David Riley

Personalized genomic analyses for cancer mutation discovery and interpretation

Analysis of matched tumor and normal DNA from the same patient improves accuracy of identification of actionable mutations, allowing better targeting of potential treatments. Will the real mutation please stand up? When a patient is diagnosed with cancer, a sample of the tumor is often analyzed to look for mutations that might guide the approach to targeted treatment of the disease. Jones et al. analyzed samples from more than 800 patients with 15 different cancer types and showed that this standard approach is not necessarily accurate without also analyzing a matched sample of normal DNA from the same patient. The authors found that, compared to analysis of paired samples, the standard tumor-only sequencing approach frequently identified mutations that were present in the patient’s normal tissues and were therefore not suitable for targeted therapy or, conversely, missed useful new mutations in the tumor. Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Direct detection of early-stage cancers using circulating tumor DNA

Noninvasive liquid biopsy analysis of circulating tumor DNA permits direct detection of early-stage cancers. Finding smaller needles in haystacks The detection and analysis of cell-free DNA in patients’ blood are becoming increasingly accepted in oncology. However, this approach has generally been applied for the monitoring of patients with existing tumors. It has not been useful for early diagnosis of cancer because of insufficient sensitivity to detect really small tumors that only shed minute quantities of DNA into the blood, as well as difficulties with identifying cancer-associated genetic changes without knowing what mutations are present in the primary tumor. A method developed by Phallen et al., called targeted error correction sequencing, addresses both of these limitations and demonstrates the feasibility of detecting circulating cell-free DNA from many early tumors, suggesting its potential use for cancer screening. Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

Plasma-based studies with intense X-ray and particle beam sources

The construction of short pulse (<200 fs) tunable X-ray laser sources based on the X-ray free electron laser (XFEL) concept will be a watershed for plasma-based and warm dense matter research. These new fourth generation light sources will have extremely high fields and short wavelengths (∼0.1 nm) with peak spectral brightnesses 10 10 greater than third generation sources. Further, the high intensity upgrade of the GSI accelerator facilities will lead to specific energy depositions up to 200 kJ/g and temperatures between 1 and 10 eV at almost solid-state densities, enabling interesting experiments in the regime of nonideal plasmas, such as the evolution of intense ion beams in the interior of a Jovian planet. Below we discuss several applications: the creation of warm dense matter (WDM) research, probing of near solid density plasmas, and laser-plasma spectroscopy of ions in plasmas. The study of dense plasmas has been severely hampered by the fact that laser-based methods have been unavailable and these new fourth generation sources will remove these restrictions.

Notch1 Mutations Are Drivers of Oral Tumorigenesis

Disruption of NOTCH1 signaling was recently discovered in head and neck cancer. This study aims to evaluate NOTCH1 alterations in the progression of oral squamous cell carcinoma (OSCC) and compare the occurrence of these mutations in Chinese and Caucasian populations. We used a high-throughput PCR-based enrichment technology and next-generation sequencing (NGS) to sequence NOTCH1 in 144 samples collected in China. Forty-nine samples were normal oral mucosa from patients undergoing oral surgery, 45 were oral leukoplakia biopsies, and 50 were chemoradiation-naïve OSCC samples with 22 paired-normal tissues from the adjacent unaffected areas. NOTCH1 mutations were found in 54% of primary OSCC and 60% of premalignant lesions. Importantly, almost 60% of patients with leukoplakia with mutated NOTCH1 carried mutations that were also identified in OSCC, indicating an important role of these clonal events in the progression of early neoplasms. We then compared all known NOTCH1 mutations identified in Chinese patients with OSCC with those reported in Caucasians to date. Although we found obvious overlaps in critical regulatory NOTCH1 domains alterations and identified specific mutations shared by both groups, possible gain-of-function mutations were predominantly seen in Chinese population. Our findings demonstrate that premalignant lesions display NOTCH1 mutations at an early stage and are thus bona fide drivers of OSCC progression. Moreover, our results reveal that NOTCH1 promotes distinct tumorigenic mechanisms in patients from different ethnical populations. Cancer Prev Res; 8(4); 277–86. ©2014 AACR. See related perspectives, p. 259 and p. 262

Zeeko/UCL process for polishing large lenses and prisms

This paper describes progress on the development of a new process for producing precision surfaces for the optics industry, and potentially for other sectors including silicon wafer fabrication and lapping and polishing of precision mechanical surfaces. The paper marks an important milestone in the development program, with the completion of the construction of the first fully-productionized machine and the first results from the commissioning process.

First aspheric form and texture results from a production machine embodying the precession process

We report on progress developing the Precession Process, that has recently been embodied for the first time in a fully-productionised aspheric polishing machine. We describe how the process uses inflated polishing tools of continuously-variable size and hardness. Despite the rapid tool rotation needed to give high removal rates, the method produce well-behaved and near-Gaussian tool influence functions, by virtue of the precession of the spin axis. We then describe how form errors are controlled. The method takes influence-function data and an error map as input, together with, uniquely, weighting factors for height and slope residuals and process time. A numerical optimisation of the cost function with variable dwell time, tool path and tool size is then performed. The advantages of this new technique are contrasted with conventional deconvolution methods. Results of form control on aspheric surfaces are presented, with an interpretation in terms of spatial frequencies. We draw particular attention to control of form at the centre and periphery of a workpiece. Finally, we describe how Precession processing gives multi- directional rubbing of surfaces, and we present the superb texture achieved on samples.

Direct measurements of compressive and tensile strain during shock breakout by use of subnanosecond x‐ray diffraction

Shock waves of order 100 kbar were launched into 50‐μm‐thick single crystals of silicon (111) by irradiation with nanosecond pulses of 1.05‐μm laser light at irradiances in the region of 2×1010 W cm−2. A separate laser beam, synchronous but delayed with respect to the shock‐driving beam, and containing approximately 25 J of 0.53‐μm laser light in a pulse of 1 ns (FWHM), was focused to a tight (<100 μm) spot on a separate titanium target to produce a plasma which was a prolific source of He‐like Ti x rays. The x rays were Bragg diffracted from the rear surface of the shocked crystal and the spectrum recorded on an x‐ray streak camera. Changes in interatomic spacings in a region within several microns of the surface were thus deduced from the resultant shift in Bragg angle with a temporal resolution of 50 ps. Shock waves with compressions of order 6% were observed. We observed the crystal in a state of dynamic tension as the two rarefaction waves met. The results are in good agreement with hydrocode simulations in conjunction with x‐ray diffraction calculations.

Precessions process for efficient production of aspheric optics for large telescopes and their instrumentation

We summarize the reasons why aspheric surfaces, including non-rotationally-symmetric surfaces, are increasingly important to ground and space-based astronomical instruments, yet challenging to produce. We mainly consider the generic problem of producing aspheres, and then lightweight segments for the primary mirror of an Extremely Large Telescope. We remark on the tension between manufacturability of spherical segments, and performance with aspheric segments. This provides the context for our presentation of the novel Precessions process for rapid polishing and form-correction of aspheric surfaces. We outline why this is a significant step beyond previous methods to automate aspheric production, and how it has resulted in a generalized, scaleable technology that does not require high capital-value tooling customized to particular types of optical form. We summarize implementation in the first two automated CNC machines of 200mm capacity, followed by the first 600mm machine, and the current status of the process-development program. We review quantitative results of polishing trials, including materials relevant to large and instrumentation optics. Finally, we comment on the potential of the technology for space optics and for removing quilting in honeycomb substrates.

Soft x-ray free electron laser microfocus for exploring matter under extreme conditions

We have focused a beam (BL3) of FLASH (Free-electron LASer in Hamburg: lambda = 13.5 nm, pulse length 15 fs, pulse energy 10-40 microJ, 5 Hz) using a fine polished off-axis parabola having a focal length of 270 mm and coated with a Mo/Si multilayer with an initial reflectivity of 67% at 13.5 nm. The OAP was mounted and aligned with a picomotor controlled six-axis gimbal. Beam imprints on poly(methyl methacrylate) - PMMA were used to measure focus and the focused beam was used to create isochoric heating of various slab targets. Results show the focal spot has a diameter of < or =1 microm. Observations were correlated with simulations of best focus to provide further relevant information.