Quantitative analysis of the cysteine redoxome by iodoacetyl tandem mass tags.

The redox conditions that reign inside a cell have a determining effect on a number of biological processes. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are key redox players and have been linked to a number of pathologies. They have also been shown to play an important regulating role in cell signaling events. On the proteome level, thiol groups of cysteinyl side chains constitute the major targets of ROS and RNS. A number of analytical techniques based on mass spectrometry have been developed to characterize the cysteine redoxome, often facing a number of technical challenges, mostly related to the lability, heterogeneity, and low abundance of the oxidized forms. Furthermore, any posttranslational modification (PTM) quantification method needs to take the parent protein's expression level into account. While taking all these limitations into consideration, we have developed a quantitative analytical strategy named OxiTMT, based on chemical labeling with iodoacetyl isobaric tandem mass tags (iodoTMT). OxiTMT allowed the generation of quantitative redox data that could be normalized by the protein's expression profile in up to three different conditions. The method was tested on Escherichia coli with or without an oxidative treatment. Results showed the method to be adequate for the analysis of cysteine PTMs with a good coverage of the cysteine redoxome, especially for the low abundant oxidized species. Some of the challenges that face reporter ion quantification of PTMs by mass spectrometry were also assessed. This study serves as a proof of concept of the established protocol and consequent data treatment step. The use of tandem mass tags opens the ways towards the application of the method to the study of tissues and sera. Graphical abstract OxiTMT workflow.

Activated phosphonated trifunctional chelates for highly sensitive lanthanide-based FRET immunoassays applied to total prostate specific antigen detection.

The first example of an activated phosphonated trifunctional chelate (TFC) is presented, which combines a non-macrocyclic coordination site for lanthanide coordination based on two aminobis-methylphosphonate coordinating arms, a central bispyrazolylpyridyl antenna and an N-hydroxysuccinimide ester in para position of the central pyridine as an activated function for the labeling of biomaterial. The synthesis of the TFC is presented together with photo-physical studies of the related Tb and Eu complexes. Excited state lifetime measurements in H2O and D2O confirmed an excellent shielding of the cation from water molecules with a hydration number of zero. The Tb complex provides a high photoluminescence (PL) quantum yield of 24% in aqueous solutions (0.01 M Tris-HCl, pH 7.4) and a very long luminescence lifetime of 2.6 ms. The activated ligand was conjugated to different biological compounds such as streptavidin, and a monoclonal antibody against total prostate specific antigen (TPSA). In combination with AlexaFluor647 (AF647) and crosslinked allophycocyanin (XL665) antibody (ABs) conjugates, homogeneous time-resolved Fluorescence Resonance Energy Transfer (FRET) immunoassays of TPSA were performed in serum samples. The Tb donor-dye acceptor FRET pairs provided large Förster distances of 5.3 nm (AF647) and 7.1 nm (XL665). A detailed time-resolved FRET analysis of Tb donor and dye acceptor PL decays revealed average donor-acceptor distances of 4.2 nm (AF647) and 6.3 nm (XL665) within the sandwich immunocomplex and FRET efficiencies of 0.79 and 0.68, respectively. Very low detection limits of 1.4 ng mL(-1) (43 pM) and 2.4 ng mL(-1) (74 pM) TPSA were determined using a KRYPTOR fluorescence immunoanalyzer. These results demonstrate the applicability of our novel Tb-bioconjugates for highly sensitive clinical diagnostics.

Setup margins and geometric uncertainties in intensity-modulated radiation therapy in treating pituitary adenomas: the experience of Lyon Sud Hospital.

PURPOSE: The authors retrospectively evaluated the setup uncertainties in Intensity-Modulated Radiation Therapy (IMRT) for pituitary adenomas and verified the margins used in daily practice (3 mm). MATERIALS AND METHODS: Craniocaudal (CC), anteroposterior (AP) and laterolateral (LL) displacements were measured during the first 3 days of treatment and then weekly by comparing two orthogonal images obtained by an electronic system of portal imaging with Digitally Reconstructed Radiographs (DRRs). Setup Margins (SM) were defined according to the International Commission on Radiation Units (ICRU)-62 formula, the Stroom equation and the van Herk equation. The systematic (Σ) and random (σ) errors of the population were calculated as standard deviation (SD) of the population mean and the mean of SDs for every patient, respectively. RESULTS: Twenty patients were treated by IMRT for pituitary adenomas, and a total of 231 measurements were obtained. Σ and σ were 0.6 and 1.3 mm, 0.8 and 1 mm, 1.2 and 1.5 mm in the AP, LL and CC direction, respectively. Larger setup margin was 2.4, 2.7 and 4 mm in the AP, LL and CC direction, respectively (van Herk formula). CONCLUSIONS: IMRT is a highly sophisticated treatment technique that requires precise definition and optimisation of local setup errors and, finally, of the irradiated volumes. The role of image-guided RT in these kinds of treatments should be prospectively evaluated.

Synthesis of an activated phosphonated bifunctional chelate with potential for PET imaging and radiotherapy.

The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity of L* towards amine functions was tested using ethylamine under different conditions of pH and concentrations, and by the labeling of two reference peptides containing both an N-terminal amino function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0, with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the labeled antibody towards its target was controlled by immunofluorescence staining experiments on human colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.

Does weight loss predict accuracy of setup in head and neck cancer patients treated with Intensity-Modulated Radiation Therapy?

PURPOSE: This prospective study reports the impact of weight loss on setup of head and neck (H&N) cancer patients treated by Intensity-Modulated Radiation Therapy (IMRT). MATERIALS AND METHODS: Setup errors of H&N cancer patients treated by IMRT from January to June 2010 were prospectively analysed and statistically related to weight loss. A mixed linear model was used for statistical evaluations. Setup margins of our institute were also calculated. RESULTS: Twenty-two patients and 128 pairs of Electronic Portal Images (EPI) were analysed. Setup errors varied between -0.6 and +0.6, -0.7 and +0.8 and -0.2 and +0.8 in the anterior-posterior, superior-inferior and right-left direction, respectively. Median and mean weight loss were 2.1 and 3.1 kg (range 0-12 kg), respectively; median and mean percent of weight loss were 2.95% and 4.64% (range 0.3-19.7%), respectively. No statistical relation was seen between weight loss and the setup errors. CONCLUSIONS: Weight loss is not a good clinical parameters for predicting an increase of setup errors. Other clinical and/or anthropometrical features should be prospectively evaluated in order to assess the need for re-planning.

Exceptional Response to Second-Line Gemcitabine/Nab-Paclitaxel Chemotherapy in Patients With Metastatic Pancreatic Adenocarcinoma.

Pancreatic cancer is an extremely lethal malignancy with the majority of patients presenting with advanced disease. Typically, fit patients with advanced unresectable disease are treated with chemotherapy, which comprises either first-line folfirinox (FNX) or gemcitabine/nab-paclitaxel (GNP) regimens based on level 1 evidence. To our knowledge, robust evidence for second-line GNP post FNX does not exist. We herein report four cases treated at our institute with second-line GNP. Amongst those were patients with durable responses lasting over a year, which is extremely rare in stage 4 pancreatic cancer.

Prognostic factors for progression in atypical meningioma.

In patients with postoperative residual atypical meningiomas, by using volumetric instead of linear measurements in follow-up imaging studies, the authors detected disease progression earlier. By using this approach, treatment for recurrent disease can be instituted promptly with potentially better tumor control and less toxicity due to smaller volume of residual disease.

Whole Abdominal-Pelvic Radiotherapy in the Management of Primary Ewing Sarcoma of the Peritoneal Cavity.

Ewing sarcoma of the abdomen is a rare entity in pediatric oncology and represents a technical challenge both for surgeons and radiation oncologists. We document the case of a young female patient with primary disseminated, intraperitoneal Ewing sarcoma who after an excellent response to chemotherapy received preoperative whole abdominal-pelvic radiotherapy with good tolerance.

Transrectal implantation and stability of gold markers in prostate bed for salvage radiotherapy of macroscopic recurrences.

BACKGROUND AND PURPOSE: The objective of the study was to verify the stability of gold markers in the prostatic bed (PB) during salvage radiotherapy. MATERIAL AND METHODS: Seven patients, diagnosed with a macroscopic nodule visible on MRI, underwent targeted MRI-guided biopsies. Three gold markers were implanted into the PB close to the relapsing nodule for CT/MRI fusion. A dose of 60Gy was delivered using IMRT to the PB followed by a dose escalation up to 72Gy to the macroscopic nodule. Daily anterior and left-lateral kV-images were acquired for repositioning. The coordinates of the center of each marker were measured on the two kV-images. The distance variations (Dvar) of the markers in the first session and the subsequent ones were compared. RESULTS: No marker was lost during treatment. The average distance between markers was 7.8mm. The average Dvar was 0.8mm, in absolute value. A total of 380/528 (72%) Dvar were ⩽1mm. A Dvar greater than 2mm was observed in 5.7% of measurements, with a maximum value of 4.8mm. CONCLUSIONS: Despite the absence of the prostate, the implantation of gold markers in the PB remains feasible, with Dvar often less than 2mm, and could be used to develop new approaches of salvage focal radiotherapy on the macroscopic relapse after prostatectomy.

[Should the treatment of anal carcinoma be adapted in the elderly? A retrospective analysis of acute toxicities in a French centre and a review of the literature]. / Faut-il adapter le traitement du carcinome du canal anal pour les patients âgés? Analyse rétrospective de la toxicité dans un centre français et revue de la littérature.

PURPOSE: To study the tolerance to radiotherapy, with or without chemotherapy, followed by brachytherapy, in elderly patients (75 years or older) suffering from anal cancer. PATIENTS AND METHODS: We treated 12 elderly patients with a curative intent. Median age was 78 years (range: 75-90). Ten patients had a stage II or IIIA (UICC 2009) tumour and six out of 12 were N+. Taking into account the age, the Eastern Cooperative Oncology Group (ECOG) performance status and comorbidities, five patients received exclusive radiotherapy ("RT group") and seven a concomitant radiochemotherapy ("RT-CT group"). All patients received a boost by interstitial brachytherapy. One patient of the "RT-CT group" presented rectorragies during brachytherapy. The irradiation was completed by external beam radiotherapy focalized on the tumour volume. RESULTS: Grade 3 acute reactions (Radiation Therapy Oncology Group [RTOG]) were reported in three out of 12 patients. One grade 2-3 leucopoenia was observed in one out of 7 patients ("RT-CT group"). After brachytherapy, one grade 3 rectal toxicity (rectorragia) (in "RT group") and one grade 4 (in "RT-CT group") were observed. One patient ("RT-CT group") presented a late grade 3 rectal toxicity (evaluated only for patients with at least 12 months of follow-up). CONCLUSIONS: Concomitant radiochemotherapy followed by brachytherapy showed an acceptable toxicity profile, and seems to be adapted in selected elderly patients. It could be recommended as reference treatment in elderly patients with a good physiological status.