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Abstract Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as ‘LamaMouse’. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

ABSTRACT: Background: Four-dimensional cardiovascular magnetic resonance flow imaging (4D flow CMR) plays an important role in assessing cardiovascular diseases. However, the manual or semi-automatic segmentation of aortic vessel boundaries in 4D flow data introduces variability and limits the reproducibility of aortic hemodynamics visualization and quantitative flow-related parameter computation. This paper explores the potential of deep learning to improve 4D flow CMR segmentation by developing models for automatic segmentation and analyzes the impact of the training data on the generalization of the model across different sites, scanner vendors, sequences, and pathologies. Methods: The study population consists of 260 4D flow CMR datasets, including subjects without known aortic pathology, healthy volunteers, and patients with bicuspid aortic valve (BAV) examined at different hospitals. The dataset was split to train segmentation models on subsets with different representations of characteristics, such as pathology, gender, age, scanner model, vendor, and field strength. An enhanced three-dimensional U-net convolutional neural network (CNN) architecture with residual units was trained for time-resolved two-dimensional aortic cross-sectional segmentation. Model performance was evaluated using Dice score, Hausdorff distance, and average symmetric surface distance on test data, datasets with characteristics not represented in the training set (model-specific), and an overall evaluation set. Standard diagnostic flow parameters were computed and compared with manual segmentation results using Bland-Altman analysis and interclass correlation. Results: The representation of technical factors, such as scanner vendor and field strength, in the training dataset had the strongest influence on the overall segmentation performance. Age had a greater impact than gender. Models solely trained on BAV patients’ datasets performed well on datasets of healthy subjects but not vice versa. Conclusion: This study highlights the importance of considering a heterogeneous dataset for the training of widely applicable automatic CNN segmentations in 4D flow CMR, with a particular focus on the inclusion of different pathologies and technical aspects of data acquisition.

Cardiac MRI is a crucial tool for assessing congenital heart disease (CHD). However, its application remains challenging in young children when performed at 3T. The aim of this retrospective single center study was to compare a non-contrast free-breathing 2D CINE T1-weighted TFE-sequence with compressed sensing (FB 2D CINE CS T1-TFE) with 3D imaging for diagnostic accuracy of CHD, image quality, and vessel diameter measurements in sedated young children. FB 2D CINE CS T1-TFE was compared with a 3D non-contrast whole-heart sequence (3D WH) and 3D contrast-enhanced MR angiography (3D CE-MRA) at 3T in 37 CHD patients (20♂, 1.5±1.4 years). Two radiologists independently assessed image quality, type of CHD, and diagnostic confidence. Diameters and measures of contrast and sharpness of the aorta and pulmonary vessels were determined. A non-parametric multi-factorial approach was used to estimate diagnostic accuracy for the diagnosis of CHD. Linear mixed models were calculated to compare contrast and vessel sharpness. Krippendorff's alpha was determined to quantify vessel diameter agreement. FB 2D CINE CS T1-TFE was rated superior regarding image quality, diagnostic confidence, and diagnostic sensitivity for both intra- and extracardiac pathologies compared to 3D WH and 3D CE-MRA (all p

Background & Aims: Cirrhosis is associated with an increased surgical morbidity and mortality. Portal hypertension and the surgery type have been established as critical determinants of postoperative outcome. We aim to evaluate the hypothesis that preoperative transjugular intrahepatic portosystemic shunt (TIPS) placement in patients with cirrhosis is associated with a lower incidence of in-house mortality/liver transplantation (LT) after surgery. Methods: A retrospective database search for the years 2010–2020 was carried out. We identified 64 patients with cirrhosis who underwent surgery within 3 months after TIPS placement and 131 patients with cirrhosis who underwent surgery without it (controls). Operations were categorised into low-risk and high-risk procedures. The primary endpoint was in-house mortality/LT. We analysed the influence of high-risk surgery, preoperative TIPS placement, age, sex, baseline creatinine, presence of ascites, Chronic Liver Failure Consortium Acute Decompensation (CLIF-C AD), American Society of Anesthesiologists (ASA), and model for end-stage liver disease (MELD) scores on in-house mortality/LT by multivariable Cox proportional hazards regression. Results: In both the TIPS and the control cohort, most patients presented with a Child-Pugh B stage (37/64, 58% vs. 70/131, 53%) at the time of surgery, but the median MELD score was higher in the TIPS cohort (14 vs. 11 points). Low-risk and high-risk procedures amounted to 47% and 53% in both cohorts. The incidence of in-house mortality/LT was lower in the TIPS cohort (12/64, 19% vs. 52/131, 40%), also when further subdivided into low-risk (0/30, 0% vs. 10/61, 16%) and high-risk surgery (12/34, 35% vs. 42/70, 60%). Preoperative TIPS placement was associated with a lower rate for postoperative in-house mortality/LT (hazard ratio 0.44, 95% CI 0.19–1.00) on multivariable analysis. Conclusions: A preoperative TIPS might be associated with reduced postoperative in-house mortality in selected patients with cirrhosis. Impact and implications: Patients with cirrhosis are at risk for more complications and a higher mortality after surgical procedures. A transjugular intrahepatic portosystemic shunt (TIPS) is used to treat complications of cirrhosis, but it is unclear if it also helps to lower the risk of surgery. This study takes a look at complications and mortality of patients undergoing surgery with or without a TIPS, and we found that patients with a TIPS develop less complications and have an improved survival. Therefore, a preoperative TIPS should be considered in selected patients, especially if indicated by ascites.

Abstract Background Four-dimensional (4D) flow cardiovascular magnetic resonance (CMR) is feasible for portal blood flow evaluation after placement of transjugular intrahepatic portosystemic shunts (TIPS) in patients with liver cirrhosis. However, clinical acceptance of 4D flow CMR in TIPS patients is limited due to the lack of validation studies. The purpose of this study was to validate 4D flow CMR-derived measurements in TIPS stent grafts using a three-dimensional (3D)-printed flow phantom. Methods A translucent flow phantom of the portal vasculature was 3D-printed. The phantom consisted of the superior mesenteric vein and the splenic vein draining into the portal vein, the TIPS-tract, and the hepatic vein. A TIPS stent graft (Gore® Viatorr®) was positioned within the TIPS-tract. Superior mesenteric vein and splenic vein served as inlets for blood-mimicking fluid. 4D flow CMR acquisitions were performed at 3T at preset flow rates of 0.8 to 2.8 l/min using velocity encoding of both 1.0 and 2.0 m/s. Flow rates and velocities were measured at predefined levels in the portal vasculature and within the stent graft. Accuracy of 4D flow CMR was assessed through linear regression with reference measurements obtained by flow sensors and two-dimensional (2D) phase contrast (PC) CMR. Intra- and interobserver agreement were assessed through Bland–Altman analyses. Results At a velocity encoding of 2.0 m/s, 4D flow CMR-derived flow rates and velocities showed an excellent correlation with preset flow rates and 2D PC CMR-derived flow velocities at all vascular levels and within the stent graft (all r ≥ 0.958, p ≤ 0.003). At a velocity encoding of 1.0 m/s, aliasing artifacts were present within the stent graft at flow rates ≥ 2.0 l/min. 4D flow CMR-derived measurements revealed high intra- and interobserver agreement. Conclusions The in vitro accuracy and precision of 4D flow CMR is unaffected by the presence of TIPS stent grafts, suggesting that 4D flow CMR may be used to monitor TIPS patency in patients with liver cirrhosis.

(1) Background: Accurate hepatic artery (HA) depiction following pediatric liver transplantation (LT) is essential for graft surveillance but challenging on ultrasound (US). This study assesses if improved HA delineation can be achieved by recording two-dimensional US volumes in Color Doppler (CD) and B-flow technique. (2) Methods: Of 42 consecutive LT, 37 cases were included, and HA delineation was retrospectively rated using a four-point score (0 = HA not detectable, 3 = HA fully detectable, separable from portal vein) within 48 h post-LT (U1) and before discharge (U2). (3) Results: Adding B-flow compared with CD alone showed superior results at neohilar (U1: 2.2 ± 1.0 vs. 1.1 ± 0.8, p < 0.0001; U2: 2.5 ± 0.8 vs. 1.5 ± 0.9, p < 0.0001) and segmental levels (U1: 2.8 ± 0.6 vs. 0.6 ± 0.8, p < 0.0001; U2: 2.8 ± 0.6 vs. 0.7 ± 0.5, p < 0.0001). (4) Conclusions: Standardized US volume recordings combining B-flow and CD can effectively delineate the HA along its vascular course in pediatric LT. The technique should be further evaluated as a standard monitoring instrument to rule out vascular complications after LT.

Abstract Transthoracic echocardiography (TTE) and cine cardiac magnetic resonance imaging (CMR) are established imaging methods of the aortic root. We aimed to evaluate the comparability of measurements in TTE and standard cine CMR sequences of the aortic root. Our study included 741 subjects (mean age 63.5 ± 8 years, 43.7% female) from the Hamburg City Health Study (HCHS). Subjects underwent CMR and TTE. Aortic root measurements were performed at the level of the aortic annulus (AoAn), sinus of Valsalva (SoV), and sinotubular junction (STJ) by standard cine CMR in left ventricular long axis and left ventricular outflow tract view. Measurements were performed applying the leading-edge to leading-edge (LL) convention and inner-edge to inner-edge (II) convention in TTE and the II convention in CMR. Inter correlation coefficients (ICCs) demonstrated high inter- and intraobserver reproducibility for CMR and TTE measurements of SoV and STJ (ICCs 0.9–0.98) and moderate reproducibility for AoAn (ICCs 0.68–0.91). CMR measurements of SoV and STJ showed strong agreement with TTE: while correlations were comparable (r = 0.75–0.85) bias was lower with TTE II (bias − 0.1 to − 0.74) versus TTE LL measurements (mean bias − 1.49 to − 2.58 mm). The agreement for AoAn was fair (r = 0.51–0.57) with variable bias (mean bias 0.39–3.9). Standard cine CMR and TTE derived aortic root measurements are reproducible and comparable with higher agreement for TTE II instead of LL measurements. These results support an interchangeable application of TTE and standard CMR for screening of aortic root diseases thereby possibly reducing redundant multimodality imaging.

BackgroundTo assess whether cardiac T1 mapping for detecting myocardial fibrosis enables preoperative identification of patients at risk for early left ventricular dysfunction after surgery of aortic regurgitation.Methods1.5 Tesla cardiac magnetic resonance imaging was performed in 40 consecutive aortic regurgitation patients before aortic valve surgery. Native and post-contrast T1 mapping was performed using a modified Look-Locker inversion-recovery sequence. Serial echocardiography was performed at baseline and 8 ± 5 days after aortic valve surgery to quantify LV dysfunction. Receiver operating characteristic analysis was performed to determine the diagnostic accuracy of native T1 mapping and extracellular volume for predicting postoperative LV ejection fraction decrease >−10% after aortic valve surgery.ResultsNative T1 was significantly increased in patients with a postoperatively decreased LVEF (n = 15) vs. patients with a preserved postoperative LV ejection fraction (n = 25) (i.e., 1,071 ± 67 ms vs. 1,019 ± 33 ms, p = .001). Extracellular volume was not significantly different between patients with preserved vs. decreased postoperative LV ejection fraction. With a cutoff-of value of 1,053 ms, native T1 yielded an area under the curve (AUC) of .820 (95% CI: .683–.958) for differentiating between patients with preserved vs. reduced LV ejection fraction with 70% sensitivity and 84% specificity.ConclusionIncreased preoperative native T1 is associated with a significantly higher risk of systolic LV dysfunction early after aortic valve surgery in aortic regurgitation patients. Native T1 could be a promising tool to optimize the timing of aortic valve surgery in patients with aortic regurgitation to prevent early postoperative LV dysfunction.

Abstract Background According to guidelines, every soft tissue tumor (STT) larger than 3 cm should be biopsied before definitive resection. Advances in magnetic resonance imaging (MRI) improve the possibility to give a provisional diagnosis of the tumor’s entity. Can lipomas and atypical lipomatous tumors (ALTs) of the extremities therefore be primarily marginally resected based on interpretation of MR images without a previous biopsy?. Methods In this retrospective, single-center study, 240 patients with the suspicion of a lipomatous tumor in MRI and surgical treatment in our institution between 2011 and 2020 were included. MR imaging was performed before surgery. All resected specimens underwent histopathological analysis. Results The collective comprised 142 tumors that were suspected as lipoma or ALT by the radiologist and underwent primary marginal resection (PMR). One case had myxoid liposarcoma that was underestimated on MRI and needed radical follow-up resection. One-hundred forty-one patients were cured after PMR. Ninety-eight patients were biopsied initially and in 93 cases resected afterwards according to the necessary oncological margins. Conclusion In our institution, PMR is performed if a lipoma or ALT is suspected on MR imaging. Our treatment method and the diagnostic algorithm are presented. Primary resection spares patients from one surgical procedure, but a slight risk for underestimation of the tumor remains.

Abstract To compare the patient radiation doses during angiographic selective adrenal vein sampling (AVS) before and after an imaging technology upgrade. In this retrospective single-center-study, cumulative air kerma (AK), cumulative dose area product (DAP), fluoroscopy time and contrast agent dosage were recorded from 70 patients during AVS. 35 procedures were performed before and 35 after an imaging processing technology upgrade. Mean values were calculated and compared using an unpaired student’s t-test. DSA image quality was assessed independently by two blinded readers using a four-point Likert scale (1 = poor; 4 = excellent) and compared using Wilcoxon signed-rank test. After the technology upgrade we observed a significant reduction of 35% in AK (1.7 ± 0.7 vs. 1.1 ± 0.7 Gy, p = 0.01) and a significant reduction of 28% in DAP (235.1 ± 113 vs. 170.1 ± 94 Gy*cm2, p = 0.01) in comparison to procedures before the upgrade. There were no significant differences between the number of exposure frames (143 ± 86 vs. 132 ± 61 frames, p = 0.53), fluoroscopy time (42 ± 23 vs. 36 ± 18 min, p = 0.22), or the amount of contrast medium used (179.5 ± 84 vs. 198.1 ± 109 ml, p = 0.41). There was also no significant difference regarding image quality (3 (2–4) vs. 3 (2–4), p = 0.67). The angiographic imaging technology upgrade significantly decreases the radiation dose during adrenal vein sampling without increasing time of fluoroscopy or contrast volume and without compromising image quality.

Abstract Background This cardiovascular magnetic resonance (CMR) study investigates the impact of trabeculae and papillary muscles (TPM) on diastolic function parameters by differentiation of the time-volume curve. Differentiation causes additional problems, which is overcome by standardization. Methods Cine steady-state free-precession imaging at 1.5 T was performed in 40 healthy volunteers stratified for age (age range 7–78y). LV time-volume curves were assessed by software-assisted delineation of endocardial contours from short axis slices applying two different methods: (1) inclusion of TPM into the myocardium and (2) inclusion of TPM into the LV cavity blood volume. Diastolic function was assessed from the differentiated time-volume curves defining the early and atrial peaks, their filling rates, filling volumes, and further dedicated diastolic measures, respectively. Results Only inclusion of TPM into the myocardium allowed precise assessment of early and atrial peak filling rates (EPFR, APFR) with clear distinction of EPFR and APFR expressed by the minimum between the early and atrial peak (EAmin) (100% vs. 36% for EAmin

Nanobodies are well suited for constructing biologics due to their high solubility. We generated nanobodies directed against CD38, a tumor marker that is overexpressed by multiple myeloma and other hematological malignancies. We then used these CD38-specific nanobodies to construct heavy chain antibodies, bispecific killer cell engagers (BiKEs), chimeric antigen receptor (CAR)-NK cells, and nanobody-displaying AAV vectors. Here we review the utility of these nanobody-based constructs to specifically and effectively target CD38-expressing myeloma cells. The promising results of our preclinical studies warrant further clinical studies to evaluate the potential of these CD38-specific nanobody-based constructs for treatment of multiple myeloma.

RationaleRecent studies have demonstrated the feasibility of CD38-specific antibody constructs for in vivo imaging of multiple myeloma. However, detecting multiple myeloma in daratumumab-pretreated patients remains difficult due to overlapping binding epitopes of the CD38-specific imaging antibody constructs and daratumumab. Therefore, the development of an alternative antibody construct targeting an epitope of CD38 distinct from that of daratumumab is needed. We report the generation of a fluorochrome-conjugated nanobody recognizing such an epitope of CD38 to detect myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo.MethodsWe conjugated the CD38-specific nanobody JK36 to the near-infrared fluorescent dye Alexa Fluor 680. The capacity of JK36AF680 to bind and detect CD38-expressing cells pretreated with daratumumab was evaluated on CD38-expressing tumor cell lines in vitro, on primary myeloma cells from human bone marrow biopsies ex vivo, and in a mouse tumor model in vivo.ResultsFluorochrome-labeled nanobody JK36AF680 showed specific binding to CD38-expressing myeloma cells pretreated with daratumumab in vitro and ex vivo and allowed for specific imaging of CD38-expressing xenografts in daratumumab-pretreated mice in vivo.ConclusionsOur study demonstrates that a nanobody recognizing a distinct, non-overlapping epitope of CD38 allows the specific detection of myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo.

Abstract Transjugular intrahepatic portosystemic shunt (TIPS) reduces portal hypertension in patients with liver cirrhosis. The exact cardiac consequences of subsequent increase of central blood volume are unknown. Cardiovascular magnetic resonance (CMR) imaging is the method of choice for quantifying cardiac volumes and ventricular function. The aim of this study was to investigate effects of TIPS on the heart using CMR, laboratory, and imaging cardiac biomarkers. 34 consecutive patients with liver cirrhosis were evaluated for TIPS. Comprehensive CMR with native T1 mapping, transthoracic echocardiography, and laboratory biomarkers were assessed before and after TIPS insertion. Follow-up (FU) CMR was obtained in 16 patients (47%) 207 (170–245) days after TIPS. From baseline (BL) to FU, a significant increase of all indexed cardiac chamber volumes was observed (all P

Germ cell tumors (GCTs) are a common malignancy in males, with variations in differentiation making different therapeutic strategies necessary. Generally, GCTs show good curation rates due to their good response to radiotherapy or chemotherapy. However, certain subtypes are resistant to these therapies and require surgery. We present a case of a 25-year-old patient with a large retroperitoneal GCT with somatic malignant transformation, where resection of large abdominal blood vessels with complex reconstruction was necessary to completely remove the tumor. The tumor was completely resected, and the patient has since been recurrence-free in the follow-up period. GCTs with somatic transformation show high resistance rates to chemo- and radiotherapy, and the patient in the presented case study did indeed show only a limited response to carboplatin-based chemotherapy. Patients suffering from these conditions should be resected whenever possible, as curation can be achieved by complete tumor resection. Infiltration of neighboring structures is no contraindication to surgery. The case presented here shows that interdisciplinary surgical planning including vascular and general surgeons as well as radiologists is vital to ensure successful tumor resection.

CD38 is a target for immunotherapy of multiple myeloma. Llama-derived CD38-specific nanobodies allow easy reformatting into mono-, bi- and multispecific proteins. To evaluate the utility of nanobodies for constructing CD38-specific nanobody-based killer cell engagers (nano-BiKEs), we generated half-life extended nano-BiKEs (HLE-nano-BiKEs) by fusing a CD38-specific nanobody to a CD16-specific nanobody for binding to the Fc-receptor on NK cells and further to an albumin-specific nanobody to extend the half-life in vivo. HLE-nano-BiKEs targeting three different epitopes (E1, E2, E3) of CD38 were expressed in transiently transfected HEK-6E cells. We verified specific and simultaneous binding to CD38 on myeloma cells, CD16 on NK cells, and to albumin. We tested the capacity of these HLE-nano-BiKEs to mediate cytotoxicity against CD38-expressing multiple myeloma cell lines and primary myeloma cells from human bone marrow biopsies in bioluminescence and flowcytometry assays with NK92 cells as effector cells. The results revealed specific time- and dose-dependent cytolysis of CD38+ myeloma cell lines and effective depletion of CD38-expressing multiple myeloma cells from primary human bone marrow samples. Our results demonstrate the efficacy of CD38-specific HLE-nano-BiKEs in vitro and ex vivo, warranting further preclinical evaluation in vivo of their therapeutic potential for the treatment of multiple myeloma.

Abstract Background Aortic valve repair has become a treatment option for adults with symptomatic bicuspid (BAV) or unicuspid (UAV) aortic valve insufficiency. Our aim was to demonstrate the feasibility of 4D flow cardiovascular magnetic resonance (CMR) to assess the impact of aortic valve repair on changes in blood flow dynamics in patients with symptomatic BAV or UAV. Methods Twenty patients with adult congenital heart disease (median 35 years, range 18–64; 16 male) and symptomatic aortic valve regurgitation (15 BAV, 5 UAV) were prospectively studied. All patients underwent 4D flow CMR before and after aortic valve repair. Aortic valve regurgitant fraction and systolic peak velocity were estimated. The degree of helical and vortical flow was evaluated according to a 3-point scale. Relative flow displacement and wall shear stress (WSS) were quantified at predefined levels in the thoracic aorta. Results All patients underwent successful aortic valve repair with a significant reduction of aortic valve regurgitation (16.7 ± 9.8% to 6.4 ± 4.4%, p

PurposeAdrenal vein sampling (AVS) is the reference standard for evaluation of lateralized hormone production in primary aldosteronism. We aimed to investigate the impact of pre-interventional right renal vein (RRV) to right adrenal vein (RAV) distance measurement on fluoroscopy time, contrast agent exposure and radiation dose during AVS.Materials and methodsForty-five patients with primary aldosteronism undergoing AVS were enrolled in our retrospective study and divided into three groups. In the group "ruler" (n = 14), RRV-RAV-distances were determined pre-interventionally by cross-sectional imaging (CT/MRI) and AVS was performed by one interventional radiologist with limited experience in AVS. CT/MRI-derived and fluoroscopy-derived RRV-RAV-distances were correlated for aimed cannulation of the RAV. Patients in group "no ruler" (n = 24, three interventional radiologists with limited experience in AVS) and in group "expert", (n = 7, one expert interventional radiologist) underwent AVS without pre-interventional estimation of RRV-RAV-distances. Procedure parameters (fluoroscopy time, contrast agent volume, radiation dose) of group "ruler" were compared to both other groups by Kruskal-Wallis rank-sum test.ResultsCorrelation of CT/MRI-derived and fluoroscopy-derived RRV-RAV-distances was good (r = 0.74;p = 0.003). The median RRV-RAV-distance was 4.5cm at CT/MRI (95%-CI:4.2-5.0cm) and 4.0cm at fluoroscopy (95%-CI:3.8-4.5cm). Fluoroscopy time (p0.05).ConclusionsCT/MRI-derived pre-interventional renal-adrenal vein distance measurements correlate well with angiographic distance measurements. Pre-interventional estimation of the RRV-RAV-distance allows for aimed cannulation of the RAV with potential reduction of fluoroscopy time, contrast agent exposure and radiation-dose during AVS.

Purpose Transabdominal ultrasound (US) and magnetic resonance enterography (MRE) are used to assess disease activity and extent in IBD, but their impact on therapeutic decisions is unclear. Therefore, our study has two goals: to compare the usefulness of US and MRE in assessing disease extent and activity in the small and large bowel, and to determine the relevance for clinical decisions in IBD. Materials and Methods We included 54 IBD patients who had undergone both MRE and US within three months. We used the construct reference standard model to compare MRE and US for detecting inflammation and examined the impact on clinical decisions in IBD patients. Results In 54 IBD patients (44 patients Crohn’s disease (CD), 5 ulcerative colitis (UC), 5 indeterminate colitis (IC)), 42 patients (77.8%) showed inflammation either in the small or large bowel. Small bowel disease was present in 34 patients (77.3%). Complications were found in 19 patients (35.2%). MRE and US both showed high sensitivity (90.5 and 88.1%) and moderate specificity (50% in MRE and US) for detecting inflammation. MRE revealed higher sensitivity than US for detecting conglomerate tumors without statistical significance (85.7 vs. 71.4%, p=1.0) and equal specificity (97.9 vs 97.7, p=1.0). Therapeutic decisions included steroids in 20 patients (47.6%) and surgery/percutaneous drainage in six patients (14.3%), these decisions were triggered by results of US or MRE in equal distribution. Conclusion US and MRE have comparable sensitivity and specificity for detecting intestinal inflammation and complications in IBD patients. Therefore, both methods are sufficient for making clinical decisions.

BackgroundTo identify magnetic resonance (MR) angiography derived predictors of progressive dilatation and surgery of the aortic root in Marfan syndrome.Material and methodsWe retrospectively included 111 patients (32.7±16.5 years, range: 7-75 years) with a total of 446 MR angiographies. Aortic diameter growth rates of the entire thoracic aorta and Z-scores were estimated from annual diameter measurements. Aortic root shape was subdivided into three different types: (T0) normal; (T1) localized dilatation; (T2) generalized aortic root dilatation. Aortic diameter, Z-score, age, and aortic root shape at baseline were tested as predictors of aortic root dilatation using a multivariate logistic regression model.ResultsThe highest aortic growth rate was observed at the level of the sinuses of Valsalva. Higher aortic root diameters and Z-scores at baseline predicted an increased growth of the aortic root (p = 0.003 and p0.05). However, significantly more patients undergoing surgery had a generalized aortic dilatation (19/28, 76.9%) than a localized aortic root dilatation (9/28, 32.1%) (p = 0.001).ConclusionLarger baseline aortic root diameter and Z-score as well as young age predict solely progressive aortic root dilatation in Marfan patients. MR angiography derived type of aortic root shape does not predict aortic growth, but patients with generalized aortic root dilatation are referred more frequently for aortic surgery.

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

Neurofibromatosis type-1 (NF1) patients suffer from cutaneous and subcutaneous neurofibromas (CNF) and large plexiform neurofibromas (PNF). Whole gene deletions of the NF1 gene can cause a more severe phenotype compared to smaller intragenic changes. Two distinct groups of NF1 whole gene deletions are type-1 deletions and atypical deletions. Our aim was to assess volumes and averaged annual growth-rates of CNF and PNF in patients with NF1 whole gene deletions and to compare these with NF1 patients without large deletions of the NF1 gene. We retrospectively evaluated 140 whole-body MR examinations of 38 patients with NF1 whole gene deletions (type-1 group: n = 27/atypical group n = 11) and an age- and sex matched collective of 38 NF1-patients. Age-dependent subgroups were created (0-18 vs >18 years). Sixty-four patients received follow-up MRI examinations (NF1whole gene deletion n = 32/control group n = 32). Whole-body tumor-volumes were semi-automatically assessed (MedX, V3.42). Tumor volumes and averaged annual growth-rates were compared. Median tumor-burden was significantly higher in the type-1 group (418ml; IQR 77 - 950ml, p = 0.012) but not in the atypical group (356ml;IQR 140-1190ml, p = 0.099) when compared to the controls (49ml; IQR 11-691ml). Averaged annual growth rates were significantly higher in both the type-1 group (14%/year; IQR 45-36%/year, p = 0.004) and atypical group (11%/year; IQR 5-23%/year, p = 0.014) compared to the controls (4%/year; IQR1-8%/year). Averaged annual growth rates were significantly higher in pediatric patients with type-1 deletions (21%/year) compared with adult patients (8%/year, p = 0.014) and also compared with pediatric patients without large deletions of the NF1 gene (3.3%/year, p = 0.0015). NF1 whole gene deletions cause a more severe phenotype of NF1 with higher tumor burden and higher growth-rates compared to NF1 patients without large deletions of the NF1 gene. In particular, pediatric patients with type-1 deletions display a pronounced tumor growth.

Appendicular soft tissue lymphoma (ASTL) is rare and is frequently misinterpreted as soft tissue sarcoma (STS). Studies investigating magnet resonance imaging (MRI) characteristics of ASTL are scarce and showed heterogenous investigation criteria and results. The purpose of this study was to systematically review clinical presentations and MRI characteristics of ASTL as described in the current literature. For that purpose, we performed a systematic literature review in compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Patient demographics, clinical presentation and MRI imaging characteristics of ASTL were investigated, resulting in a total of nine included studies reporting a total of 77 patients. Signal intensity of lymphoma compared to muscle tissue was mostly described as isointense (53%) or slightly hyperintense (39%) in T1-weighted images and always as hyperintense in proton-and T2-weighted images. Multicompartmental involvement was reported in 59% of cases and subcutaneous stranding in 74%. Long segmental involvement was present in 80% of investigated cases. Involvement of neurovascular structures was reported in 41% of cases and the presence of traversing vessels in 83% of patients. The presence of these findings should lead to the inclusion of ASTL in the differential diagnosis of soft tissue masses.

Intramuscular myxomas (IMMs) are benign tumors. Evidence regarding diagnostic and therapeutic pathways is rare, and guidelines do not exist due to their low incidence. The aim of this study was a retrospective analysis at a university cancer center and the interdisciplinary re-evaluation of the individual diagnostic and therapeutic procedures. Overall, 38 patients were included in the study. IMMs occurred mostly in middle-aged women. At the time of first consultation, 57.9% had few symptoms or were asymptomatic. In 92.1% of the cases, the tumor was localized in the extremities. The lower extremity was affected in 73.7%. The average size of IMMs was 5.0 cm. The proximally located tumors in the gluteus, thighs, and upper arms were significantly larger (p = 0.02) than the distally-located tumors in the forearms and lower legs. An MRI was performed in 97.4%. Based on imaging, an IMM was suspected in 5.6% by radiologists and in 54.1% by musculoskeletal surgeons. An incision biopsy was performed in 68.4% and led in 100.0% to the right histopathological diagnosis. In total, 89.5% of IMMs were resected. Postoperative complications requiring revision occurred in 8.8%. Recurrences or degenerations of IMMs were not reported in any of these cases.

Abstract Background Contrast-enhanced cardiovascular magnetic resonance angiography (CE-CMRA) is the established imaging modality for patients with Marfan syndrome requiring life-long annual aortic imaging before and after aortic root replacement. Contrast-free CMRA techniques avoiding side-effects of contrast media are highly desirable for serial imaging but have not been evaluated in the postoperative setup of Marfan patients. The purpose of this study was to assess the feasibility of non-contrast balanced steady-state free precession (bSSFP) magnetic resonance imaging for aortic monitoring of postoperative patients with Marfan syndrome. Methods Sixty-four adult Marfan patients after aortic root replacement were prospectively included. Fourteen patients (22%) had a residual aortic dissection after surgical treatment of type A dissection. bSSFP imaging and CE-CMRA were performed at 1.5 Tesla. Two radiologists evaluated the images regarding image quality (1 = poor, 4 = excellent), artifacts (1 = severe, 4 = none) and aortic pathologies. Readers measured the aortic diameters at defined levels in both techniques. Statistics included observer agreement for image scoring and diameter measurements and ROC analyses for comparison of the diagnostic performance of bSSFP and CE-CMRA. Results Both readers observed no significant differences in image quality between bSSFP and CE-CMRA and found a median image quality score of 4 for both techniques (all p > .05). No significant differences were found regarding the frequency of image artifacts in both sequences (all p > .05). Sensitivity and specificity for detection of aortic dissections was 100% for both readers and techniques. Compared to bSSFP imaging, CE-CMRA resulted in higher diameters (mean bias, 0.9 mm; p .05), except for the distal graft anastomosis (p = .001). Using both techniques, the readers correctly identified a graft suture dehiscence with aneurysm formation requiring surgery. Conclusion Unenhanced bSSFP CMR imaging allows for riskless aortic monitoring with high diagnostic accuracy in Marfan patients after aortic root surgery.

INTRODUCTION:Patients with Neurofibromatosis type 1 (NF1) develop plexiform neurofibromas (PNF) and cutaneous neurofibromas. These tumors are a major cause of the patient's morbidity and mortality. An influence of estrogen and progesterone on tumor growth has been suggested but reports on growth or malignant transformation of tumors during pregnancy remain anecdotal. The purpose of this study was to quantify growth of cutaneous and plexiform neurofibromas in NF1 patients during pregnancy, and to assess the onset of NF1 related symptoms. MATERIAL AND METHODS:Retrospectively, 13 mothers with NF1 were included and compared to nullipara, nulligravida, age-matched women with NF1. All women received whole-body magnetic resonance imaging (MRI) before and after pregnancy or after a matched time period. Presence of plexiform and cutaneous neurofibromas was evaluated. PNF were subjected to semi-automated volumetry (MedX). The sum of the longest diameters (SLD) of representative cutaneous neurofibromas was determined for both groups. Clinical symptoms and subjective tumor growth were assessed. RESULTS:PNF were identified in 12/26 women (46.2%). Follow up showed neither new PNF nor a significant difference in growth rate (median tumor-growth/year: pregnant group-0.38% (IQR -1.1-5.4%) vs control group 3.59% (IQR -2.1-5.5%; P = 0.69). Malignant transformation of PNF was not observed. There was a significant growth of cutaneous neurofibromas in both groups (median SLD increase: pregnant group 17mm; P = 0.0026 / control group 12mm; P = 0.0004) The difference in increase of SLD was not significant (P = 0.48). Singular cutaneous neurofibromas in the pregnant group displayed high levels of tumor growth (>20%/year). NF1-associated symptoms and subjective tumor growth were not significantly increased in pregnant patients. CONCLUSIONS:Growth of plexiform and cutaneous neurofibromas in pregnant patients is not significantly different compared to non-pregnant patients. Cutaneous neurofibromas show a significant increase in growth over time in both, pregnant and non-pregnant patients and NF1 related clinical symptoms do not significantly aggravate during the course of pregnancy.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

The NAD+-metabolizing ectoenzyme CD38 is an established therapeutic target in multiple myeloma. The CD38-specific monoclonal antibodies daratumumab and isatuximab show promising results in the clinic. Nanobodies correspond to the single variable domains (VHH) derived from heavy chain antibodies that naturally occur in camelids. VHHs display high solubility and excellent tissue penetration in vivo. We recently generated a panel of CD38-specific nanobodies, some of which block or enhance the enzymatic activity of CD38. Fusion of such a nanobody to the hinge, CH2, and CH3 domains of human IgG1 generates a chimeric llama/human hcAb of about half the size of a conventional moAb (75 vs. 150 kDa). Similarly, a fully human CD38-specific hcAb can be generated using a CD38-specific human VH3 instead of a CD38-specific camelid nanobody. Here we discuss the advantages and disadvantages of CD38-specific hcAbs vs. conventional moAbs and provide an outlook for the potential use of CD38-specific hcAbs as novel therapeutics for multiple myeloma.

Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.

The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated.

The objective of this study was to quantify enzymatic activity on the surface of T cells by magnetic resonance imaging (MRI) using R2 and R2* relaxometry. Lymphoma cells expressing adenosine diphosphate (ADP)-ribosyltransferase 2 (ART2) were incubated with increasing doses of its substrate etheno–nicotinamide adenine dinucleotide (NAD), resulting in increasing amounts of surface protein ADP-ribosylation. Etheno-ADP-ribosylated proteins were detected with monoclonal antibody 1G4 and superparamagnetic iron oxide conjugated secondary antibodies (Ab-SPIO). Labeling efficiency was determined with R2 and R2* relaxometry on a clinical 3.0 T scanner. Parallel aliquots of cells were analyzed by flow cytometry. Cell-bound SPIO conjugates were detected by immunofluorescence and electron microscopy and quantified by atomic absorption spectroscopy. To mimic an inflammatory site in vivo, Ab-SPIO-labeled cells were injected subcutaneously in mice and analyzed by MRI. Immunofluorescence and electron microscopy confirmed cell-surface localization of Ab-SPIO. MRI of Ab-SPIO-labeled cells showed a corresponding signal reduction. Increases in R2 and R2* determined by magnetic resonance relaxometry correlated linearly with the expression level of ART2 and the concentration of the ART2 substrate etheno-NAD. R2 and R2* increases correlated linearly with the results from flow cytometry and atomic absorption spectroscopy analyses. Quantitative R2 and R2* mapping enable noninvasive determination of enzymatic activity on T cells and holds promise for characterization of inflammatory sites in vivo by MRI.