A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3-7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.

Assessment of Minimal Residual Disease in Standard-Risk AML.

BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Consensus on BCR-ABL1 reporting in chronic myeloid leukaemia in the UK.

For patients with chronic myeloid leukaemia (CML), treatment guidelines recommend monitoring response to treatment with tyrosine kinase inhibitors (TKIs) by testing the BCR-ABL1 fusion gene transcript level using reverse transcriptase quantitative polymerase chain reaction. Despite recent efforts to standardise protocols for BCR-ABL1 testing, some variability remains among laboratories in the UK regarding the techniques used and the approach to reporting results. This increases the risk of misinterpretation of results by both clinicians and patients. An expert panel met to discuss current issues surrounding BCR-ABL1 testing in the UK and to develop guidance for laboratories, with emphasis on the optimal approach to reporting laboratory results. Topics included the minimum required information to include in the laboratory report, units of measurement, test sensitivity and BCR-ABL1 transcript variants. To aid communication between laboratories and clinics, standard forms were generated that could be used by (i) clinics when submitting samples to laboratories, and (ii) laboratories when reporting results to clinics. Standardising the way in which BCR-ABL1 test results are reported from laboratories to clinics should help to improve communication, interpretation of results and patient care.

Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project.

PURPOSE: Fresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. METHODS: We conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. RESULTS: We found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). CONCLUSION: We present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.

Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL.

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the 'discoverability' of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group.

Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.

NUP98-NSD1 fusion in association with FLT3-ITD mutation identifies a prognostically relevant subgroup of pediatric acute myeloid leukemia patients suitable for monitoring by real time quantitative PCR.

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment.

Simultaneous sequencing of genetic and epigenetic bases in DNA.

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.

Draft genome sequences of Helicobacter pylori isolates from Malaysia, cultured from patients with functional dyspepsia and gastric cancer.

Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.

Molecular epidemiologic investigation of an anthrax outbreak among heroin users, Europe.

In December 2009, two unusual cases of anthrax were diagnosed in heroin users in Scotland. A subsequent anthrax outbreak in heroin users emerged throughout Scotland and expanded into England and Germany, sparking concern of nefarious introduction of anthrax spores into the heroin supply. To better understand the outbreak origin, we used established genetic signatures that provided insights about strain origin. Next, we sequenced the whole genome of a representative Bacillus anthracis strain from a heroin user (Ba4599), developed Ba4599-specific single-nucleotide polymorphism assays, and genotyped all available material from other heroin users with anthrax. Of 34 case-patients with B. anthracis-positive PCR results, all shared the Ba4599 single-nucleotide polymorphism genotype. Phylogeographic analysis demonstrated that Ba4599 was closely related to strains from Turkey and not to previously identified isolates from Scotland or Afghanistan, the presumed origin of the heroin. Our results suggest accidental contamination along the drug trafficking route through a cutting agent or animal hides used to smuggle heroin into Europe.

Magnetic discontinuities in magnetohydrodynamic turbulence and in the solar wind.

Recent measurements of solar wind turbulence report the presence of intermittent, exponentially distributed angular discontinuities in the magnetic field. In this Letter, we study whether such discontinuities can be produced by magnetohydrodynamic (MHD) turbulence. We detect the discontinuities by measuring the fluctuations of the magnetic field direction, Δθ, across fixed spatial increments Δx in direct numerical simulations of MHD turbulence with an imposed uniform guide field B(0). A large region of the probability density function (pdf) for Δθ is found to follow an exponential decay, proportional to exp(-Δθ/θ(*)), with characteristic angle θ(*)≈(14°)(b(rms)/B(0))(0.65) for a broad range of guide-field strengths. We find that discontinuities observed in the solar wind can be reproduced by MHD turbulence with reasonable ratios of b(rms)/B(0). We also observe an excess of small angular discontinuities when Δx becomes small, possibly indicating an increasing statistical significance of dissipation-scale structures. The structure of the pdf in this case closely resembles the two-population pdf seen in the solar wind. We thus propose that strong discontinuities are associated with inertial-range MHD turbulence, while weak discontinuities emerge from dissipation-range turbulence. In addition, we find that the structure functions of the magnetic field direction exhibit anomalous scaling exponents, which indicates the existence of intermittent structures.

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.

Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.

Numerical measurements of the spectrum in magnetohydrodynamic turbulence.

We report the results of an extensive set of direct numerical simulations of forced, incompressible, magnetohydrodynamic (MHD) turbulence with a strong guide field. The aim is to resolve the controversy regarding the power-law exponent (alpha, say) of the field-perpendicular energy spectrum E(k) proportional variant k(alpha). The two main theoretical predictions alpha=-3/2 and alpha=-5/3 have both received some support from differently designed numerical simulations. Our calculations have a resolution of 512(3) mesh points, a strong guide field, and an anisotropic simulation domain and implement a broad range of large-scale forcing routines, including those previously reported in the literature. Our findings indicate that the spectrum of well-developed, strong incompressible MHD turbulence with a strong guide field is E(k) proportional variant k(-3/2).

Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL.

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

BACKGROUND: Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. RESULTS: TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. CONCLUSIONS: Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.

Whole-genome sequencing investigation of animal-skin-drum-associated UK anthrax cases reveals evidence of mixed populations and relatedness to a US case.

There have been two anthrax cases affecting people that played and/or made animal-skin drums in the UK during the last 10 years, with single fatal occurrences in Scotland in 2006 and London in 2008. Investigations by the Health Protection Agency (now Public Health England) employing multi-locus-variable number tandem repeat analysis had previously linked the clinical cases to spores associated with animal skins and drums the patients had been in contact with. In this study, whole-genome sequencing of 23 Bacillus anthracis isolates harvested during the investigations was performed. High-quality draft assemblies of these genomes provided greater characterization of the B. anthracis strains present and placed them all upon a new branch of the global phylogeny. Although closely related, the clinical isolates from the two events, and another isolated from a drum-skin-associated case in New York in 2006, were distinct from each other. Multiple distinct genotypes were found during both investigations, implying either multiple contamination events or a single heterogeneous contamination. One environmental isolate from the Scottish incident was more closely related to London isolates than to the other Scottish isolates. As B. anthracis of this subgroup was present at both geographically and temporally distinct events, it may be more widespread at the source of contamination. All isolates were distinct from currently characterized West African strains, despite this being the likely origin of the drums and hides, therefore adding to our knowledge of B. anthracis diversity in the region.

Origin and subset distribution of peripheral blood dendritic cells in patients with chronic graft-versus-host disease.

BACKGROUND: After allogeneic hematopoietic stem cell transplantation, donor T cells interact with an antigen-presenting cell environment that is distorted in number, level of activation, and origin. The role of antigen presentation in the development of chronic graft-versus host disease (cGVHD) is unknown. METHODS: The number and origin of peripheral blood immature myeloid (CD19- CD1c+) and plasmacytoid (BDCA-2+) dendritic cells (DCs) was determined in 30 patients at more than 100 days after allogeneic hematopoietic stem cell transplantation. RESULTS: Patients with cGVHD had significantly higher plasmacytoid DC numbers than individuals without this complication (9.1+/-2.0 x 10(6)/L versus 3.8+/-0.6 x 10(6)/L, =0.025). Chimerism studies demonstrated that DCs in patients with cGVHD were exclusively of donor origin, whereas persistence of host DCs was observed in some control patients. CONCLUSIONS: The antigen-presenting cell environment in patients with cGVHD, as represented by immature blood DCs, is of donor origin but distorted in terms of subset distribution.