A clinically annotated post-mortem approach to study multi-organ somatic mutational clonality in normal tissues.

Recent research on normal human tissues identified omnipresent clones of cells, driven by somatic mutations known to be responsible for carcinogenesis (e.g., in TP53 or NOTCH1). These new insights are fundamentally changing current tumor evolution models, with broad oncological implications. Most studies are based on surgical remnant tissues, which are not available for many organs and rarely in a pan-organ setting (multiple organs from the same individual). Here, we describe an approach based on clinically annotated post-mortem tissues, derived from whole-body donors that are routinely used for educational purposes at human anatomy units. We validated this post-mortem approach using UV-exposed and unexposed epidermal skin tissues and confirm the presence of positively selected NOTCH1/2-, TP53- and FAT1-driven clones. No selection signals were detected in a set of immune genes or housekeeping genes. Additionally, we provide the first evidence for smoking-induced clonal changes in oral epithelia, likely underlying the origin of head and neck carcinogenesis. In conclusion, the whole-body donor-based approach provides a nearly unlimited healthy tissue resource to study mutational clonality and gain fundamental mutagenic insights in the presumed earliest stages of tumor evolution.

Digital body preservation: Technique and applications.

High-fidelity anatomical models can be produced with three-dimensional (3D) scanning techniques and as such be digitally preserved, archived, and subsequently rendered through various media. Here, a novel methodology-digital body preservation-is presented for combining and matching scan geometry with radiographic imaging. The technique encompasses joining layers of 3D surface scans in an anatomical correct spatial relationship. To do so, a computed tomography (CT) volume is used as template to join and merge different surface scan geometries by means of nonrigid registration into a single environment. In addition, the use and applicability of the generated 3D models in digital learning modalities is presented. Finally, as computational expense is usually the main bottleneck in extended 3D applications, the influence of mesh simplification in combination with texture mapping on the quality of 3D models was investigated. The physical fidelity of the simplified meshes was evaluated in relation to their resolution and with respect to key anatomical features. Large- and medium-scale features were well preserved despite extensive 3D mesh simplification. Subtle fine-scale features, particular in curved areas demonstrated the major limitation to extensive mesh size reduction. Depending on the local topography, workable mesh sizes ranging from 10% to 3% of the original size could be obtained, making them usable in various learning applications and environments.

Chromatographic clarification overcomes chromatin-mediated hitch-hiking interactions on Protein A capture column.

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHOBC ® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.

Comparison of supine or prone crawl photon or proton breast and regional lymph node radiation therapy including the internal mammary chain.

We report on a dosimetrical study comparing supine (S) and prone-crawl (P) position for radiotherapy of whole breast (WB) and loco-regional lymph node regions, including the internal mammary chain (LN_IM). Six left sided breast cancer patients were CT-simulated in S and P positions and four patients only in P position. Treatment plans were made using non-coplanar volumetric modulated arc photon therapy (VMAT) or pencil beam scanning intensity modulated proton therapy (IMPT). Dose prescription was 15*2.67 Gy(GyRBE). The average mean heart doses for S or P VMAT were 5.6 or 4.3 Gy, respectively (p = 0.16) and 1.02 or 1.08 GyRBE, respectively for IMPT (p = 0.8; p < 0.001 for IMPT versus VMAT). The average mean lung doses for S or P VMAT were 5.91 or 2.90 Gy, respectively (p = 0.002) and 1.56 or 1.09 GyRBE, respectively for IMPT (p = 0.016). In high-risk patients, average (range) thirty-year mortality rates from radiotherapy-related cardiac injury and lung cancer were estimated at 6.8(5.4-9.4)% or 3.8(2.8-5.1)% for S or P VMAT (p < 0.001), respectively, and 1.6(1.1-2.0)% or 1.2(0.8-1.6)% for S or P IMPT (p = 0.25), respectively. Radiation-related mortality risk could outweigh the ~8% disease-specific survival benefit of WB + LN_IM radiotherapy for S VMAT but not P VMAT. IMPT carries the lowest radiation-related mortality risks.

The relation between patient discomfort and uncompensated forces of a patient support device for breast and regional lymph node radiotherapy.

Although many authors stated that a user-centred design approach in medical device development has added values, the most common research approach within healthcare is evidence-based medicine, which tend to focus on functional data rather than patient wellbeing and comfort. End user comfort is well addressed in literature for commercial products such as seats and hand tools but no data was found for medical devices. A commercial patient support device for breast radiotherapy was analysed and a relation was found between discomfort and uncompensated internal body forces. Derived from CT-images, simplified patient free-body diagrams were analysed and pain and comfort evaluated. Subsequently, a new patient position was established and prototypes were developed. Patient comfort- and prototype optimization was done through iterative prototyping. With this approach, we were able to compensate all internal body forces and establish a force neutral patient free-body diagram. This resulted in comfortable patient positioning and favourable medical results.

Potential benefits of crawl position for prone radiation therapy in breast cancer.

PURPOSE: To investigate crawl position with the arm at the treated side alongside the body and at the opposite side above the head for prone treatment in patients requiring breast and regional lymph node irradiation. METHODS: Patient support devices for crawl position were built for CT simulation and treatment. An asymmetric fork design resulted from an iterative process of prototype construction and testing. The fork's large horn supports the hemi-thorax, shoulder, and elevated arm at the nontreated side and the head. The short, narrow horn supports the arm at the treated side. Between both horns, the treated breast and its regional lymph nodes are exposed. Endpoints were pain, comfort, set-up precision, beam access to the breast and lymph nodes, and plan dose metrics. Pain and comfort were tested by volunteers (n = 9); set-up precision, beam access, and plan dose metrics were tested by means of a patient study (n = 10). The AIO™ (Orfit, Wijnegem, Belgium) prone breastboard (AIO™) was used as a reference regarding comfort and set-up precision. RESULTS: Pain at the sternum, the ipsilateral shoulder, upper arm, and neck was lower in crawl position than with bilateral arm elevation on AIO™. Comfort and set-up precision were better on the crawl prototype than on AIO™. In crawl position, beam directions in the coronal and near-sagittal planes have access to the breast or regional lymph nodes without traversing device components. Plan comparison between supine and crawl positions showed better dose homogeneity for the breast and lymph node targets and dose reductions to all organs at risk for crawl position. CONCLUSIONS: Radiation therapy for breast and regional lymph nodes in crawl position is feasible. Good comfort and set-up precision were demonstrated. Planning results support the hypothesis that breast and regional lymph nodes can be treated in crawl position with less dose to organs at risk and equal or better dose distribution in the target volumes than in supine position. The crawl technique is a candidate methodology for further investigation for patients requiring breast and regional lymph node irradiation.

Whole breast and regional nodal irradiation in prone versus supine position in left sided breast cancer.

BACKGROUND: Prone whole breast irradiation (WBI) leads to reduced heart and lung doses in breast cancer patients receiving adjuvant radiotherapy. In this feasibility trial, we investigated the prone position for whole breast + lymph node irradiation (WB + LNI). METHODS: A new support device was developed for optimal target coverage, on which patients are positioned in a position resembling a phase from the crawl swimming technique (prone crawl position). Five left sided breast cancer patients were included and simulated in supine and prone position. For each patient, a treatment plan was made in prone and supine position for WB + LNI to the whole axilla and the unoperated part of the axilla. Patients served as their own controls for comparing dosimetry of target volumes and organs at risk (OAR) in prone versus in supine position. RESULTS: Target volume coverage differed only slightly between prone and supine position. Doses were significantly reduced (P < 0.05) in prone position for ipsilateral lung (Dmean, D2, V5, V10, V20, V30), contralateral lung (Dmean, D2), contralateral breast (Dmean, D2 and for total axillary WB + LNI also V5), thyroid (Dmean, D2, V5, V10, V20, V30), oesophagus (Dmean and for partial axillary WB + LNI also D2 and V5), skin (D2 and for partial axillary WB + LNI V105 and V107). There were no significant differences for heart and humeral head doses. CONCLUSIONS: Prone crawl position in WB + LNI allows for good breast and nodal target coverage with better sparing of ipsilateral lung, thyroid, contralateral breast, contralateral lung and oesophagus when compared to supine position. There is no difference in heart and humeral head doses. TRIAL REGISTRATION: No trial registration was performed because there were no therapeutic interventions.

Optimal number of atlases and label fusion for automatic multi-atlas-based brachial plexus contouring in radiotherapy treatment planning.

BACKGROUND: The present study aimed to define the optimal number of atlases for automatic multi-atlas-based brachial plexus (BP) segmentation and to compare Simultaneous Truth and Performance Level Estimation (STAPLE) label fusion with Patch label fusion using the ADMIRE® software. The accuracy of the autosegmentations was measured by comparing all of the generated autosegmentations with the anatomically validated gold standard segmentations that were developed using cadavers. MATERIALS AND METHODS: Twelve cadaver computed tomography (CT) atlases were used for automatic multi-atlas-based segmentation. To determine the optimal number of atlases, one atlas was selected as a patient and the 11 remaining atlases were registered onto this patient using a deformable image registration algorithm. Next, label fusion was performed by using every possible combination of 2 to 11 atlases, once using STAPLE and once using Patch. This procedure was repeated for every atlas as a patient. The similarity of the generated automatic BP segmentations and the gold standard segmentation was measured by calculating the average Dice similarity (DSC), Jaccard (JI) and True positive rate (TPR) for each number of atlases. These similarity indices were compared for the different number of atlases using an equivalence trial and for the two label fusion groups using an independent sample-t test. RESULTS: DSC's and JI's were highest when using nine atlases with both STAPLE (average DSC = 0,532; JI = 0,369) and Patch (average DSC = 0,530; JI = 0,370). When comparing both label fusion algorithms using 9 atlases for both, DSC and JI values were not significantly different. However, significantly higher TPR values were achieved in favour of STAPLE (p < 0,001). When fewer than four atlases were used, STAPLE produced significantly lower DSC, JI and TPR values than did Patch (p = 0,0048). CONCLUSIONS: Using 9 atlases with STAPLE label fusion resulted in the most accurate BP autosegmentations (average DSC = 0,532; JI = 0,369 and TPR = 0,760). Only when using fewer than four atlases did the Patch label fusion results in a significantly more accurate autosegmentation than STAPLE.

Brachial plexus 3D reconstruction from MRI with dissection validation: a baseline study for clinical applications.

PURPOSE: The present study aimed to establish a baseline for detailed 3D brachial plexus reconstruction from magnetic resonance imaging (MRI). Concretely, the goal was to determine the individual brachial plexus anatomy with maximum detail and accuracy achievable, as yet irrespective of whether the methods used could be economically and practically applied in the clinical setting. MATERIALS AND METHODS: Six embalmed cadavers were randomly taken for MRI imaging of the brachial plexus. Detailed two-dimensional (2D) segmentation for all brachial plexus parts was done. The 2D brachial plexus segmentations were 3D reconstructed using Mimics(®) software. Then, these 3D reconstructions were anatomically validated by dissection of the cadavers. After finalising the cadaver experiments, brachial plexus MRIs were obtained in three healthy male volunteers and the same reconstruction procedure as in vitro was followed. RESULTS: A procedure was developed for brachial plexus 3D reconstruction based on MRI without the use of any contrast agent. Anatomical validation of six cadaver brachial plexus reconstructions showed high correspondence with the dissected brachial plexuses. Anatomical variations of the main branches were equally present in the 3D reconstructions generated. However, there were also some differences that related to the difference between the surface anatomy of the nerve and the internal nerve structure. In vivo, it was possible to reconstruct the complete brachial plexus in such a manner that normal-appearing BPs were derived in a reproducible way. CONCLUSIONS: This study showed that the described procedure results in accurate and reproducible brachial plexus 3D reconstructions.

The effect of morphometric atlas selection on multi-atlas-based automatic brachial plexus segmentation.

PURPOSE: The present study aimed to measure the effect of a morphometric atlas selection strategy on the accuracy of multi-atlas-based BP autosegmentation using the commercially available software package ADMIRE® and to determine the optimal number of selected atlases to use. Autosegmentation accuracy was measured by comparing all generated automatic BP segmentations with anatomically validated gold standard segmentations that were developed using cadavers. MATERIALS AND METHODS: Twelve cadaver computed tomography (CT) atlases were included in the study. One atlas was selected as a patient in ADMIRE®, and multi-atlas-based BP autosegmentation was first performed with a group of morphometrically preselected atlases. In this group, the atlases were selected on the basis of similarity in the shoulder protraction position with the patient. The number of selected atlases used started at two and increased up to eight. Subsequently, a group of randomly chosen, non-selected atlases were taken. In this second group, every possible combination of 2 to 8 random atlases was used for multi-atlas-based BP autosegmentation. For both groups, the average Dice similarity coefficient (DSC), Jaccard index (JI) and Inclusion index (INI) were calculated, measuring the similarity of the generated automatic BP segmentations and the gold standard segmentation. Similarity indices of both groups were compared using an independent sample t-test, and the optimal number of selected atlases was investigated using an equivalence trial. RESULTS: For each number of atlases, average similarity indices of the morphometrically selected atlas group were significantly higher than the random group (p < 0,05). In this study, the highest similarity indices were achieved using multi-atlas autosegmentation with 6 selected atlases (average DSC = 0,598; average JI = 0,434; average INI = 0,733). CONCLUSIONS: Morphometric atlas selection on the basis of the protraction position of the patient significantly improves multi-atlas-based BP autosegmentation accuracy. In this study, the optimal number of selected atlases used was six, but for definitive conclusions about the optimal number of atlases and to improve the autosegmentation accuracy for clinical use, more atlases need to be included.

Deep inspiration breath hold in the prone position retracts the heart from the breast and internal mammary lymph node region.

Deep inspiration breath hold in the prone position causes a caudal shift of the heart away from the nearly unmodified breast and internal mammary lymph node region, suggesting better heart protection for left-sided whole breast irradiation with or without regional nodal radiotherapy.

Morphometric Atlas Selection for Automatic Brachial Plexus Segmentation.

PURPOSE: The purpose of this study was to determine the effects of atlas selection based on different morphometric parameters, on the accuracy of automatic brachial plexus (BP) segmentation for radiation therapy planning. The segmentation accuracy was measured by comparing all of the generated automatic segmentations with anatomically validated gold standard atlases developed using cadavers. METHODS AND MATERIALS: Twelve cadaver computed tomography (CT) atlases (3 males, 9 females; mean age: 73 years) were included in the study. One atlas was selected to serve as a patient, and the other 11 atlases were registered separately onto this "patient" using deformable image registration. This procedure was repeated for every atlas as a patient. Next, the Dice and Jaccard similarity indices and inclusion index were calculated for every registered BP with the original gold standard BP. In parallel, differences in several morphometric parameters that may influence the BP segmentation accuracy were measured for the different atlases. Specific brachial plexus-related CT-visible bony points were used to define the morphometric parameters. Subsequently, correlations between the similarity indices and morphometric parameters were calculated. RESULTS: A clear negative correlation between difference in protraction-retraction distance and the similarity indices was observed (mean Pearson correlation coefficient = -0.546). All of the other investigated Pearson correlation coefficients were weak. CONCLUSIONS: Differences in the shoulder protraction-retraction position between the atlas and the patient during planning CT influence the BP autosegmentation accuracy. A greater difference in the protraction-retraction distance between the atlas and the patient reduces the accuracy of the BP automatic segmentation result.

Hair-sparing whole brain radiotherapy with volumetric arc therapy in patients treated for brain metastases: dosimetric and clinical results of a phase II trial.

PURPOSE: To report the dosimetric results and impact of volumetric arc therapy (VMAT) on temporary alopecia and hair-loss related quality of life (QOL) in whole brain radiotherapy (WBRT). METHODS: The potential of VMAT-WBRT to reduce the dose to the hair follicles was assessed. A human cadaver was treated with both VMAT-WBRT and conventional opposed field (OF) WBRT, while the subcutaneously absorbed dose was measured by radiochromic films and calculated by the planning system. The impact of these dose reductions on temporary alopecia was examined in a prospective phase II trial, with the mean score of hair loss at 1 month after VMAT-WBRT (EORTC-QOL BN20) as a primary endpoint and delivering a dose of 20 Gy in 5 fractions. An interim analysis was planned after including 10 patients to rule out futility, defined as a mean score of hair loss exceeding 56.7. A secondary endpoint was the global alopecia areata severity score measured with the "Severity of Alopecia Tool" (SALT) with a scale of 0 (no hair loss) to 100 (complete alopecia). RESULTS: For VMAT-WBRT, the cadaver measurements demonstrated a dose reduction to the hair follicle volume of 20.5% on average and of 41.8% on the frontal-vertex-occipital medial axis as compared to OF-WBRT. In the phase II trial, a total of 10 patients were included before the trial was halted due to futility. The EORTC BN20 hair loss score following WBRT was 95 (SD 12.6). The average median dose to the hair follicle volume was 12.6 Gy (SD 0.9), corresponding to a 37% dose reduction compared to the prescribed dose. This resulted in a mean SALT-score of 75. CONCLUSIONS: Compared to OF-WBRT, VMAT-WBRT substantially reduces hair follicle dose. These dose reductions could not be related to an improved QOL or SALT score.

Reliability and accuracy assessment of Radiation Therapy Oncology Group-endorsed guidelines for brachial plexus contouring.

PURPOSE: The goal of this work was to validate the Radiation Therapy Oncology Group (RTOG)-endorsed guidelines for brachial plexus (BP) contouring by determining the intra- and interobserver agreement. Accuracy of the delineation process was determined using anatomically validated imaging datasets as a gold standard. MATERIALS AND METHODS: Five observers delineated the right BP on three cadaver computed tomography (CT) datasets. To assess intraobserver variation, every observer repeated each delineation three times with a time interval of 2 weeks. The BP contours were divided into four regions for detailed analysis. Inter- and intraobserver variation was verified using the Computerized Environment for Radiation Research (CERR) software. Accuracy was measured using anatomically validated fused CT-magnetic resonance imaging (MRI) datasets by measuring the BP inclusion of the delineations. RESULTS: The overall kappa (κ) values were rather low (mean interobserver overall κ: 0.29, mean intraobserver overall κ: 0.45), indicating poor inter- and intraobserver reliability. In general, the κ coefficient decreased gradually from the medial to lateral BP regions. The total agreement volume (TAV) was much smaller than the union volume (UV) for all delineations, resulting in a low Jaccard index (JI; interobserver agreement 0-0.124; intraobserver agreement 0.004-0.636). The overall accuracy was poor, with an average total BP inclusion of 38%. Inclusions were insufficient for the most lateral regions (region 3: 21.5%; region 4: 12.6%). CONCLUSION: The inter- and intraobserver reliability of the RTOG-endorsed BP contouring guidelines was poor. BP inclusion worsened from the medial to lateral regions. Accuracy assessment of the contours showed an average BP inclusion of 38%. For the first time, this was assessed using the original anatomically validated BP volume. The RTOG-endorsed BP guidelines have insufficient accuracy and reliability, especially for the lateral head-and-neck regions.

An anatomically validated brachial plexus contouring method for intensity modulated radiation therapy planning.

PURPOSE: To develop contouring guidelines for the brachial plexus (BP) using anatomically validated cadaver datasets. Magnetic resonance imaging (MRI) and computed tomography (CT) were used to obtain detailed visualizations of the BP region, with the goal of achieving maximal inclusion of the actual BP in a small contoured volume while also accommodating for anatomic variations. METHODS AND MATERIALS: CT and MRI were obtained for 8 cadavers positioned for intensity modulated radiation therapy. 3-dimensional reconstructions of soft tissue (from MRI) and bone (from CT) were combined to create 8 separate enhanced CT project files. Dissection of the corresponding cadavers anatomically validated the reconstructions created. Seven enhanced CT project files were then automatically fitted, separately in different regions, to obtain a single dataset of superimposed BP regions that incorporated anatomic variations. From this dataset, improved BP contouring guidelines were developed. These guidelines were then applied to the 7 original CT project files and also to 1 additional file, left out from the superimposing procedure. The percentage of BP inclusion was compared with the published guidelines. RESULTS: The anatomic validation procedure showed a high level of conformity for the BP regions examined between the 3-dimensional reconstructions generated and the dissected counterparts. Accurate and detailed BP contouring guidelines were developed, which provided corresponding guidance for each level in a clinical dataset. An average margin of 4.7 mm around the anatomically validated BP contour is sufficient to accommodate for anatomic variations. Using the new guidelines, 100% inclusion of the BP was achieved, compared with a mean inclusion of 37.75% when published guidelines were applied. CONCLUSION: Improved guidelines for BP delineation were developed using combined MRI and CT imaging with validation by anatomic dissection.