TU-E-BRB-02: A Decision Support Tool for SBRT Planning Using a Searchable DVH Database.

PURPOSE: Develop a decision support tool that aids dosimetrists, physicians, and physicists in assessing and improving plan quality through comparison to plans previously used in similar clinical situations. METHODS: Software was developed to capture and store DVHs and other clinically relevant treatment plan characteristics in a database. In addition to the plan DVH, the database contains a total of 24 plan characteristics including fractionation, prescribed dose, treatment volume, prior surgery, tumor position, and smoking history. DVH and other plan data was captured from the treatment planning system via exported dicom RT files. Structures in the plan were automatically matched by name to a list of standard structures using a system of regular expressions. Additional fields were entered manually using a simple java interface. As a support tool, a plan under development can be quickly compared to similar plans in the database based on selected plan characteristics. A plot displaying the current and historical DVHs provides an easy visual comparison. Our interface also provides statistics for comparison for each dose/volume level such as average, minimum, maximum and standard deviation. RESULTS: DVHs from 111 lung SBRT plans treated from 2009-2011 were imported in accordance with an approved IRB protocol. As an example of data comparisons that can be easily performed to guide plan evaluation, we examined plans prescribing 5400cGy in 3 fractions and found that tumors >7.5cc (n=34) had an average PTV coverage of 94.2% (range: 73.5-95.0%), and tumors =7.5cc (n=35) had an average PTV coverage of 94.9% (range: 81.6-99.6%). CONCLUSION: A searchable DVH database was constructed to provide planners, physicists, and physicians with a straightforward means of comparing plans against historic distributions of DVHs. In the future, outcome data will be included in the database to strengthen its functionality as a decision support and research tool.

Src homology domain-containing phosphatase 2 suppresses cellular senescence in glioblastoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) signalling is frequently altered during glioblastoma de novo pathogenesis. An important downstream modulator of this signal cascade is SHP2 (Src homology domain-containing phosphatase 2). METHODS: We examined the The Cancer Genome Atlas (TCGA) database for SHP2 mutations. We also examined the expression of a further 191 phosphatases in the TCGA database and used principal component and comparative marker analysis available from the Broad Institute to recapitulate the TCGA-defined subgroups and identify the specific phosphatases defining each subgroup. We identified five siRNAs from two independent commercial sources that were reported by the vendor to be pre-optimised in their specificity of SHP2 silencing. The specificity and physiological effects of these siRNAs were tested using an in vitro glioma model. RESULTS: TCGA data demonstrate SHP2 to be mutated in 2% of the glioblastoma multiforme's studied. Both mutations identified in this study are likely to be activating mutations. We found that the four subgroups of GBM as defined by TCGA differ significantly with regard to the expression level of specific phosphatases as revealed by comparative marker analysis. Surprisingly, the four subgroups can be defined solely on the basis of phosphatase expression level by principal component analysis. This result suggests that critical phosphatases are responsible for the modulation of specific molecular pathways within each subgroup. Src homology domain-containing phosphatase 2 constitutes one of the 12 phosphatases that define the classical subgroup. We confirmed the biological significance by siRNA knockdown of SHP2. All five siRNAs tested reduced SHP2 expression by 70-100% and reduced glioblastoma cell line growth by up to 80%. Profiling the established molecular targets of SHP2 (ERK1/2 and STAT3) confirmed specificity of these siRNAs. The loss of cell viability induced by SHP2 silencing could not be explained by a significant increase in apoptosis alone as demonstrated by terminal deoxyribonucleotidyl transferase-mediated nick-end labelling and propidium iodide staining. Src homology domain-containing phosphatase 2 silencing, however, did induce an increase in ß-galactosidase staining. Propidium iodide staining also showed that SHP2 silencing increases the population of glioblastoma cells in the G1 phase of the cell cycle and reduces the population of such cells in the G2/M- and S-phase. CONCLUSION: Src homology domain-containing phosphatase 2 promotes the growth of glioblastoma cells by suppression of cellular senescence, a phenomenon not described previously. Selective inhibitors of SHP2 are commercially available and may be considered as a strategy for glioblastoma therapy.

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

Functional reconstitution and characterization of AqpZ, the E. coli water channel protein.

Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.