Predicting personalised and progressive adaptive dose escalation to gross tumour volume using knowledge-based planning models for inoperable advanced-stage non-small cell lung cancer patients treated with volumetric modulated arc therapy.

Objectives. Increased radiation doses could improve local control and overall survival of lung cancer patients, however, this could be challenging without exceeding organs at risk (OAR) dose constraints, especially for patients with advanced-stage disease. Increasing OAR doses could reduce the therapeutic ratio and quality of life. It is therefore important to investigate methods to increase the dose to target volume without exceeding OAR dose constraints.Methods. Gross tumour volume (GTV) was contoured on synthetic computerised tomography (sCT) datasets produced using the Velocity adaptive radiotherapy software for eleven patients. The fractions where GTV volume decreased compared to that prior to radiotherapy (reference plan) were considered for personalised progressive dose escalation. The dose to the adapted GTV (GTVAdaptive) was increased until OAR doses were affected (as compared to the original clinical plan). Planning target volume (PTV) coverage was maintained for all plans. Doses were also escalated to the reference plan (GTVClinical) using the same method. Adapted, dose-escalated, plans were combined to estimate accumulated dose, D99(dose to 99%) of GTVAdapted, PTV D99and OAR doses and compared with those in the original clinical plans. Knowledge-based planning (KBP) model was developed to predict D99of the adapted GTV with OAR doses and PTV coverage kept similar to the original clinical plans; prediction accuracy and model verification were performed using further data sets.Results. Compared to the original clinical plan, the dose to GTV was significantly increased without exceeding OAR doses. Adaptive dose-escalation increased the average D99to GTVAdaptiveby 15.1Gy and 8.7Gy compared to the clinical plans. The KBP models were verified and demonstrated prediction accuracy of 0.4% and 0.7% respectively.Conclusion. Progressive adaptive dose escalation can significantly increase the dose to GTV without increasing OAR doses or compromising the dose to microscopic disease. This may increase overall survival without increasing toxicities.

Predicting personalised optimal arc parameter using knowledge-based planning model for inoperable locally advanced lung cancer patients to reduce organ at risk doses.

Objectives. Volumetric modulated arc therapy (VMAT) allows for reduction of organs at risk (OAR) volumes receiving higher doses, but increases OAR volumes receiving lower radiation doses and can subsequently increasing associated toxicity. Therefore, reduction of this low-dose-bath is crucial. This study investigates personalizing the optimization of VMAT arc parameters (gantry start and stop angles) to decrease OAR doses.Materials and Methods. Twenty previously treated locally advanced non-small cell lung cancer (NSCLC) patients treated with half-arcs were randomly selected from our database. These plans were re-optimized with seven different arcs parameters; optimization objectives were kept constant for all plans. All resulting plans were reviewed by two clinicians and the optimal plan (lowest OAR doses and adequate target coverage) was selected. Furthermore, knowledge-based planning (KBP) model was developed using these plans as 'training data' to predict optimal arc parameters for individual patients based on their anatomy. Treatment plan complexity scores and deliverability measurements were performed for both optimal and original clinical plans.Results.The results show that different arc geometries resulted in different dose distributions to the OAR but target coverage was mostly similar. Different arc geometries were required for different patients to minimize OAR doses. Comparison of the personalized against the standard (2 half-arcs) plans showed a significant reduction in lung V5(lung volume receiving 5 Gy), mean lung dose and mean heart doses. Reduction in lung V20and heart V30were statistically insignificant. Plan complexity and deliverability measurements show the test plans can be delivered as planned.Conclusions.Our study demonstrated that personalizing arc parameters based on an individual patient's anatomy significantly reduces both lung and heart doses. Dose reduction is expected to reduce toxicity and improve the quality of life for these patients.

Validation of in-house knowledge-based planning model for predicting change in target coverage during VMAT radiotherapy to in-operable advanced-stage NSCLC patients.

Objectives. anatomical changes are inevitable during the course of radiotherapy treatments and, if significant, can severely alter expected dose distributions and affect treatment outcome. Adaptive radiotherapy (ART) is employed to maintain the planned distribution and minimise detriment to predicted treatment outcome. Typically, patients who may benefit from adaptive planning are identified via a re-planning process, i.e., re-simulation, re-contouring, re-planning and treatment plan quality assurance (QA). This time-intensive process significantly increases workload, can introduce delays and increases unnecessary stress to those patients who will not actually gain benefit. We consider it crucial to develop efficient models to predict changes to target coverage and trigger ART, without the need for re-planning.Methods.knowledge-based planning (KBP) models were developed using data for 20 patients' (400 fractions) to predict changes in PTV V95coverageΔV95PTV.Initially, this change in coverage was calculated on the synthetic computerised tomography (sCT) images produced using the Velocity adaptive radiotherapy software. Models were developed using patient (cell death bio-marker) and treatment fraction (PTV characteristic) specific parameters to predictΔV95PTVand verified using five patients (100 fractions) data.Results. three models were developed using combinations of patient and fraction specific terms. The prediction accuracy of the model developed using biomarker (PD-L1 expression) and the difference in 'planning' and 'fraction' PTV centre of the mass (characterised by mean square difference, MSD) had the higher prediction accuracy, predicting theΔV95PTVwithin ± 1.0% for 77% of the total fractions; with 59% for the model developed using, PTV size, PD-L1 and MSD and 48% PTV size and MSD respectively.Conclusion. the KBP models can predictΔV95PTVvery effectively and efficiently for advanced-stage NSCLC patients treated using volumetric modulated arc therapy and to identify patients who may benefit from adaption for a specific fraction.

Development and characterization of a eukaryotic expression system for human type II procollagen.

BACKGROUND: Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. RESULTS: Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. CONCLUSIONS: Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

BACKGROUND: The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. RESULTS: We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli ß-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli ß-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. CONCLUSIONS: The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis.

BACKGROUND: The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. RESULTS: Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli ß-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and ß-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. CONCLUSIONS: We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals.