The methylotrophic yeast Hansenula polymorpha is a recognised model system for investigation of peroxisomal function, special metabolic pathways like methanol metabolism, of nitrate assimilation or thermostability. Strain RB11, an odc1 derivative of the particular H. polymorpha isolate CBS4732 (synonymous to ATCC34438, NRRL-Y-5445, CCY38-22-2) has been developed as a platform for heterologous gene expression. The scientific and industrial significance of this organism is now being met by the characterisation of its entire genome. The H. polymorpha RB11 genome consists of approximately 9.5 Mb and is organised as six chromosomes ranging in size from 0.9 to 2.2 Mb. Over 90% of the genome was sequenced with concomitant high accuracy and assembled into 48 contigs organised on eight scaffolds (supercontigs). After manual annotation 4767 out of 5933 open reading frames (ORFs) with significant homologies to a non-redundant protein database were predicted. The remaining 1166 ORFs showed no significant similarity to known proteins. The number of ORFs is comparable to that of other sequenced budding yeasts of similar genome size.

The therascreen PITX2 RGQ PCR assay is an optimized in vitro DNA methylation-specific quantitative real-time PCR test intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) transcription factor gene promoter 2. The test uses bisulfite-converted genomic DNA derived from formalin-fixed paraffin-embedded tumor tissues of breast cancer patients. The PMR will aid clinicians in selecting adjuvant systemic chemotherapy, e.g. anthracycline-based chemotherapy in high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients. The assay is intended to be used by qualified users, such as technicians, molecular biologists/clinical chemists, or physicians, trained in molecular biology techniques and in vitro diagnostic procedures. The complete workflow is streamlined for medium sample throughput with highly reliable and robust readout and can be performed in two working days.

Background: Gene expression profiling is a useful tool for the diagnosis and basic research of cancer. One of the major limitations of this approach is that already a short-term storage of native specimens of peripheral blood (PB) or bone marrow (BM) and/or the method of RNA isolation has significant influence on gene expression due to gene induction, repression and RNA degradation. The objective of the current study was to investigate the influence of a newly developed RNA stabilization and preparation system for BM specimens (PAXgeneTM Bone Marrow RNA System), focusing on RNA-yield, RNA-integrity and stability of gene expression profiles over time of BM sample storage. Patients and methods: 180 RNA samples that have been processed from 45 heparinized BM specimens of unselected children with acute leukemia at initial diagnosis, during treatment or without pathological findings in hematopoiesis were analyzed. Immediately after collection, heparinized BM specimens were divided into two PAXgeneTM RNA tubes and two standard tubes, each. RNA isolation using either the PAXgeneTM protocol (P) or a reference protocol (R) was performed after 2 hours (P0 and R0) or after 48 hours (P2 and R2). Results: The overall RNA yield (normalized to 1×107 leukocytes) was not different in all four isolation procedures, however the integrity of the RNA using the PAXgeneTM system was significantly higher at both time-points than that of the reference system: 8.6±0.2 (P0) vs. 6.8±0.4 (R0), p=0.0003 and 8.1±0.2 (P2) vs. 6.7±0.5 (R2), p=0.008. For the stabilized samples, we found very good pairwise correlation in gene expression for either gene at P2 compared to P0: GATA1 89±6%, RUNX1 83±10%, NCAM 69±12% and SPI1 89±6%. However, there were significant differences in two of the analyzed genes using the reference RNA isolation procedure: 40±6%, p<0.001 (GATA1) and 47±8%, p=0.005 (NCAM), respectively. Comparing the two RNA isolation procedures, there are significant differences in the expression levels of the analyzed genes. Discussion: The PAXgeneTM system is appropriate for the stabilization of gene expression levels in BM samples after short-term storage. However, as in some genes the reference RNA isolation procedure resulted in similar gene expression levels, the use of a stabilization reagent has to be carefully evaluated within the preanalytical handling of the samples. Our analysis has shown that it is not suitable to compare RNA expression levels derived from different isolation procedures. In conclusion, the PAXgeneTM system is able to stabilize RNA from clinical BM samples while being suitable to isolate high quality and quantity RNA.

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Introduction Somatic mutations acquired in key signalling pathway, transcription factor, spliceosome, epigenetic and tumor suppressor genes are of central importance in the development and progression of myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To date, in order to evaluate all relevant genetic alterations, multiple tests are needed, requiring large amounts of DNA. As tests are typically performed sequentially this unnecessarily extends the time between sample acquisition and mutation detection. The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System provides a single solution to simultaneously test for actionable mutations, whilst also saving sample material (only 40ng DNA input required per sample), shortening test time and enabling simplification of lab operations. The QIAact Myeloid DNA UMI Panel is a multi-gene targeted sequencing panel designed to detect complex mutations throughout the most informative genes linked to myeloid disease. This allows reliable and sensitive detection of single nucleotide variants (SNV) and large Insertion/Deletion (InDel) mutations. Methods The QIAact Myeloid DNA UMI Panel targets 25 genes known to be important in myeloid leukemia. A key feature of the panel is the addition of a unique molecular index (UMI) to tag each individual original DNA molecule prior to target enrichment by PCR. UMIs enable sequencing and PCR bias corrections, allowing sensitive detection of mutations. To assess the assay performance, reference standards and blood and bone marrow samples were used. Following target enrichment, libraries were sequenced on the GeneReader NGS System and mutations analyzed using the QIAGEN Clinical Insight (QCI) Analyze software suite. Results To confirm DNA mutation detection, Horizon Discovery and SeraCare Reference Standards containing variants typical of myeloid malignancies were used. The anticipated DNA mutations were consistently identified both within and between runs. The samples used, including blood and bone marrow samples, demonstrated the ability of the assay to detect important large indels (52 bp deletion CALR type 1 variant) and key SNVs down to a minor allele fraction (MAF) of 1% for JAK2 (e.g. exon 12, 13, 14 & 15) and KIT (exon 8, 9, 10, 11 & 17). A sensitive variant detection of allele frequency <0.5% for the KIT D816V was also achieved. For the other genes covered by the panel, including ASXL1, RUNX1, NPM1, DNMT3A, IDH1/2, results show sufficiently uniform amplification and sequencing coverage to support mutation detection with a MAF of 5%. Conclusion The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System offer a fully integrated DNA to variant detection and interpretation solution. The optimized chemistry allows superior analytical sensitivity resulting in accurate and efficient mutation detection of highly relevant genetic alterations for myeloid malignancy research. Disclosures Laloux: QIAGEN France S.A.S: Employment. Biglia:HalioDx: Employment. Bona:HalioDx: Employment. Lafi:HalioDx: Employment. Charifi:HalioDx: Employment. Larsen:QIAGEN Aarhus: Employment. Lueerssen:QIAGEN Manchester Ltd: Employment. Gupta:QIAGEN Aarhus: Employment. Lauber:QIAGEN GmbH: Employment. Hughes:QIAGEN Manchester Ltd: Employment.