Predicting Survival with Brain Metastases in the Stereotactic Radiosurgery Era: are Existing Prognostic Scores Still Relevant? Or Can we do Better?

Predicting survival is essential to tailoring treatment for patients diagnosed with brain metastases. We have evaluated the performance of widely used, validated prognostic scoring systems (Graded Prognostic Assessment and diagnosis-specific Graded Prognostic Assessment) in over 1000 'real-world' patients treated with stereotactic radiosurgery to the brain, selected according to National Health Service commissioning criteria. Survival outcomes from our dataset were consistent with those predicted by the prognostic systems, but with certain cancer subtypes showing a significantly better survival than predicted. Although performance status remains the simplest tool for prediction, total brain tumour volume emerges as an independent prognostic factor, and a new, improved, prognostic scoring system incorporating this has been developed.

Clinical Implementation of Robust Multi-isocentric Volumetric Modulated Arc Radiotherapy for Craniospinal Irradiation.

AIMS: To determine if multi-isocentric volumetric modulated arc radiotherapy for craniospinal irradiation (CSI-VMAT) can be implemented safely and accurately using robust optimisation in a commercially available treatment planning system. Our initial clinical experience is reported for the first 20 patients treated with the technique. MATERIALS AND METHODS: Patients received between 23.4 and 39.6 Gy (mode 23.4 Gy) in 13-22 fractions with CSI-VMAT. The heart mean dose was 4.2-10.3 Gy (median 5.3 Gy) for patients prescribed up to 24 Gy and 6.5-16.3 Gy (median 10.1 Gy) for patients receiving 35 Gy or more. The lung mean dose was 5.5-7.6 Gy (median 6.8 Gy) for patients prescribed up to 24 Gy and 6.9-11.1 Gy (median 10.0 Gy) for patients receiving 35 Gy or more. The robustness of the planning target volume D0.1cm3 and D99% to systematic errors in the isocentre superoinferior position of up to 5 mm was evaluated. These remained acceptable but were correlated to the length of the available beam overlap through the neck. RESULTS: As of January 2021, one patient was deceased after 508 days and one patient was lost to follow-up after completing treatment. The median follow-up was 399 days (range 175-756 days) and progression-free survival was 131 days (34-490 days). Acute toxicities at Common Terminology Criteria for Adverse Events v5.0 grade 3+ included lowered white blood cell count (16/20), decreased platelet count (8/20), nausea (5/20), vomiting (2/20), pharyngeal mucositis (1/20) and oral mucositis (1/20). Three patients developed grade 4 neutropenia or decreased white blood cell count. CONCLUSIONS: CSI-VMAT can be implemented safely and accurately using robust optimisation functions in a commercially available treatment planning system.

Normal Tissue Complication Probability (NTCP) Modelling of Severe Acute Mucositis using a Novel Oral Mucosal Surface Organ at Risk.

AIMS: A normal tissue complication probability (NTCP) model of severe acute mucositis would be highly useful to guide clinical decision making and inform radiotherapy planning. We aimed to improve upon our previous model by using a novel oral mucosal surface organ at risk (OAR) in place of an oral cavity OAR. MATERIALS AND METHODS: Predictive models of severe acute mucositis were generated using radiotherapy dose to the oral cavity OAR or mucosal surface OAR and clinical data. Penalised logistic regression and random forest classification (RFC) models were generated for both OARs and compared. Internal validation was carried out with 100-iteration stratified shuffle split cross-validation, using multiple metrics to assess different aspects of model performance. Associations between treatment covariates and severe mucositis were explored using RFC feature importance. RESULTS: Penalised logistic regression and RFC models using the oral cavity OAR performed at least as well as the models using mucosal surface OAR. Associations between dose metrics and severe mucositis were similar between the mucosal surface and oral cavity models. The volumes of oral cavity or mucosal surface receiving intermediate and high doses were most strongly associated with severe mucositis. CONCLUSIONS: The simpler oral cavity OAR should be preferred over the mucosal surface OAR for NTCP modelling of severe mucositis. We recommend minimising the volume of mucosa receiving intermediate and high doses, where possible.

Neurocognitive function after (chemo)-radiotherapy for head and neck cancer.

Radical radiotherapy has a pivotal role in the treatment of head and neck cancer (HNC) and cures a significant proportion of patients while simultaneously sparing critical normal organs. Some patients treated with radical radiotherapy for HNC receive significant radiation doses to large volumes of brain tissue. In fact, intensity-modulated radiotherapy techniques for HNC have been associated with a net increase in irradiated brain volumes. The increasing use of chemoradiotherapy for HNC has additionally exposed this patient population to potential neurotoxicity due to cytotoxic drugs. Patients with HNC may be particularly at risk for adverse late brain effects after (chemo)-radiotherapy, such as impaired neurocognitive function (NCF), as risk factors for the development of HNC, such as smoking, excess alcohol consumption and poor diet, are also associated with impaired NCF. The relatively good survival rates with modern treatment for HNC, and exposure to multiple potentially neurotoxic factors, means that it is important to understand the impact of (chemo)-radiotherapy for HNC on NCF, and to consider what measures can be taken to minimise treatment-related neurotoxicity. Here, we review evidence relating to the late neurotoxicity of radical (chemo)-radiotherapy for HNC, with a focus on studies of NCF in this patient population.

Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X-ray fibre diffraction and solid-state NMR data supports specific models of phage assembly at the bacterial membrane.

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of virion assembly.

The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus.

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.

The molecular structure and structural transition of the alpha-helical capsid in filamentous bacteriophage Pf1.

The major coat protein in the capsid of Pf1 filamentous bacteriophage (Inovirus) forms a helical assembly of about 7000 identical protein subunits, each of which contains 46 amino-acid residues and can be closely approximated by a single gently curved alpha-helix. Since the viral DNA occupies the core of the tubular capsid and appears to make no significant specific interactions with the capsid proteins, the capsid is a simple model system for the study of the static and dynamic properties of alpha-helix assembly. The capsid undergoes a reversible temperature-induced structural transition at about 283 K between two slightly different helix forms. The two forms can coexist without an intermediate state, consistent with a first-order structural phase transition. The molecular model of the higher temperature form was refined using improved X-ray fibre diffraction data and new refinement and validation methods. The refinement indicates that the two forms are related by a change in the orientation of the capsid subunits within the virion, without a significant change in local conformation of the subunits. On the higher temperature diffraction pattern there is a region of observed intensity that is not consistent with a simple helix of identical subunits; it is proposed that the structure involves groups of three subunits which each have a slightly different orientation within the group. The grouping of subunits suggests that a change in subunit libration frequency could be the basis of the Pf1 structural transition; calculations from the model are used to explore this idea.

Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution.

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered.

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Matching electrostatic charge between DNA and coat protein in filamentous bacteriophage. Fibre diffraction of charge-deletion mutants.

The virion of Ff (fd, f1, M13) filamentous bacteriophage consists of a long tube of coat protein subunits in a shingled, helical array, surrounding a genome of circular single-stranded DNA. Modified fd virions have been generated by a mutation (K48A) that removes one positive charge from each coat protein subunit in the C-terminal region of the polypeptide chain facing the DNA. The number of nucleotides in the mutant DNA is unchanged, but the K48A virions are 35% longer than wild-type. We have measured the X-ray diffraction attributable to single virions in hydrated gels of wild-type and K48A bacteriophages. Most of the diffraction pattern shows no significant difference between wild-type and K48A. Since the DNA is only about 12% by weight of the wild-type virion, the diffraction pattern is dominated by the protein contribution, and the absence of significant differences indicates that there are no significant changes in the symmetry or structure of the protein coat. But there is a change in the diffraction pattern in a region where the DNA and protein contributions are comparable. The diffraction pattern of the K48A mutant shows an increase in intensity of one of the weaker equatorial peaks, relative to wild-type, in a region where the protein contribution has negative sign but the DNA contribution has positive sign. This is consistent with a decrease in the ratio of DNA:protein per unit length of the K48A mutant. The results support the view that the protein forms a sheath lined with positive charges interacting electrostatically and non-specifically with a negatively charged DNA core of matching charge density. The lower positive charge density lining the capsid in the K48A mutant means that correspondingly fewer nucleotides can be packaged per coat protein subunit, which in turn requires an elongation of the DNA inside the virion. A longer virion is thus required to package the same amount of DNA. Within the error of measurement, the number of positive charges on the protein interacting with the DNA is the same in K48A as in the wild-type, despite the fact that the mutant is 35% longer than the wild-type.