Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods

Reproducibility of the lung anatomy under active breathing coordinator control:Dosimetric consequences for scanned proton treatments

Purpose The treatment of moving targets with scanned proton beams is challenging. For motion mitigation, an Active Breathing Coordinator (ABC) can be used to assist breath-holding. The delivery of pencil beam scanning fields often exceeds feasible breath-hold durations, requiring high breath-hold reproducibility. We evaluated the robustness of scanned proton therapy against anatomical uncertainties when treating nonsmall-cell lung cancer (NSCLC) patients during ABC controlled breath-hold. Methods Four subsequent MRIs of five healthy volunteers (3 male, 2 female, age: 25-58, BMI: 19-29) were acquired under ABC controlled breath-hold during two simulated treatment fractions, providing both intrafractional and interfractional information about breath-hold reproducibility. Deformation vector fields between these MRIs were used to deform CTs of five NSCLC patients. Per patient, four or five cases with different tumor locations were modeled, simulating a total of 23 NSCLC patients. Robustly optimized (3 and 5 mm setup uncertainty respectively and 3% density perturbation) intensity-modulated proton plans (IMPT) were created and split into subplans of 20 s duration (assumed breath-hold duration). A fully fractionated treatment was recalculated on the deformed CTs. For each treatment fraction the deformed CTs representing multiple breath-hold geometries were alternated to simulate repeated ABC breath-holding during irradiation. Also a worst-case scenario was simulated by recalculating the complete treatment plan on the deformed CT scan showing the largest deviation with the first deformed CT scan, introducing a systematic error. Both the fractionated breath-hold scenario and worst-case scenario were dosimetrically evaluated. Results Looking at the deformation vector fields between the MRIs of the volunteers, up to 8 mm median intra- and interfraction displacements (without outliers) were found for all lung segments. The dosimetric evaluation showed a median difference in D-98% between the planned and breath-hold scenarios of -0.1 Gy (range: -4.1 Gy to 2.0 Gy). D-98% target coverage was more than 57.0 Gy for 22/23 cases. The D-1 cc of the CTV increased for 21/23 simulations, with a median difference of 0.9 Gy (range: -0.3 to 4.6 Gy). For 14/23 simulations the increment was beyond the allowed maximum dose of 63.0 Gy, though remained under 66.0 Gy (110% of the prescribed dose of 60.0 Gy). Organs at risk doses differed little compared to the planned doses (difference in mean doses <0.9 Gy for the heart and lungs, <1.4% difference in V-35 [%] and V-20 [%] to the esophagus and lung). Conclusions When treating under ABC controlled breath-hold, robustly optimized IMPT plans show limited dosimetric consequences due to anatomical variations between repeated ABC breath-holds for most cases. Thus, the combination of robustly optimized IMPT plans and the delivery under ABC controlled breath-hold presents a safe approach for PBS lung treatments

Health-related quality of life after prophylactic cranial irradiation for stage III non-small cell lung cancer patients:Results from the NVALT-11/DLCRG-02 phase III study

BACKGROUND AND PURPOSE: The NVALT-11/DLCRG-02 phase III trial (clinicaltrials.gov identifier: NCT01282437) showed that, after standard curative intent treatment, prophylactic cranial irradiation (PCI) decreased the incidence of symptomatic brain metastases (BM) in stage III non-small cell lung cancer (NSCLC) patients compared to observation. In this study we assessed the impact of PCI on health-related quality of life (HRQoL). In addition, an exploratory analysis was performed to assess the impact of neurocognitive symptoms and symptomatic BM on HRQoL. MATERIALS AND METHODS: Stage III NSCLC patients were randomized between PCI and observation. HRQoL was measured using the EuroQol 5D (EQ-5D-3L), EORTC QLQ-C30 and QLQ-BN20 instruments at completion of standard curative intent treatment and 4 weeks, 3, 6, 12, 24 and 36 months thereafter. Generalized linear mixed effects (GLM) models were used to assess the impact of PCI compared to observation over time on three HRQoL metrics: the EORTC QLQ-C30 global health status and the EQ-5D-3L utility and visual analogue scale (EQ VAS) scores. RESULTS: In total, 86 and 88 patients were included in the PCI and observation arm, with a median follow-up of 48.5 months (95% CI 39-54 months). Baseline mean HRQoL scores were comparable between the PCI and observation arm for the three HRQoL metrics. In the GLM models, none of the HRQoL metrics were clinically relevant or statistically significantly different between the PCI and the observation arm (p-values ranged between 0.641 and 0.914). CONCLUSION: No statistically significant nor a clinically relevant impact of PCI on HRQoL was observed

External validation of NTCP-models for radiation pneumonitis in lung cancer patients treated with chemoradiotherapy

PURPOSE: Normal tissue complication probability (NTCP) models can be used to estimate the risk of radiation pneumonitis (RP). The aim of this study was to externally validate the most frequently used prediction models for RP, i.e., the QUANTEC and APPELT models, in a large cohort of lung cancer patients treated with IMRT or VMAT. [1-2] METHODS AND MATERIALS: This prospective cohort study, included lung cancer patients treated between 2013 and 2018. A closed testing procedure was performed to test the need for model updating. To improve model performance, modification or removal of variables was considered. Performance measures included tests for goodness of fit, discrimination, and calibration.RESULTS: In this cohort of 612 patients, the incidence of RP ≥ grade 2 was 14.5%. For the QUANTEC-model, recalibration was recommended which resulted in a revised intercept and adjusted regression coefficient (from 0.126 to 0.224) of the mean lung dose (MLD),. The APPELT-model needed revision including model updating with modification and elimination of variables. After revision, the New RP-model included the following predictors (and regression coefficients): MLD (B = 0.250), age (B = 0.049, and smoking status (B = 0.902). The discrimination of the updated APPELT-model was higher compared to the recalibrated QUANTEC-model (AUC: 0.79 vs. 0.73).CONCLUSIONS: This study demonstrated that both the QUANTEC- and APPELT-model needed revision. Next to changes of the intercept and regression coefficients, the APPELT model improved further by model updating and performed better than the recalibrated QUANTEC model. This New RP-model is widely applicable containing non-tumour site specific variables, which can easily be collected.</p

18F-FDG PET during stereotactic body radiotherapy for stage I lung tumours cannot predict outcome: a pilot study

(18)F-Fluorodeoxyglucose positron emission tomography (FDG PET) has been used to assess metabolic response several months after stereotactic body radiotherapy (SBRT) for early-stage non-small cell lung cancer. However, whether a metabolic response can be observed already during treatment and thus can be used to predict treatment outcome is undetermined. Ten medically inoperable patients with FDG PET-positive lung tumours were included. SBRT consisted of three fractions of 20 Gy delivered at the 80% isodose at days 1, 6 and 11. FDG PET was performed before, on day 6 immediately prior to administration of the second fraction of SBRT and 12 weeks after completion of SBRT. Tumour metabolism was assessed semi-quantitatively using the maximum standardized uptake value (SUV(max)) and SUV(70%). After the first fraction, median SUV(max) increased from 6.7 to 8.1 (p = 0.07) and median SUV(70%) increased from 5.7 to 7.1 (p = 0.05). At 12 weeks, both median SUV(max) and median SUV(70%) decreased by 63% to 3.1 (p = 0.008) and to 2.5 (p = 0.008), respectively. SUV increased during treatment, possibly due to radiation-induced inflammation. Therefore, it is unlikely that (18)F-FDG PET during SBRT will predict treatment success

Organ sparing potential and inter-fraction robustness of adaptive intensity modulated proton therapy for lung cancer

Background: The aim of this study was to compare adaptive intensity modulated proton therapy (IMPT) robustness and organ sparing capabilities with that of adaptive volumetric arc photon therapy (VMAT). Material and methods: Eighteen lung cancer patients underwent a planning 4DCT (p4DCT) and 5 weekly repeated 4DCT (r4DCT) scans. Target volumes and organs at risk were manually delineated on the three-dimensional (3D) average scans of the p4DCT (av_p4DCT) and of the r4DCT scans (av_r4DCT). Planning target volume (PTV)-based VMAT plans and internal clinical target volume (ICTV)-based robust IMPT plans were optimized in 3D on the av_p4DCT and re-calculated on the av_r4DCTs. Re-planning on av_r4DCTs was performed when indicated and accumulated doses were evaluated on the av_p4DCT. Results: Adaptive VMAT and IMPT resulted in adequate ICTV coverage on av_r4DCT in all patients and adequate accumulated-dose ICTV coverage on av_p4DCT in 17/18 patients (due to a shrinking target in one patient). More frequent re-planning was needed for IMPT than for VMAT. The average mean heart dose reduction with IMPT compared with VMAT was 4.6 Gy (p = .001) and it was >5 Gy for five patients (6, 7, 8, 15, and 22 Gy). The average mean lung dose reduction was 3.2 Gy (p < .001). Significant reductions in heart and lung V5 Gy were observed with IMPT. Conclusion: Robust-planned IMPT required re-planning more often than VMAT but resulted in similar accumulated ICTV coverage. With IMPT, heart and lung mean dose values and low dose regions were significantly reduced. Substantial cardiac sparing was obtained in a subgroup of five patients (28%)