A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood

Introduction: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. Methods: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. Results: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. Conclusions: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible

MDS/MPN-Unclassifiable with t(X;17)(q28;q21) and KANSL1-MTCP1/CMC4 Fusion Gene

Myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U) is a poorly characterized entity among overlap myeloid syndromes. Recent studies have shown heterogeneous mutational profiles in this group being able to subclassify them into entities closely related to the more well-established disorders under the umbrella term of the MDS/MPN group. Recurrent cytogenetic alterations are, nonetheless, rare in MDS/MPN-U. Here, for the first time, we report a case of MDS/MPN-U with a t(X;17)(q28;q21) chromosomal rearrangement leading to the KANSL1-MTCP1 fusion gene

Three-Color FISH Analysis of TMPRSS2/ERG Fusions in Prostate Cancer Indicates That Genomic Microdeletion of Chromosome 21 Is Associated with Rearrangement

The recent description of novel recurrent gene fusions in ~80% of prostate cancer (PCa) cases has generated increased interest in the search for new translocations in other epithelial cancers and emphasizes the importance of understanding the origins and biologic implications of these genomic rearrangements. Analysis of 15 PCa cases by reverse transcription-polymerase chain reaction was used to detect six ERG-related gene fusion transcripts with TMPRSS2. No TMPRSS2/ETV1 chimeric fusion was detected in this series. Three-color fluorescence in situ hybridization confirms that TMPRSS2/ERG fusion may be accompanied by a small hemizygous sequence deletion on chromosome 21 between ERG and TMPRSS2 genes. Analysis of genomic architecture in the region of genomic rearrangement suggests that tracts of microhomology could facilitate TMPRSS2/ERG fusion events

Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores

Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores.A major challenge is low quantity and quality of extracted nucleic acids, which can limit ourability to perform genetic analyses, and have a significant influence on overall study design.This study was aimed at identifying the most reliable and reproducible method of obtainingsufficient high-quality nucleic acids from FFPE tissues. We compared the yield and qualityof nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extractionprotocols, using 14 commercially available kits. Nucleic acid yield was determined byfluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleicacids in quantities that are compatible with downstream molecular applications. However,the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and fiveDNA extraction protocols, including protocols from two kits for dual-extraction of RNA andDNA from the same tissue source, were prioritized for further quality assessment based onthe yield and purity of their products. Specifically, their compatibility with downstream reactionswas assessed using both NanoString nCounter gene expression assays and reversetranscriptasereal-time PCR for RNA, and methylation-specific PCR assays for DNA. [...

Bilingualism and language management in Czech minority in Croatia

This thesis attends to the issue of a Czech minority in Croatia, local bilingualism and language management on various levels: from the level of an individual, over the level of the entire Czech minority living there and local government, up to the state level and its laws. In the first chapter I have summarized the history of Czech migration to the territory of today's Croatia, mainly during the 19th century, and their gradual organization after they found themselves located out of their home country when the Austria-Hungary dismembered. The second chapter describes current situation in the minority - its organizational structure, cultural life, school system and its information and publishing activities. The safeguard of its existence by law and constitution is also of importance. The third chapter dedicates to the language situation in the Czech minority in Croatia: Czech-croatian and Croatian-czech bilingualism, differentness in dialectological origin of the language, language as a system and factors that influence both, the language and its users. The chapter is supplemented by the examples of spoken and written discourse of Czech fellow countrymen. The last chapter describes the progression and results of a research that was performed in minor companies with mixed language employees in the town of..

Additional file 3 of Molecular characterization of Gleason patterns 3 and 4 prostate cancer using reverse Warburg effect-associated genes

MTE primer sequences. Table S3. MTE primer sequences. (XLSX 45.9 kb

Yield and purity of RNA and DNA extracted using various kits.

<p>All extractions were performed strictly according to the respective manufacturer’s protocols (links in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179732#pone.0179732.t001" target="_blank">Table 1</a>). Shown are nucleic acid yields, standardized for tissue input; absorbance ratios of 260 nm/280 nm and 260 nm/230 nm; and Bioanalyzer DV₂₀₀ (i.e., the percentage of RNA fragments between 200 and 4000 bp).</p

DNA and RNA extraction kits compared in this study.

<p>Provided are the detailed kit names plus the names of their respective manufacturers and hyperlinks to online protocols.</p