The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

1.° Collectio juris veteris canonici juxta rerum ordinem distributa et ex canonibus Conciliorum et summorum Pontificum epistolis Decretalibus concinnata : praemittitur notitia provinciarum et civitatum Imperii Romani. — 2.° Epistola formata, data anno 867. — 3.° Capitulatio de nominibus regionum ; sive notitia provinciarum Imperii Romani. — 4.° Epistola Gregorii ad Anatolium, Diaconum Constantinopolitanum. — 5.° Fragmentum Decreti cujusdam Papae, de pace. — 6.° Felicis, Papae, epistola ad Episcopos Aegypti

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Na 50 Nachlass Arthur Schopenhauer - 'Schopenhauer-Archiv', 867 - Glückwünsche zum Geburtstag

abgedruckt in: Schopenhauer-Jahrbuch 30 (1943), S. 212-213

Project Neos; a generalized study of processes for the realization of esign configurations in materials. [Final] technical documentary report no. ML-TDR-64-311. MTD project no.7-867.

Mode of access: Internet.

The genesis of science education research in Australasia

Although the sciences were being taught in Australian schools well before the Second World War, the only evidence of research studies of this teaching is to be found in the report, published by ACER in 1932 of Roy Stanhope’s survey of the teaching of chemistry in New South Wales and a standardized test he had developed. Roy Stanhope was a science teacher with a research masters degree in chemistry. He had won a scholarship to go to Stanford University for doctoral studies, but returned after one year when his scholarship was not extended. He went on to be a founder in 1943 of the Australian Science Teachers Association (ASTA), which honours this remarkable pioneer through its annual Stanhope Oration. In his retirement Stanhope undertook a comparative study of science

Knowledge and attitudes about Vitamin D and impact on sun protection practices among urban office workers in Brisbane, Australia

Background: Sun exposure is the main source of vitamin D. Increasing scientific and media attention to the potential health benefits of sun exposure may lead to changes in sun exposure behaviors. Methods: To provide data that might help frame public health messages, we conducted an online survey among office workers in Brisbane, Australia, to determine knowledge and attitudes about vitamin D and associations of these with sun protection practices. Of the 4,709 people invited to participate, 2,867 (61%) completed the questionnaire. This analysis included 1,971 (69%) participants who indicated that they had heard about vitamin D. Results: Lack of knowledge about vitamin D was apparent. Eighteen percent of people were unaware of the bone benefits of vitamin D but 40% listed currently unconfirmed benefits. Over half of the participants indicated that more than 10 minutes in the sun was needed to attain enough vitamin D in summer, and 28% indicated more than 20 minutes in winter. This was significantly associated with increased time outdoors and decreased sunscreen use. People believing sun protection might cause vitamin D deficiency (11%) were less likely to be frequent sunscreen users (summer odds ratio, 0.63; 95% confidence interval, 0.52-0.75). Conclusions: Our findings suggest that there is some confusion about sun exposure and vitamin D, and that this may result in reduced sun-protective behavior. Impact: More information is needed about vitamin D production in the skin. In the interim, education campaigns need to specifically address the vitamin D issue to ensure that skin cancer incidence does not increase.