Implantation of a biodegradable rectum balloon implant: Tips, Tricks and Pitfalls

<div><p>ABSTRACT Introduction: A rectum balloon implant (RBI) is a new device to spare rectal structures during prostate cancer radiotherapy. The theoretical advantages of a RBI are to reduce the high radiation dose to the anterior rectum wall, the possibility of a post-implant correction, and their predetermined shape with consequent predictable position. Objective: To describe, step-by-step, our mini-invasive technique for hands-free transperineal implantation of a RBI before start of radiotherapy treatment. Materials and Methods: We provide step-by-step instructions for optimization of the transperineal implantation procedure performed by urologists and/or radiation oncologists experienced with prostate brachytherapy and the use of the real-time bi-plane transrectal ultrasonography (TRUS) probe. A RBI was performed in 15 patients with localised prostate cancer. Perioperative side-effects were reported. Results: We provide ‘tips and tricks’ for optimizing the procedure and proper positioning of the RBI. Please watch the animation, see video in https://vimeo.com/205852376/789df4fae4. The side-effects included mild discomfort to slight pain at the perineal region in 8 out of 15 patients. Seven patients (47%) had no complaints at all. Two patients developed redness of the skin, where prompt antibiotic regimen was started with no further sequelae. One patient revealed a temporary urine retention, which resolved in a few hours following conservative treatment. Further no perioperative complications occurred. Conclusion: This paper describes in detail the implantation procedure for an RBI. It is a feasible, safe and very well-tolerated procedure.</p></div

Additional file 1 of Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Supplementary Material

Feature selection methodology for longitudinal cone-beam CT radiomics

<p><b>Background:</b> Cone-beam CT (CBCT) scans are typically acquired daily for positioning verification of non-small cell lung cancer (NSCLC) patients. Quantitative information, derived using radiomics, can potentially contribute to (early) treatment adaptation. The aims of this study were to (1) describe and investigate a methodology for feature selection of a longitudinal radiomics approach (2) investigate which time-point during treatment is potentially useful for early treatment response assessment.</p> <p><b>Material and methods:</b> For 90 NSCLC patients CBCT scans of the first two fractions of treatment (considered as ‘test-retest’ scans) were analyzed, as well as weekly CBCT images. One hundred and sixteen radiomic features were extracted from the GTV of all scans and subsequently absolute and relative differences were calculated between weekly CBCT images and the CBCT of the first fraction. Test-retest scans were used to determine the smallest detectable change (C = 1.96 * SD) allowing for feature selection by choosing a minimum number of patients for which a feature should change more than ‘C’ to be considered as relevant. Analysis of which features change at which moment during treatment was used to investigate which time-point is potentially relevant to extract longitudinal radiomics information for early treatment response assessment.</p> <p><b>Results:</b> A total of six absolute delta features changed for at least ten patients at week 2 of treatment and increased to 61 at week 3, 79 at week 4 and 85 at week 5. There was 93% overlap between features selected at week 3 and the other weeks.</p> <p><b>Conclusions:</b> This study describes a feature selection methodology for longitudinal radiomics that is able to select reproducible delta radiomics features that are informative due to their change during treatment, which can potentially be used for treatment decisions concerning adaptive radiotherapy. Nonetheless, the prognostic value of the selected delta radiomic features should be investigated in future studies.</p

Defining the hypoxic target volume based on positron emission tomography for image guided radiotherapy – the influence of the choice of the reference region and conversion function

<p><b>Background:</b> Hypoxia imaged by positron emission tomography (PET) is a potential target for optimization in radiotherapy. However, the implementation of this approach with respect to the conversion of intensities in the images into oxygenation and radiosensitivity maps is not straightforward. This study investigated the feasibility of applying two conversion approaches previously derived for ¹⁸F-labeled fluoromisonidazole (¹⁸F-FMISO)-PET images for the hypoxia tracer ¹⁸F-flortanidazole (¹⁸F-HX4).</p> <p><b>Material and methods:</b> Ten non-small-cell lung cancer patients imaged with ¹⁸F-HX4 before the start of radiotherapy were considered in this study. PET image uptake was normalized to a well-oxygenated reference region and subsequently linear and non-linear conversions were used to determine tissue oxygenations maps. These were subsequently used to delineate hypoxic volumes based partial oxygen pressure (pO₂) thresholds. The results were compared to hypoxic volumes segmented using a tissue-to-background ratio of 1.4 for ¹⁸F-HX4 uptake.</p> <p><b>Results:</b> While the linear conversion function was not found to result in realistic oxygenation maps, the non-linear function resulted in reasonably sized sub-volumes in good agreement with uptake-based segmented volumes for a limited range of pO₂ thresholds. However, the pO₂ values corresponding to this range were significantly higher than what is normally considered as hypoxia. The similarity in size, shape, and relative location between uptake-based sub-volumes and volumes based on the conversion to pO₂ suggests that the relationship between uptake and pO₂ is similar for ¹⁸F-FMISO and ¹⁸F-HX4, but that the model parameters need to be adjusted for the latter.</p> <p><b>Conclusions:</b> A non-linear conversion function between uptake and oxygen partial pressure for ¹⁸F-FMISO-PET could be applied to ¹⁸F-HX4 images to delineate hypoxic sub-volumes of similar size, shape, and relative location as based directly on the uptake. In order to apply the model for e.g., dose-painting, new parameters need to be derived for the accurate calculation of dose-modifying factors for this tracer.</p

Cells Deficient in K63-Ub Chain Formation Are Sensitized to Cisplatin Treatment while UV Sensitivity Is Revealed only upon POLη Knockdown

<div><p>(A and B) Clonogenic survival assays were used to determine sensitivity to 1 h acute treatment with cisplatin in untransfected A549 cells or in A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub.</i> The mean values of three independent experiments are shown with standard error of the mean (error bars). Cells expressing <i>K33R-Ub</i> or cells that lost <i>K63R-Ub</i> expression revert to WT-Ub cisplatin sensitivity.</p><p>(C) Cells were treated for 24 h with 100 μM cisplatin followed by Hoechst staining to detect apoptosis. The mean values of three independent experiments are shown with standard deviation.</p><p>(D) Clonogenic survival assays were used to determine sensitivity to UV irradiation in untransfected A549 cells or in A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub.</i></p><p>(E) Clonogenic survival of A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> with or without POLη RNAi following 10 J/m² UV treatment.</p></div

Model of the DDT Pathway in Mammalian Cells

<p>Recovery from a stalled replication fork at sites of DNA damage can occur by one of two alternative pathways. Previous work has shown that PCNA mono-ubiquitination by the RAD6/RAD18 complex stimulates lesion bypass through recruitment of the error-prone TLS polymerases. Here we show that an alternative error-free pathway requires formation of K63-polyUb chains. Blockade of this error-free pathway results in increased use of the TLS polymerases after DNA damage and a corresponding increase in mutations. As the TLS polymerases POLη and POLι both bind directly and avidly to polyUb chains [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-b020" target="_blank">20</a>], it is hypothesized that the interaction with K63-polyUb causes a disengagement of the polymerase from the DNA, allowing other proteins to migrate to the site of damage to perform error-free repair. This model predicts that K63-polyubiquitination acts to suppress environmental carcinogenesis by preventing genomic instability that would otherwise be introduced by the TLS polymerases.</p

Cells Deficient in K63-Ub Chain Formation Are Mutagenic in Response to UV Treatment

<div><p>(A and B) Cells were treated with cisplatin for 1 h or UV irradiation and subcultured for 7 d. Cells were then plated and grown in 6-TG to select for <i>HPRT</i> mutants. The mean values of three independent experiments are shown with standard deviation.</p><p>(C) Normal fibroblasts stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> were UV-irradiated (10 J/m²) and cultured for 5 d. Cells were then plated and grown in 6-TG to select for <i>HPRT</i> mutants.</p><p>(D) The number of <i>HPRT</i> mutants was quantitated for A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> with or without POLη RNAi. Cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g003" target="_blank">Figure 3</a>C.</p><p>(E) Cells were UV-irradiated and plated in the absence or presence of 0.4 mM caffeine. The mean values of three independent experiments are shown with standard error of the mean (error bars).</p></div

Disrupting K63-PolyUb Chain Formation Increases Reliance of Cells on the Error-Prone TLS Pathway

<div><p>(A) HeLa cells stably expressing <i>WT-Ub-puro</i> or <i>K63R-Ub-puro</i> were transiently transfected with a plasmid expressing a <i>POLη-GFP</i> fusion. Twenty-four hours post-transfection, cells were UV-irradiated (10 J/m²). POLη (green) and PCNA (red) were detected using antibodies. Shown are representative confocal photographs of cells 6 h post-UV treatment.</p><p>(B) Kinetics of POLη foci formation in <i>WT-Ub–</i> and <i>K63R-Ub–</i>expressing HeLa cell lines.</p><p>(C) <i>HPRT</i> mutation spectra. RNA was isolated from 6-TG resistant 10 J/m² UV-treated clones followed by RT-PCR and sequence analysis of the <i>HPRT</i> locus. The UV-induced mutations are shown in the upper table. Most of the point mutations were G→A or C→T transitions indicated as G/C→A/T. The lower table in (C) shows the same mutants in sequence context.</p><p>(D) Foci were quantitated 6 h post-UV treatment using a live-cell imaging fluorescent microscope.</p><p>(E) The number of <i>HPRT</i> mutants was quantitated for A549 cells stably expressing <i>K63R-Ub</i> with or without RAD18 RNAi. Cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g004" target="_blank">Figure 4</a>B.</p></div

Modification of PCNA by Polyubiquitin in Human Cells after DNA Damage

<div><p>(A) A549, 293T, and Hela cells were irradiated with 0 or 30 J/m² UV and lysed 6 h posttreatment followed by immunoblotting for PCNA.</p><p>(B) 293T cells were transfected with 100 nM of either control siRNA, siRNA Ubc13, or siRNA RAD18. Seventy-two hours post-transfection, cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g001" target="_blank">Figure 1</a>A. A darker and lighter exposure of the PCNA immunoblot is shown.</p><p>(C) A549, 293T, and Hela cells were irradiated with 30 J/m² UV and lysed in boiling SDS, diluted in lysis buffer and subjected to immunoprecipitation with a PCNA antibody and detected with PCNA or Ub antibodies. The controls in the immunoprecipitations were “no 1”, in which lysates were incubated with beads but no PCNA antibody, and “1 B” in which PCNA antibody was incubated with beads alone.</p><p>(D) 293T cells were transfected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g005" target="_blank">Figure 5</a>B. Seventy-two hours post-transfection, cells were irradiated with 30 J/m² of UV and lysed 6 h later in boiling SDS, diluted in lysis buffer, and subjected to immunoprecipitation with a PCNA antibody and immunoblotted for PCNA (upper panel) and Ub (lower panel). A lighter exposure of the PCNA IP immunoblotted for Ub is also shown. A PCNA immunoblot with darker and lighter exposure performed on protein lysates from the same samples used in the immunoprecipitations is also shown. Asterisks denote immunoglobulin heavy and light chains as detected on the immunoprecipitations.</p></div

Radiomics.PET.features

PET features used in model development: imaging features computed for tumor (Tumor) and merging structure including involved lymph nodes (Nodes). Survival information (in years) is available for all patients under analysis (status: 1 - deceased, 0 - alive)