Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test

Testing the portal imager GLAaS algorithm for machine quality assurance

<p>Abstract</p> <p>Background</p> <p>To report about enhancements introduced in the GLAaS calibration method to convert raw portal imaging images into absolute dose matrices and to report about application of GLAaS to routine radiation tests for linac quality assurance procedures programmes.</p> <p>Methods</p> <p>Two characteristic effects limiting the general applicability of portal imaging based dosimetry are the over-flattening of images (eliminating the "horns" and "holes" in the beam profiles induced by the presence of flattening filters) and the excess of backscattered radiation originated by the detector robotic arm supports. These two effects were corrected for in the new version of GLAaS formalism and results are presented to prove the improvements for different beams, detectors and support arms. GLAaS was also tested for independence from dose rate (fundamental to measure dynamic wedges).</p> <p>With the new corrections, it is possible to use GLAaS to perform standard tasks of linac quality assurance. Data were acquired to analyse open and wedged fields (mechanical and dynamic) in terms of output factors, MU/Gy, wedge factors, profile penumbrae, symmetry and homogeneity. In addition also 2D Gamma Evaluation was applied to measurement to expand the standard QA methods. GLAaS based data were compared against calculations on the treatment planning system (the Varian Eclipse) and against ion chamber measurements as consolidated benchmark. Measurements were performed mostly on 6 MV beams from Varian linacs. Detectors were the PV-as500/IAS2 and the PV-as1000/IAS3 equipped with either the robotic R- or Exact- arms.</p> <p>Results</p> <p>Corrections for flattening filter and arm backscattering were successfully tested. Percentage difference between PV-GLAaS measurements and Eclipse calculations relative doses at the 80% of the field size, for square and rectangular fields larger than 5 × 5 cm²showed a maximum range variation of -1.4%, + 1.7% with a mean variation of <0.5%. For output factors, average percentage difference between GLAaS and Eclipse (or ion chamber) data was -0.4 ± 0.7 (-0.2 ± 0.4) respectively on square fields. Minimum, maximum and average percentage difference between GLAaS and Eclipse (or ion chamber) data in the flattened field region were: 0.1 ± 1.0, 0.7 ± 0.8, 0.1 ± 0.4 (1.0 ± 1.4, -0.3 ± 0.2, -0.1 ± 0.2) respectively. Similar minimal deviations were observed for flatness and symmetry.</p> <p>For Dynamic wedges, percentage difference of MU/Gy between GLAaS and Eclipse (or ion chamber) was: -1.1 ± 1.6 (0.4 ± 0.7). Minimum, maximum and average percentage difference between GLAaS and Eclipse (or ion chamber) data in the flattened field region were: 0.4 ± 1.6, -1.5 ± 1.8, -0.1 ± 0.3 (-2.2 ± 2.3, 2.3 ± 1.2, 0.8 ± 0.3) respectively.</p> <p>For mechanical wedges differences of transmission factors were <1.6% (Eclipse) and <1.1% (ion chamber) for all wedges. Minimum, maximum and average percentage difference between GLAaS and Eclipse (or ion chamber) data in the flattened field region were: -1.3 ± 0.7, -0.7 ± 0.7, -0.2 ± 0.2 (-0.8 ± 0.8, 0.7 ± 1.1, 0.2 ± 0.3) respectively.</p> <p>Conclusion</p> <p>GLAaS includes now efficient methods to correct for missing "horns" and "holes" induced by flattening filter in the beam and to compensate for excessive backscattering from the support arm. These enhancements allowed to use GLAaS based dosimetric measurement to perform standard tasks of Linac quality assurance with reliable and consistent results. This fast method could be applied to routine practice being also fast in usage and because it allows the introduction of new analysis tools in routine QA by means, e.g., of the Gamma Index analysis.</p

Early clinical experience of radiotherapy of prostate cancer with volumetric modulated arc therapy

<p>Abstract</p> <p>Background</p> <p>To report about initial clinical experience in radiation treatment of carcinoma of prostate with volumetric modulated arcs with the RapidArc (RA) technology.</p> <p>Methods</p> <p>Forty-five patients with a median age of 72 ± 3, affected by prostate carcinoma (T1c: 22 patients, T2a-b: 17 patients, T3a-b: 6 patients. N0: 43 patients, N1-Nx: 2 patients, all M0), with initial PSA of 10.0 ± 3.0 ng/mL, were treated with RapidArc in a feasibility study. All patients were treated with single arc using 6MV photons. Dose prescription ranged between 76 (7 patients) and 78 Gy (38 patients) in 2Gy/fraction. Plan quality was assessed by means of Dose Volume Histogram (DVH) analysis. Technical parameters of arcs and pre-treatment quality assurance results (Gamma Agreement Index, GAI) are reported to describe delivery features. Early toxicity was scored (according to the Common Terminology Criteria of Adverse Effects scale, CTCAE, scale) at the end of treatment together with biochemical outcome (PSA).</p> <p>Results</p> <p>From DVH data, target coverage was fulfilling planning objectives: V_95%was in average higher than 98% and V_107%~0.0% (D_2%~104.0% in average). Homogeneity D_5%-D_95%ranged between 6.2 ± 1.0% to 6.7 ± 1.3%. For rectum, all planning objectives were largely met (e.g. V_70Gy= 10.7 ± 5.5% against an objective of < 25%) similarly for bladder (e.g. D_2%= 79.4 ± 1.2Gy against an objective of 80.0Gy). Maximum dose to femurs was D_2%= 36.7 ± 5.4Gy against an objective of 47Gy. Monitor Units resulted: MU/Gy = 239 ± 37. Average beam on time was 1.24 ± 0.0 minutes. Pre-treatment GAI resulted in 98.1 ± 1.1%. Clinical data were recorded as PSA at 6 weeks after RT, with median values of 0.4 ± 0.4 ng/mL. Concerning acute toxicity, no patient showed grade 2-3 rectal toxicity; 5/42 (12%) patients experienced grade 2 dysuria; 18/41 (44%) patients preserved complete or partial erectile function.</p> <p>Conclusion</p> <p>RapidArc proved to be a safe, qualitative and advantageous treatment modality for prostate cancer.</p

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin

FusionArc optimization: A hybrid volumetric modulated arc therapy (VMAT) and intensity modulated radiation therapy (IMRT) planning strategy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134837/1/mp8153.pd

Single fraction radiosurgery using Rapid Arc for treatment of intracranial targets

<p>Abstract</p> <p>Background</p> <p>Stereotactic-Radio-Surgery (SRS) using Conformal-Arc-Therapy (CAT) is a well established irradiation technique for treatment of intracranial targets. Although small safety margins are required because of very high accuracy of patient positioning and exact online localisation, there are still disadvantages like long treatment time, high number of monitor units (MU) and covering of noncircular targets. This planning study analysed whether Rapid Arc (RA) with stereotactic localisation for single-fraction SRS can solve these problems.</p> <p>Methods</p> <p>Ten consecutive patients were treated with Linac-based SRS. Eight patients had one or more brain metastases. The other patients presented a symptomatic vestibularis schwannoma and an atypic meningeoma. For all patients, two plans (CAT/RA) were calculated and analysed.</p> <p>Results</p> <p>Conformity was higher for RA with additional larger low-dose areas. Furthermore, RA reduced the number of MU and the treatment time for all patients. Dose to organs at risk were equal or slightly higher using RA in comparison to CAT.</p> <p>Conclusions</p> <p>RA provides a new alternative for single-fraction SRS irradiation combining advantages of short treatment time with lower number of MU and better conformity in addition to accuracy of stereotactic localisation in selected cases with uncomplicated clinical realization.</p

Evaluation of volumetric modulated arc therapy (VMAT) with Oncentra MasterPlan® for the treatment of head and neck cancer

Background Several comparison studies have shown the capability of VMAT to achieve similar or better plan quality as IMRT, while reducing the treatment time. The experience of VMAT in a multi vendor environment is limited. We compared the plan quality and performance of VMAT to IMRT and we investigate the effects of varying various user-selectable parameters. Methods IMRT, single arc VMAT and dual arc VMAT were compared for four different head-and-neck tumors. For VMAT, the effect of varying gantry angle spacing and treatment time on the plan quality was investigated. A comparison of monitor units and treatment time was performed. Results IMRT and dual arc VMAT achieved a similar plan quality, while single arc could not provide an acceptable plan quality. Increasing the number of control points does not improve the plan quality. Dual arc VMAT delivery time is about 30% of IMRT delivery time. Conclusions Dual arc VMAT is a fast and accurate technique for the treatment of head and neck cancer. It applies similar number of MUs as IMRT, but the treatment time is strongly reduced, maintaining similar or better dose conformity to the PTV and OAR sparing