The impact of next generation sequencing technologies on haematological research - A review

Next-generation sequencing (NGS) technologies have begun to revolutionize the field of haematological malignancies through the assessment of a patient's genetic makeup with a minimal cost. Significant discoveries have already provided a unique insight into disease initiation, risk stratification and therapeutic intervention. Sequencing analysis will likely form part of the routine diagnostic testing in the future. However, a number of important issues need to be addressed for that to become a reality with regard to result interpretation, laboratory workflow, data storage and ethical issues. In this review we summarize the contribution that NGS has already made to the field of haematological malignancies. Finally, we discuss the challenges that NGS technologies will bring in relation to data storage, ethical and legal issues and laboratory validation. Despite these challenges, we predict that high-throughput DNA sequencing will redefine haematological malignancies based on individualized genomic analysis.

Electron impact excitation of Be-like ions: a comparison of DARC and ICFT results

Emission lines of Be-like ions are frequently observed in astrophysical plasmas, and many are useful for density and temperature diagnostics. However, accurate atomic data for energy levels, radiative rates (A-values) and effective electron excitation collision strengths ($\Upsilon$) are required for reliable plasma modelling. In general it is reasonably straightforward to calculate energy levels and A- values to a high level of accuracy. By contrast, considerable effort is required to calculate $\Upsilon$, and hence it is not always possible to assess the accuracy of available data. Recently, two independent calculations (adopting the $R$-matrix method) but with different approaches (DARC and ICFT) have appeared for a range of Be-like ions. Therefore, in this work we compare the two sets of $\Upsilon$, highlight the large discrepancies for a significant number of transitions and suggest possible reasons for these.

Characterization of x-ray lens for use in probing high energy density states of matter

By using polycapillary lenses to focus laser-produced x-ray sources to high intensities, an improvement in signal-to-noise ratio can be achieved. Here the He-alpha line emission produced by driving a titanium backlighter target is focused by a polycapillary lens and the output characterized. The x-ray spot is measured to have a peak intensity of 4.5 x 10(7) photons, with a total photon count of 8.8 x 10(8) in 0.13 +/- 0.01 mm(2). This setup is equivalent to placing the backlighter target 3 mm from the sample with a 600 mu m diameter pinhole. The polycapillary lens enables the placement of the backlighter target at a much larger distance from the sample to be studied and therefore has the ability to greatly improve the signal-to-noise ratio on detectors. We demonstrate this with two simple diffraction experiments using pyrolytic graphite and polycrystalline aluminium.

From Sensing to Molecular Logic: A Journey

A personal account of the establishment of luminescent PET (photoinduced electron transfer) sensing and its development into molecular logic is given. Several applications of these two research areas, e.g. blood electrolyte diagnostics, ‘lab-on-amolecule’ systems and molecular computational identification (MCID) are illustrated.

Unconventional Computing: A Boolean Chemical Perspective

Elementary computing operations can be arranged within molecules so that problems in chemical, biochemical, and biological situations can be addressed. Problems that are found in small and/or living spaces, where the corresponding semiconductor logic devices cannot operate conveniently, are particularly amenable to this approach. The visualization and monitoring of intracellular species is one such category. Problems in medical diagnostics and therapy form additional categories. Chemists and biologists employ chemical synthesis and molecular biology techniques to build molecular logic devices. The photochemical approach to molecular logic devices is particularly prevalent. The fluorescent photoinduced electron transfer (PET) switching principle is particularly useful for designing logic functions into small molecules.

Automated tumor analysis for molecular profiling in lung cancer

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

Electron dynamics and frequency coupling in a radio-frequency capacitively biased planar coil inductively coupled plasma system

The electron dynamics in a planar coil inductively coupled plasma (ICP) system with a capacitively biased electrode is investigated using space and phase resolved optical emission spectroscopy. The two power source frequencies are exact multiple of each other and phase-locked. In this configuration, the system is investigated when the coil is operated in both E-mode and H-mode. The results show that in a phase synchronized RF biased ICP, the electrode bias power couples with the capacitive contribution of the coil, in both E-mode and H-modes, similar to dual-frequency capacitively coupled plasmas (2f-CCPs). It is also demonstrated that in H-mode, the phase between the electrode bias frequency and the ICP coil frequency influences the electron heating, similar to the electrical asymmetry effect in 2f-CCPs.

Effectiveness of a biomarker-based exclusion of ventilator-acquired pneumonia to reduce antibiotic use: protocol for a randomised controlled trial (VAPrapid-2):Study protocol for a randomised controlled trial

Background Ventilator-acquired pneumonia (VAP) is a common reason for antimicrobial therapy in the intensive care unit (ICU). Biomarker-based diagnostics could improve antimicrobial stewardship through rapid exclusion of VAP. Bronchoalveloar lavage (BAL) fluid biomarkers have previously been shown to allow the exclusion of VAP with high confidence. Methods/Design This is a prospective, multi-centre, randomised, controlled trial to determine whether a rapid biomarker-based exclusion of VAP results in fewer antibiotics and improved antimicrobial management. Patients with clinically suspected VAP undergo BAL, and VAP is confirmed by growth of a potential pathogen at > 104 colony-forming units per millilitre (CFU/ml). Patients are randomised 1:1, to either a ‘biomarker-guided recommendation on antibiotics’ in which BAL fluid is tested for IL-1β and IL-8 in addition to routine microbiology testing, or to ‘routine use of antibiotics’ in which BAL undergoes routine microbiology testing only. Clinical teams are blinded to intervention until 6 hours after randomisation, when biomarker results are reported to the clinician. The primary outcome is a change in the frequency distribution of antibiotic-free days (AFD) in the 7 days following BAL. Secondary outcome measures include antibiotic use at 14 and 28 days; ventilator-free days; 28-day mortality and ICU mortality; sequential organ failure assessment (SOFA) at days 3, 7 and 14; duration of stay in critical care and the hospital; antibiotic-associated infections; and antibiotic-resistant pathogen cultures up to hospital discharge, death or 56 days. A healthcare-resource-utilisation analysis will be calculated from the duration of critical care and hospital stay. In addition, safety data will be collected with respect to performing BAL. A sample size of 210 will be required to detect a clinically significant shift in the distribution of AFD towards more patients having fewer antibiotics and therefore more AFD. Discussion This trial will test whether a rapid biomarker-based exclusion of VAP results in rapid discontinuation of antibiotics and therefore improves antibiotic management in patients with suspected VAP.

Quiescent and flaring X-ray emission from the nearby M/T dwarf binary SCR 1845-6357

Aims: X-ray emission is an important diagnostics to study magnetic activity in very low mass stars that are presumably fully convective and have an effectively neutral photosphere. Methods: We investigate an XMM-Newton observation of SCR 1845-6357, a nearby, ultracool M 8.5 / T 5.5 dwarf binary. The binary is unresolved in the XMM detectors, but the X-ray emission is very likely from the M 8.5 dwarf. We compare its flaring emission to those of similar very low mass stars and additionally present an XMM observation of the M 8 dwarf VB 10. Results: We detect quasi-quiescent X-ray emission from SCR 1845-6357 at soft X-ray energies in the 0.2-2.0 keV band, as well as a strong flare with a count rate increase of a factor of 30 and a duration of only 10 min. The quasi-quiescent X-ray luminosity of log LX = 26.2 erg/s and the corresponding activity level of log LX/Lbol = -3.8 point to a fairly active star. Coronal temperatures of up to 5 MK and frequent minor variability support this picture. During the flare, which is accompanied by a significant brightening in the near-UV, plasma temperatures of 25-30 MK are observed and an X-ray luminosity of LX = 8 × 1027 erg/s is reached. Conclusions: The source SCR 1845-6357 is a nearby, very low mass star that emits X-rays at detectable levels in quasi-quiescence, implying the existence of a corona. The high activity level, coronal temperatures and the observed large flare point to a rather active star, despite its estimated age of a few Gyr.

Diagnostic value of increased sensitivity by massively parallel sequencing

Routine molecular diagnostics modalities are unable to confidently detect low frequency mutations (<5-15%) that may indicate response to targeted therapies. We confirm the presence of a low frequency NRAS mutation in a rectal cancer patient using massively parallel sequencing when previous Sanger sequencing results proved negative and Q-PCR testing inconclusive. There is increasing evidence that these low frequency mutations may confer resistance to anti-EGFR therapy. In view of negative/inconclusive Sanger sequencing and Q-PCR results for NRAS mutations in a KRAS wt rectal case, the diagnostic biopsy and 4 distinct subpopulations of cells in the resection specimen after conventional chemo/radiotherapy were massively parallel sequenced using the Ion Torrent PGM. DNA was derived from FFPE rectal cancer tissue and amplicons produced using the Cancer Hotspot Panel V2 and sequenced using semiconductor technology. NRAS mutations were observed at varying frequencies in the patient biopsy (12.2%) and all four subpopulations of cells in the resection with an average frequency of 7.3% (lowest 2.6%). The results of the NGS also provided the mutational status of 49 other genes that may have prognostic or predictive value, including KRAS and PIK3CA. NGS technology has been postulated in diagnostics because of its capability to generate results in large panels of clinically meaningful genes in a cost-effective manner. This case illustrates another potential advantage of this technology: its use for detecting low frequency mutations that may influence therapeutic decisions in cancer treatment.

An interview with Manuel Salto-Tellez on diagnostic pathology: the future is morphomolecular

Professor Manuel Salto-Tellez of Queen’s University, Belfast, Northern Ireland is an expert histopathologist and molecular diagnostician. Professor Salto-Tellez is a lead investigator at the Northern Ireland Molecular Pathology Laboratory and also serves as a member of the Editorial Advisory Board for Expert Review of Molecular Diagnostics. In this interview, he proposes directions for the future of molecular pathology and molecular diagnostics, integrating all aspects of pathology toward a common goal.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

Hα Line Profile Asymmetries and the Chromospheric Flare Velocity Field

The asymmetries observed in the line profiles of solar flares can provide important diagnostics of the properties and dynamics of the flaring atmosphere. In this paper the evolution of the Hα and Ca ii λ8542 lines are studied using high spatial, temporal, and spectral resolution ground-based observations of an M1.1 flare obtained with the Swedish 1 m Solar Telescope. The temporal evolution of the Hα line profiles from the flare kernel shows excess emission in the red wing (red asymmetry) before flare maximum and excess in the blue wing (blue asymmetry) after maximum. However, the Ca ii λ8542 line does not follow the same pattern, showing only a weak red asymmetry during the flare. RADYN simulations are used to synthesize spectral line profiles for the flaring atmosphere, and good agreement is found with the observations. We show that the red asymmetry observed in Hα is not necessarily associated with plasma downflows, and the blue asymmetry may not be related to plasma upflows. Indeed, we conclude that the steep velocity gradients in the flaring chromosphere modify the wavelength of the central reversal in the Hα line profile. The shift in the wavelength of maximum opacity to shorter and longer wavelengths generates the red and blue asymmetries, respectively.