predicted MIR-15a/16-1 target genes in CLL patients and in normal controls. We identified 35 genes that are deregulated in CLL patients, 5 of which appear to be specific targets of the MIR-15a/16-1 cluster. These targets included 2 genes (BAZ2A and RNF41) that were significantly up-regulated (p<0.05) and 3 genes (RASSF5, MKK3 and LRIG1) that were significantly down-regulated (p<0.05) in CLL patients with down-regulated MIR-15a/16-1 expression. SIGNIFICANCE: The genes identified here as being subject to MIR-15a/16-1 regulation could represent direct or indirect targets of these miRNAs. Many of these are good biological candidates for involvement in tumorigenesis and as such, may be important in the aetiology of CLL.

Evaluation of 13q14 status in multiple myeloma by digital single nucleotide polymorphism technology.

Chromosome 13q deletions are common in multiple myeloma and other cancers, demonstrating the importance of this region in tumorigenesis. We used a novel single nucleotide polymorphism (SNP)-based technique, digital SNP (dSNP), to identify loss of heterozygosity (LOH) at chromosome 13q in paraffin-embedded bone marrow biopsies from 22 patients with multiple myeloma. We analyzed heterozygous SNPs at 13q for the presence of allelic imbalances and examined the results by sequential probability ratio analysis. Where possible, dSNP results were confirmed by fluorescence in situ hybridization. Using dSNP, we identified 13q LOH in 16/18 (89%) (95% Confidence Interval; 65%, 99%) patients without the need for neoplastic cell enrichment. In 8/16 (50%) cases, either partial or interstitial patterns of LOH were observed. Both fluorescence in situ hybridization and dSNP data proved concordant in just 3/9 cases. Five of the six discrepancies showed LOH by dSNP occurring beyond the boundaries of the fluorescence in situ hybridization probes. Our findings show that dSNP represents a useful technique for the analysis of LOH in archival tissue with minimal infiltration of neoplastic cells. The high-resolution screening afforded by the dSNP technology allowed for the identification of complex chromosomal rearrangements, resulting in either partial or interstitial LOH. Digital SNP represents an attractive approach for the investigation of tumors not suitable for genomic-array analysis.

Evaluation of 13q14 status in patients with chronic lymphocytic leukemia using single nucleotide polymorphism-based techniques.

Deletions of chromosome 13q14 are common in chronic lymphocytic leukemia and other cancers, demonstrating the importance of this region in tumorigenesis. We report the use of two single-nucleotide polymorphism (SNP)-based techniques to determine 13q loss of heterozygosity (LOH) status in 15 patients with CLL: (i) digital SNP (dSNP), where analysis of heterozygous SNPs detects allelic imbalances, and (ii) DNA sequencing, where LOH is identified by comparison of allelic peak heights in normal and neoplastic cells. The SNP-based techniques were compared with established molecular techniques, fluorescence in situ hybridization and multiplex ligation-dependent probe amplification, to determine their utility and relative sensitivity. dSNP proved to be the most sensitive technique, identifying 13q14 LOH in 11 of 13 (85%) patients (95% CI: 55%, 98%) without the need for neoplastic cell enrichment. Three cases showed evidence of LOH by dSNP that was not apparent by other techniques. In 8 of 13 (62%) cases, partial or interstitial patterns of LOH were observed by dSNP. Our findings demonstrate that dSNP represents a useful, sensitive technique for the analysis of chromosomal aberrations that result in LOH. It may have applications for the analysis of other malignancies that are difficult to assess by conventional molecular techniques.

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study.

BACKGROUND: Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. DESIGN AND METHODS: JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. RESULTS: A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean +/- 2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -- one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean +/- 2SD -- with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2V617F. CONCLUSIONS: Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.

Development of a quantitative real-time polymerase chain reaction assay for the detection of the JAK2 V617F mutation.

Achieving a specific diagnosis of polycythemia vera (PV) and other myeloproliferative disorders (MPDs) is often costly and complex. However, the recent identification of a V617F mutation in the JH2 domain of the JAK2 gene in a high proportion of patients suffering from MPDs may provide confirmation of a diagnosis. This is an acquired mutation and, as such, may only be present in a small number of cells within a sample. There is therefore a clinical need for highly sensitive detection techniques. We have developed a sensitive real-time polymerase chain reaction (PCR)-based approach for both detection and quantification of the JAK2 V671F mutation load, which allows determination of mutation status without the need for prior purification of granulocytes. We have performed a comparison of this assay with two previously published detection methods. Although an amplification refractory mutation system (ARMS) was shown to be slightly superior in terms of sensitivity, our real-time PCR method provides the potential for quantification of the JAK2 V617F mutation, having potential future applications in the monitoring of minimal residual disease or predicting outcome of disease severity.