Comparison of the performance of two targeted metagenomic virus capture probe-based methods using reference control materials and clinical samples.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50-500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.

A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2.

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting.

Bounded distributions place limits on skewness and larger moments.

Distributions of strictly positive numbers are common and can be characterized by standard statistical measures such as mean, standard deviation, and skewness. We demonstrate that for these distributions the skewness D3 is bounded from below by a function of the coefficient of variation (CoV) δ as D3 > δ - 1/δ. The results are extended to any distribution that is bounded with minimum value xmin and/or bounded with maximum value xmax. We build on the results to provide bounds for kurtosis D4, and conjecture analogous bounds exists for higher statistical moments.

Deformable vector fields warping for modelling of irregular breathing.

Purpose 4D computed tomography (4DCT) is the clinical standard to image organ motion in radiotherapy, although it is limited in imaging breathing variability. We propose a method to transfer breathing motion across longitudinal imaging datasets to include intra-patient variability and verify its performance in lung cancer patients. Methods Five repeated control 4DCTs for 6 non-small cell lung cancer patients were combined into multi-breath datasets (m4DCT) by merging stages of deformable image registration to isolate respiratory motion. The displacement of the centre of mass of the primary tumour and its volume changes were evaluated to quantify intra-patient differences. Internal target volumes defined on the m4DCT were compared with those conventionally drawn on the 4DCT. Results Motion analysis suggests no discontinuity at the junction between successive breaths, confirming the method's ability to merge repeated imaging into a continuum. Motion (variability) is primarily in superior-inferior direction and goes from 14.4 mm (8.7 mm) down to 0.1 mm (0.6 mm), respectively for tumours located in the lower lobes or most apical ones. On average, up to 65% and 74% of the tumour volume was subject to expansion or contraction in the inhalation and exhalation phases. These variations lead to an enlargement of the ITV up to 8% of its volume in our dataset. Conclusion 4DCT can be extended to model variable breathing motion by adding synthetic phases from multiple time-resolved images. The inclusion of this improved knowledge of patients' breathing allows better definition of treatment volumes and their margins for radiation therapy. .

Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr-/-.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

A bi-directional beam-line energy ramping for efficient patient treatment with scanned proton therapy.

Objective.The treatment of mobile tumours using Pencil Beam Scanning (PBS) has become more prevalent in the last decade. However, to achieve the same beam delivery quality as for static tumours, treatments have to be combined with motion mitigation techniques, not limited but including, breath hold, gating and re-scanning, which typically prolong treatment time. In this article we present a novel method of bi-directional energy modulation and demonstrate our initial experience in improvement of treatment efficiency. Approach.At Paul Scherrer Institute Gantry 2 mobile tumours are treated by combining PBS with gating and volumetric re-scanning (VR), where the target volume is irradiated multiple times. Initial implementation of VR used only descending beam energies, creating a substantial dead time due to the beam-line initialization (ramping) before each re-scan. In 2019 we commissioned an energy meandering strategy that allows us to avoid beam line ramping in-between energy series while maintaining beam delivery quality.Main results.The measured beam parameters difference for both energy sequence are in the order of the typical daily variations: 0.2 mm in beam position and 0.2 mm in range. Using machine log files, we performed point-to-point dose difference calculations between original and new applications where we observed dose differences of less than 2%. After three years of operation employing bi-directional energy modulation, we have analysed the individual beam delivery time for 181 patients and have compared this to simulations of the timing behaviour assuming uni-directional energy sequence application. Depending on treatment complexity, we obtained plan delivery time reductions of up to 55%, with a median time gain of 17% for all types of treatments.Significance. Bi-directional energy modulation can help improving patient treatment efficiency by reducing delivery times especially for complex and specialised irradiations. It could be implemented in many existing facilities without significant additional hardware upgrades.

Exploring beamline momentum acceptance for tracking respiratory variability in lung cancer proton therapy: a simulation study.

Objective. Investigating the aspects of proton beam delivery to track organ motion with pencil beam scanning therapy. Considering current systems as a reference, specify requirements for next-generation units aiming at real-time image-guided treatments.Approach. Proton treatments for six non-small cell lung cancer (NSCLC) patients were simulated using repeated 4DCTs to model respiratory motion variability. Energy corrections required for this treatment site were evaluated for different approaches to tumour tracking, focusing on the potential for energy adjustment within beamline momentum acceptance (dp/p). A respiration-synchronised tracking, taking into account realistic machine delivery limits, was compared to ideal tracking scenarios, in which unconstrained energy corrections are possible. Rescanning and the use of multiple fields to mitigate residual interplay effects and dose degradation have also been investigated.Main results. Energy correction requirements increased with motion amplitudes, for all patients and tracking scenarios. Higher dose degradation was found for larger motion amplitudes, rescanning has beneficial effects and helped to improve dosimetry metrics for the investigated limited dp/pof 1.2% (realistic) and 2.4%. The median differences between ideal and respiratory-synchronised tracking show minimal discrepancies, 1% and 5% respectively for dose coverage (CTV V95) and homogeneity (D5-D95). Multiple-field planning improves D5-D95 up to 50% in the most extreme cases while it does not show a significant effect on V95.Significance. This work shows the potential of implementing tumour tracking in current proton therapy units and outlines design requirements for future developments. Energy regulation within momentum acceptance was investigated to tracking tumour motion with respiratory-synchronisation, achieving results in line with the performance of ideal tracking scenarios. ±5% Δp/p would allow to compensate for all range offsets in our NSCLC patient cohort, including breathing variability. However, the realistic momentum of 1.2% dp/prepresentative of existing medical units limitations, has been shown to preserve plan quality.

Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis.

The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is an important regulator of hepatic lipid metabolism. While PPARalpha is known to activate transcription of numerous genes, no comprehensive picture of PPARalpha binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARalpha agonist GW7647. We found that GW7647 increased PPARalpha binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARalpha, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARalpha binding to their promoter. A GW7647-induced PPARalpha-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARalpha and SREBP signaling. Our data furthermore demonstrate interaction between PPARalpha and STAT transcription factors in PPARalpha-mediated transcriptional repression, and suggest interaction between PPARalpha and TBP, and PPARalpha and C/EBPalpha in PPARalpha-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARalpha in human liver and highlight the importance of cross-talk with other transcription factors.

Ultra-fast pencil beam scanning proton therapy for locally advanced non-small-cell lung cancers: Field delivery within a single breath-hold.

PURPOSE: The use of motion mitigation techniques such as breath-hold can reduce the dosimetric uncertainty of lung cancer proton therapy. We studied the feasibility of pencil beam scanning (PBS) proton therapy field delivery within a single breath-hold at PSI's Gantry 2. METHODS: In PBS proton therapy, the delivery time for a field is determined by the beam-on time and the dead time between proton spots (the time required to change the energy and/or lateral position). We studied ways to reduce beam-on and lateral scanning time, without sacrificing dosimetric plan quality, aiming at a single field delivery time of 15 seconds at maximum. We tested this approach on 10 lung cases with varying target volumes. To reduce the beam-on time, we increased the beam current at the isocenter by developing new beam optics for PSI's PROSCAN beamline and Gantry 2. To reduce the dead time between the spots, we used spot-reduced plan optimization. RESULTS: We found that it is possible to achieve conventional fractionated (2 Gy(RBE)/fraction) and hypofractionated (6 Gy(RBE)/fraction) field delivery times within a single breath-hold (<15 sec) for a variety non-small-cell lung cancer cases. CONCLUSION: In summary, the combination of spot reduction and improved beam line transmission is a promising approach for the treatment of mobile tumours within clinically achievable breath-hold durations.

Universal and dynamic ridge filter for pencil beam scanning particle therapy: a novel concept for ultra-fast treatment delivery.

Objective.In pencil beam scanning particle therapy, a short treatment delivery time is paramount for the efficient treatment of moving targets with motion mitigation techniques (such as breath-hold, rescanning, and gating). Energy and spot position change time are limiting factors in reducing treatment time. In this study, we designed a universal and dynamic energy modulator (ridge filter, RF) to broaden the Bragg peak, to reduce the number of energies and spots required to cover the target volume, thus lowering the treatment time.Approach. Our RF unit comprises two identical RFs placed just before the isocenter. Both RFs move relative to each other, changing the Bragg peak's characteristics dynamically. We simulated different Bragg peak shapes with the RF in Monte Carlo simulation code (TOPAS) and validated them experimentally. We then delivered single-field plans with 1 Gy/fraction to different geometrical targets in water, to measure the dose delivery time using the RF and compare it with the clinical settings.Main results.Aligning the RFs in different positions produces different broadening in the Bragg peak; we achieved a maximum broadening of 2.5 cm. With RF we reduced the number of energies in a field by more than 60%, and the dose delivery time by 50%, for all geometrical targets investigated, without compromising the dose distribution transverse and distal fall-off.Significance. Our novel universal and dynamic RF allows for the adaptation of the Bragg peak broadening for a spot and/or energy layer based on the requirement of dose shaping in the target volume. It significantly reduces the number of energy layers and spots to cover the target volume, and thus the treatment time. This RF design is ideal for ultra-fast treatment delivery within a single breath-hold (5-10 s), efficient delivery of motion mitigation techniques, and small animal irradiation with ultra-high dose rates (FLASH).

The lifetime of charged dust in the atmosphere.

Wind-blown dust plays a critical role in numerous geophysical and biological systems, yet current models fail to explain the transport of coarse-mode particles (>5 µm) to great distances from their sources. For particles larger than a few microns, electrostatic effects have been invoked to account for longer-than-predicted atmospheric residence times. Although much effort has focused on elucidating the charging processes, comparatively little effort has been expended understanding the stability of charge on particles once electrified. Overall, electrostatic-driven transport requires that charge remain present on particles for days to weeks. Here, we present a set of experiments designed to explore the longevity of electrostatic charge on levitated airborne particles after a single charging event. Using an acoustic levitator, we measured the charge on particles of different material compositions suspended in atmospheric conditions for long periods of time. In dry environments, the total charge on particles decayed in over 1 week. The decay timescale decreased to days in humid environments. These results were independent of particle material and charge polarity. However, exposure to UV radiation could both increase and decrease the decay time depending on polarity. Our work suggests that the rate of charge decay on airborne particles is solely determined by ion capture from the air. Furthermore, using a one-dimensional sedimentation model, we predict that atmospheric dust of order 10 µm will experience the largest change in residence time due to electrostatic forces.

Increase of the transmission and emittance acceptance through a cyclotron-based proton therapy gantry.

PURPOSE: In proton therapy, the gantry, as the final part of the beamline, has a major effect on beam intensity and beam size at the isocenter. Most of the conventional beam optics of cyclotron-based proton gantries have been designed with an imaging factor between 1 and 2 from the coupling point (CP) at the gantry entrance to the isocenter (patient location) meaning that to achieve a clinically desirable (small) beam size at isocenter, a small beam size is also required at the CP. Here we will show that such imaging factors are limiting the emittance which can be transported through the gantry. We, therefore, propose the use of large beam size and low divergence beam at the CP along with an imaging factor of 0.5 (2:1) in a new design of gantry beam optics to achieve substantial improvements in transmission and thus increase beam intensity at the isocenter. METHODS: The beam optics of our gantry have been re-designed to transport higher emittance without the need of any mechanical modifications to the gantry beamline. The beam optics has been designed using TRANSPORT, with the resulting transmissions being calculated using Monte Carlo simulations (BDSIM code). Finally, the new beam optics have been tested with measurements performed on our Gantry 2 at PSI. RESULTS: With the new beam optics, we could maximize transmission through the gantry for a fixed emittance value. Additionally, we could transport almost four times higher emittance through the gantry compared to conventional optics, whilst achieving good transmissions through the gantry (>50%) with no increased losses in the gantry. As such, the overall transmission (cyclotron to isocenter) can be increased by almost a factor of 6 for low energies. Additionally, the point-to-point imaging inherent to the optics allows adjustment of the beam size at the isocenter by simply changing the beam size at the CP. CONCLUSION: We have developed a new gantry beam optics which, by selecting a large beam size and low divergence at the gantry entrance and using an imaging factor of 0.5 (2:1), increases the emittance acceptance of the gantry, leading to a substantial increase in beam intensity at low energies. We expect that this approach could easily be adapted for most types of existing gantries.

Beam properties within the momentum acceptance of a clinical gantry beamline for proton therapy.

PURPOSE: Energy changes in pencil beam scanning proton therapy can be a limiting factor in delivery time, hence, limiting patient throughput and the effectiveness of motion mitigation techniques requiring fast irradiation. In this study, we investigate the feasibility of performing fast and continuous energy modulation within the momentum acceptance of a clinical beamline for proton therapy. METHODS: The alternative use of a local beam degrader at the gantry coupling point has been compared with a more common upstream regulation. Focusing on clinically relevant parameters, a complete beam properties characterization has been carried out. In particular, the acquired empirical data allowed to model and parametrize the errors in range and beam current to deliver clinical treatment plans. RESULTS: For both options, the local and upstream degrader, depth-dose curves measured in water for off-momentum beams were only marginally distorted (γ(1%, 1 mm) > 90%) and the errors in the spot position were within the clinical tolerance, even though increasing at the boundaries of the investigated scan range. The impact on the beam size was limited for the upstream degrader, while dedicated strategies could be required to tackle the beam broadening through the local degrader. Range correction models were investigated for the upstream regulation. The impaired beam transport required a dedicated strategy for fine range control and compensation of beam intensity losses. Our current parameterization based on empirical data allowed energy modulation within acceptance with range errors (median 0.05 mm) and transmission (median -14%) compatible with clinical operation and remarkably low average 27 ms dead time for small energy changes. The technique, tested for the delivery of a skull glioma treatment, resulted in high gamma pass rates at 1%, 1 mm compared to conventional deliveries in experimental measurements with about 45% reduction of the energy switching time when regulation could be performed within acceptance. CONCLUSIONS: Fast energy modulation within beamline acceptance has potential for clinical applications and, when realized with an upstream degrader, does not require modification in the beamline hardware, therefore, being potentially applicable in any running facility. Centers with slow energy switching time can particularly profit from such a technique for reducing dead time during treatment delivery.

Catalytic activity imperative for nanoparticle dose enhancement in photon and proton therapy.

Nanoparticle-based radioenhancement is a promising strategy for extending the therapeutic ratio of radiotherapy. While (pre)clinical results are encouraging, sound mechanistic understanding of nanoparticle radioenhancement, especially the effects of nanomaterial selection and irradiation conditions, has yet to be achieved. Here, we investigate the radioenhancement mechanisms of selected metal oxide nanomaterials (including SiO2, TiO2, WO3 and HfO2), TiN and Au nanoparticles for radiotherapy utilizing photons (150 kVp and 6 MV) and 100 MeV protons. While Au nanoparticles show outstanding radioenhancement properties in kV irradiation settings, where the photoelectric effect is dominant, these properties are attenuated to baseline levels for clinically more relevant irradiation with MV photons and protons. In contrast, HfO2 nanoparticles retain some of their radioenhancement properties in MV photon and proton therapies. Interestingly, TiO2 nanoparticles, which have a comparatively low effective atomic number, show significant radioenhancement efficacies in all three irradiation settings, which can be attributed to the strong radiocatalytic activity of TiO2, leading to the formation of hydroxyl radicals, and nuclear interactions with protons. Taken together, our data enable the extraction of general design criteria for nanoparticle radioenhancers for different treatment modalities, paving the way to performance-optimized nanotherapeutics for precision radiotherapy.

Comparing radiolytic production of H2O2 and development of Zebrafish embryos after ultra high dose rate exposure with electron and transmission proton beams.

The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos.

Preliminary investigations for the option to use fast uniform scanning with compensators on a gantry designed for IMPT.

PURPOSE: In this experimental study, the authors explored the possibility to deliver the dose for proton therapy with fast uniform scanning on a gantry primarily designed for the delivery of conformal beam scanning and IMPT. The uniform scanning submode has been realized without equipment modifications by using the same small pencil beam used for conformal scanning, resulting in reduced realization costs. Uniform scanning has recently been adopted in a few proton therapy centers, as a basic beam delivery solution, and as an alternative to the use of scattering foils. The option to use such a mode to mimic scattering on a full-fledged scanning gantry could be of interest for treating some specific indications and as a possible solution for treating moving targets. METHODS: Uniform iso-energy dose layers were painted by fast magnetic scanning alternated with fast energy changes with the gantry beam line. The layers were stacked and repainted appropriately to produce homogeneous three-dimensional dose distributions. A collimator∕compensator was used to adjust the dose to coincide laterally∕distally with the target volume. In addition, they applied volumetric repainting, since they are confident that this will further mitigate the effects of organ motion as compared with the presently used clinical scanning solutions. With the approach presented in this paper, they can profit from the higher flexibility of the scanning system to obtain additional advantages. For instance the shape of the energy layers can be adjusted to the projected target shape in order to reduce treatment time and neutrons produced in the collimator. The shape of the proximal layers can be shrunk, according to the cross section of the target at the corresponding range. This provides variable range modulation (proximal conformity) while standard scattering only provides fixed range modulation with unnecessary 100% dose proximal to the target. The field-specific hardware for a spherical target volume was mounted on the Gantry 2 nozzle. One field with proximal field size shrinking and one without, each of 1 Gy, were delivered. The dose distributions at different depths were recorded as CCD images of a scintillating screen. RESULTS: The time to scan the volume once was about 4 s and the total delivery time was approximately 30 s. For the field with proximal conformity, dose sparing of up to 25% was measured in the region proximal to the target. A repainting capability of 48 times was achieved on the most distal layer. The proximal layers were repainted more due to the contribution of the plateau dose from the deeper layers. CONCLUSIONS: The flexibility of a fast scanning gantry with very fast energy changes can easily provide beam delivery by uniform layer stacking with a significant degree of volumetric repainting and with the benefit of a dose reduction proximal to the target volume.

A new emittance selection system to maximize beam transmission for low-energy beams in cyclotron-based proton therapy facilities with gantry.

PURPOSE: In proton therapy, the potential of using high-dose rates in the cancer treatment is being explored. High-dose rates could improve efficiency and throughput in standard clinical practice, allow efficient utilization of motion mitigation techniques for moving targets, and potentially enhance normal tissue sparing due to the so-called FLASH effect. However, high-dose rates are difficult to reach when lower energy beams are applied in cyclotron-based proton therapy facilities, because they result in large beam sizes and divergences downstream of the degrader, incurring large losses from the cyclotron to the patient position (isocenter). In current facilities, the emittance after the degrader is reduced using circular collimators; however, this does not provide an optimal matching to the acceptance of the following beamline, causing a low transmission for these energies. We, therefore, propose to use a collimation system, asymmetric in both beam size and divergence, resulting in symmetric emittance in both beam transverse planes as required for a gantry system. This new emittance selection, together with a new optics design for the following beamline and gantry, allows a better matching to the beamline acceptance and an improvement of the transmission. METHODS: We implemented a custom method to design the collimator sizes and shape required to select high emittance, to be transported by the following beamline using new beam optics (designed with TRANSPORT) to maximize acceptance matching. For predicting the transmission in the new configuration (new collimators + optics), we used Monte Carlo simulations implemented in BDSIM, implementing a model of PSI Gantry 2 which we benchmarked against measurements taken in the current clinical scenario (circular collimators + clinical optics). RESULTS: From the BDSIM simulations, we found that the new collimator system and matching beam optics results in an overall transmission from the cyclotron to the isocenter for a 70 MeV beam of 0.72%. This is an improvement of almost a factor of 6 over the current clinical performance (0.13% transmission). The new optics satisfies clinical beam requirements at the isocenter. CONCLUSIONS: We developed a new emittance collimation system for PSI's PROSCAN beamline which, by carefully selecting beam size and divergence asymmetrically, increases the beam transmission for low-energy beams in current state-of-the-art cyclotron-based proton therapy gantries. With these improvements, we could predict almost 1% transmission for low-energy beams at PSI's Gantry 2. Such a system could easily be implemented in facilities interested in increasing dose rates for efficient motion mitigation and FLASH experiments alike.

Commissioning of a clinical pencil beam scanning proton therapy unit for ultra-high dose rates (FLASH).

PURPOSE: The purpose of this work was to provide a flexible platform for FLASH research with protons by adapting a former clinical pencil beam scanning gantry to irradiations with ultra-high dose rates. METHODS: PSI Gantry 1 treated patients until December 2018. We optimized the beamline parameters to transport the 250 MeV beam extracted from the PSI COMET accelerator to the treatment room, maximizing the transmission of beam intensity to the sample. We characterized a dose monitor on the gantry to ensure good control of the dose, delivered in spot-scanning mode. We characterized the beam for different dose rates and field sizes for transmission irradiations. We explored scanning possibilities in order to enable conformal irradiations or transmission irradiations of large targets (with transverse scanning). RESULTS: We achieved a transmission of 86% from the cyclotron to the treatment room. We reached a peak dose rate of 9000 Gy/s at 3 mm water equivalent depth, along the central axis of a single pencil beam. Field sizes of up to 5 × 5 mm2 were achieved for single-spot FLASH irradiations. Fast transverse scanning allowed to cover a field of 16 × 1.2 cm2 . With the use of a nozzle-mounted range shifter, we are able to span depths in water ranging from 19.6 to 37.9 cm. Various dose levels were delivered with precision within less than 1%. CONCLUSIONS: We have realized a proton FLASH irradiation setup able to investigate continuously a wide dose rate spectrum, from 1 to 9000 Gy/s in single-spot irradiation as well as in the pencil beam scanning mode. As such, we have developed a versatile test bench for FLASH research.

Al2O3:C optically stimulated luminescence dosimeters (OSLDs) for ultra-high dose rate proton dosimetry.

The response of Al2O3:C optically stimulated luminescence detectors (OSLDs) was investigated in a 250 MeV pencil proton beam. The OSLD response was mapped for a wide range of average dose rates up to 9000 Gy s-1, corresponding to a ∼150 kGy s-1instantaneous dose rate in each pulse. Two setups for ultra-high dose rate (FLASH) experiments are presented, which enable OSLDs or biological samples to be irradiated in either water-filled vials or cylinders. The OSLDs were found to be dose rate independent for all dose rates, with an average deviation <1% relative to the nominal dose for average dose rates of (1-1000) Gy s-1when irradiated in the two setups. A third setup for irradiations in a 9000 Gy s-1pencil beam is presented, where OSLDs are distributed in a 3 × 4 grid. Calculations of the signal averaging of the beam over the OSLDs were in agreement with the measured response at 9000 Gy s-1. Furthermore, a new method was presented to extract the beam spot size of narrow pencil beams, which is in agreement within a standard deviation with results derived from radiochromic films. The Al2O3:C OSLDs were found applicable to support radiobiological experiments in proton beams at ultra-high dose rates.

Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.