CD19-immunoPET for noninvasive visualization of CD19 expression in B-cell lymphoma patients.

Cell- and antibody-based CD19-directed therapies have demonstrated great potential for treating B-cell non-Hodgkin lymphoma (B-NHL). However, all these approaches suffer from limited response rates and considerable toxicity. Until now, therapy decisions have been routinely based on histopathological CD19 staining of a single lesion at initial diagnosis or relapse, disregarding heterogeneity and temporal alterations in antigen expression. To visualize in vivo CD19 expression noninvasively, we radiolabeled anti-human CD19 monoclonal antibodies with copper-64 (64Cu-αCD19) for positron emission tomography (CD19-immunoPET). 64Cu-αCD19 specifically bound to subcutaneous Daudi xenograft mouse models in vivo. Importantly, 64Cu-αCD19 did not affect the anti-lymphoma cytotoxicity of CD19 CAR-T cells in vitro. Following our preclinical validation, 64Cu-αCD19 was injected into four patients with follicular lymphoma, diffuse large B-cell lymphoma or mantle zone lymphoma. We observed varying 64Cu-αCD19 PET uptake patterns at different lymphoma sites, both within and among patients, correlating with ex vivo immunohistochemical CD19 expression. Moreover, one patient exhibited enhanced uptake in the spleen compared to that in patients with prior B-cell-depleting therapy, indicating that 64Cu-αCD19 is applicable for identifying B-cell-rich organs. In conclusion, we demonstrated the specific targeting and visualization of CD19+ B-NHL in mice and humans by CD19-immunoPET. The intra- and interindividual heterogeneous 64Cu-αCD19 uptake patterns of lymphoma lesions indicate variability in CD19 expression, suggesting the potential of CD19-immunoPET as a novel tool to guide CD19-directed therapies.

Translational immunoPET imaging using a radiolabeled GD2-specific antibody in neuroblastoma.

Background: Antibodies targeting surface expressed disialoganglioside GD2 are increasingly used in neuroblastoma immunotherapy and might also have potential for use in radioimmunotherapy. As such targeted treatments might benefit from a dedicated theranostic approach, we studied the influence of radiolabeling on the binding characteristics of ch14.18 antibodies produced by Chinese hamster ovary (CHO) cells and evaluated the benefit of GD2-ImmunoPET as a potential tool for therapy planning. Methods:64Cu was used to reduce radiation burden, which is of high importance especially in a pediatric patient population. 64Cu-labeling was accomplished using the chelators NOTA- or DOTAGA-NCS. Radiolabeled antibodies were characterized in vitro. [64Cu]Cu-DOTAGA-ch14.18/CHO was studied in a neuroblastoma mouse model (subcutaneous CHP-134 xenografts). In vivo PET and MR images were acquired at 3 h, 24 h, and 48 h p.i. The specificity of binding was verified using GD2-negative tumors (HEK-293 xenografts), a control antibody and in vivo blocking. A first translational application was performed by PET/MRI in a patient with metastasized neuroblastoma. Results: Radiolabeling at an antibody-to-chelator ratio ≥1:10 yielded a product with a radiochemical purity of ≥90% and a specific activity of 0.2-1.0 MBq/µg. Radiochelation was stable over 48 h in PBS, mouse serum or EDTA, and 50.8 ± 3.5% and 50.8 ± 2.0% of the radiolabeled conjugates, prepared at antibody-to-chelator ratios of 1:10 or 1:15, were immunoreactive. In vivo, highly specific accumulation (31.6 ± 5.8% ID/g) in neuroblastoma was shown preclinically. Clinical PET/MR scans using [64Cu]Cu-NOTA-ch14.18/CHO (NOTA used for safety reasons) could visualize neuroblastoma metastases. Conclusions:In vivo,64Cu-labeled ch14.18/CHO is suitable for specific identification of neuroblastoma in PET. A first patient PET indicated the feasibility of the method for clinical translation and the potential utility in image-guided therapy.

Using Human-Centered Design to Improve a Surgery Resident Well-Being Program.

INTRODUCTION: Surgery resident mental health, burnout, and overall well-being are constantly scrutinized, and improving surgery resident well-being programs continuously requires refinement. We sought to evaluate the effectiveness of human-centered design (HCD) sprints to enhance our surgery resident well-being program. METHODS: An HCD sprint was conducted with 34 surgery residents in a single session using seven separate domains, including Mental Health/Reflection and Therapy; Mentoring or Faculty Engagement; Physical Well-being; Retreats; Scheduled Breaks or Free Time; Social Connection; and Well-being Lectures, Emails, or Curriculum. Responses were characterized as: "How might we", Suggestions, Useful, and Not Useful. RESULTS: Well-being Lectures, Emails, or Curriculum were overwhelmingly viewed, as Not Useful (77%), as was Mental Health/Reflection and Therapy (42%). Scheduled Breaks or Free Time was viewed as the most Useful (42%). This category also had the most suggestions and "How might we" ideas for improvement (41%). Lastly, Suggestions and "How might we" ideas were also common for improving Mentoring or Faculty Engagement (31% and 29%, respectively). These results were incorporated into multiple strategies to improve surgery resident well-being and also shared in a Department of Surgery Grand Rounds. CONCLUSIONS: Surgery resident well-being and a targeted approach by a well-being program are critical to a residency program, particularly with the arduous nature of surgical training during the pandemic resulting in periods of prolonged social isolation. HCD sprints are an effective means to refine a surgery resident well-being program and to involve the residents themselves in that process.

Impact of TSPO Receptor Polymorphism on [18F]GE-180 Binding in Healthy Brain and Pseudo-Reference Regions of Neurooncological and Neurodegenerative Disorders.

TSPO-PET tracers are sensitive to a single-nucleotide polymorphism (rs6971-SNP), resulting in low-, medium- and high-affinity binders (LABs, MABs and HABS), but the clinical relevance of [18F]GE-180 is still unclear. We evaluated the impact of rs6971-SNP on in vivo [18F]GE-180 binding in a healthy brain and in pseudo-reference tissue in neuro-oncological and neurodegenerative diseases. Standardized uptake values (SUVs) of [18F]GE-180-PET were assessed using a manually drawn region of interest in the frontoparietal and cerebellar hemispheres. The SUVs were compared between the LABs, MABs and HABs in control, glioma, four-repeat tauopathy (4RT) and Alzheimer's disease (AD) subjects. Second, the SUVs were compared between the patients and controls within their rs6971-subgroups. After excluding patients with prior therapy, 24 LABs (7 control, 5 glioma, 6 4RT and 6 AD) were analyzed. Age- and sex-matched MABs (n = 38) and HABs (n = 50) were selected. The LABs had lower frontoparietal and cerebellar SUVs when compared with the MABs and HABs, but no significant difference was observed between the MABs and HABs. Within each rs6971 group, no SUV difference between the patients and controls was detected in the pseudo-reference tissues. The rs6971-SNP affects [18F]GE-180 quantification, revealing lower binding in the LABs when compared to the MABs and HABs. The frontoparietal and cerebellar ROIs were successfully validated as pseudo-reference regions.

Dual-Phase ß-Amyloid PET Captures Neuronal Injury and Amyloidosis in Corticobasal Syndrome.

Objectives: In recent years several 18F-labeled amyloid PET (Aß-PET) tracers have been developed and have obtained clinical approval. There is evidence that Aß-PET perfusion can provide surrogate information about neuronal injury in neurodegenerative diseases when compared to conventional blood flow and glucose metabolism assessment. However, this paradigm has not yet been tested in neurodegenerative disorders with cortical and subcortical affection. Therefore, we investigated the performance of early acquisition 18F-flutemetamol Aß-PET in comparison to 18F-fluorodeoxyglucose (FDG)-PET in corticobasal syndrome (CBS). Methods: Subjects with clinically possible or probable CBS were recruited within the prospective Activity of Cerebral Networks, Amyloid and Microglia in Aging and Alzheimer's Disease (ActiGliA) observational study and all CBS cases with an available FDG-PET prior to Aß-PET were selected. Aß-PET was acquired 0-10 min p.i. (early-phase) and 90-110 min p.i. (late-phase) whereas FDG-PET was recorded statically from 30 to 50 min p.i. Semiquantitative regional values and asymmetry indices (AI) were compared between early-phase Aß-PET and FDG-PET. Visual assessments of hypoperfusion and hypometabolism were compared between both methods. Late-phase Aß-PET was evaluated visually for assessment of Aß-positivity. Results: Among 20 evaluated patients with CBS, 5 were Aß-positive. Early-phase Aß-PET and FDG-PET SUVr correlated highly in cortical (mean R = 0.86, range 0.77-0.92) and subcortical brain regions (mean R = 0.84, range 0.79-0.90). Strong asymmetry was observed in FDG-PET for the motor cortex (mean |AI| = 2.9%), the parietal cortex (mean |AI| = 2.9%), and the thalamus (mean |AI| = 5.5%), correlating well with AI of early-phase Aß-PET (mean R = 0.87, range 0.62-0.98). Visual assessments of hypoperfusion and hypometabolism were highly congruent. Conclusion: Early-phase Aß-PET facilitates assessment of neuronal injury in CBS for cortical and subcortical areas. Known asymmetries in CBS are captured by this method, enabling assessment of Aß-status and neuronal injury with a single radiation exposure at a single visit.

Circadian rhythm impacts preclinical FDG-PET quantification in the brain, but not in xenograft tumors.

The inner clock of biological organisms plays a pivotal role and has strong effects on metabolic processes such as glucose consumption. Since the commonly used positron emission tomography (PET) tracer 18F-flourodeoxygucose (FDG) is a glucose analogue, it is not surprising that the FDG distribution in mice and humans has been shown to succumb to daily rhythms. In preclinical studies, the circadian rhythm of animals is often not considered, and studies are performed at different times of day. Only a few studies have analyzed the effect of the circadian rhythm on FDG uptake in mice, and none of these studies included human tumor xenografts. Therefore, it is not known how strongly a preclinical tumor study is influenced by the time of day. In this work, the effect of the circadian rhythm on FDG uptake in human tumor xenografts and other organs was analyzed. CD1 nu/nu mice were kept for three weeks under a 12 h light/12 h dark rhythm and then injected s.c. with PC3 or A431 tumor cells. When the tumors had reached an appropriate volume, FDG-PET scans were performed on different animal groups (n = 4-5) every 4 h over a time period from 8 A.M. to 8 P.M. Tracer uptake in the tumors and in other organs was determined based on the PET scans and biodistribution studies. The standardized uptake value and %injected dose/cc of the tumors remained constant over the whole observed time period, and no statistically significant differences were determined according to the PET analysis. In the brain, we found a small but statistically significant increase from noon to 4 P.M., which led to a decrease in the tumor-to-brain ratio. No evidence for an effect of the circadian rhythm on FDG uptake could be found in subcutaneous tumors, however, in brain studies the circadian rhythm needs to be considered.

LESS ovariohysterectomy in cats using a new homemade multiport / Ovário-histerectomia por LESS em gatas com um novo multiportal artesanal

ABSTRACT: The purpose of this study was to assess the use of a homemade multiport for LESS (laparoendoscopic single-site surgery) ovariohysterectomy (OVH) in cats. Intra and postoperative variables of the surgery steps, technical challenges, complications and evolution of surgical time by a surgeon in training were evaluated. Twenty queens were selected for LESS OVH. The multiport device was manufactured of a conical-shaped ethylene polytereftalate (PET) bottle, urethral catheter no. 8, latex balloon no. 11, a 3.5mm and a 6mm laparoscopic trocars. Hemostasis was carried out using bipolar diathermy. Mean total surgical time was 14.54±5.12 minutes. Approach to right and left ovarian pedicles and abdominal access for insertion of the multiport device were the most time consuming surgical steps. LESS ovariohysterectomy using a new homemade multiport device is feasible and safe. Thus, the proposed technique may be considered as a minimally invasive alternative to ovariohysterectomy in the feline specie.

RESUMO: Objetivou-se avaliar o emprego do novo dispositivo multiportal artesanal para realização de ovariohisterectomia por LESS (cirurgia laparoendoscópica por único acesso) em gatas, analisando variáveis intraoperatórias de tempo cirúrgico das diferentes etapas que compõem o procedimento, suas possíveis dificuldades técnicas de execução e complicações trans e pós-operatórias. Avaliou-se ainda a curva de aprendizado deste procedimento, realizado por um cirurgião não proficiente na técnica. Vinte gatas foram submetidas à laparoscopia. Para a confecção do multiportal foi utilizado um recipiente de politereftalato de etileno (PET), uma sonda uretral nº8, um balão de látex nº11, um portal de videocirurgia de 3,5mm, e um de 6mm. O sistema de coagulação foi o bipolar. O tempo cirúrgico médio foi de 14,54±5,12 minutos. A abordagem aos pedículos ovarianos direito e esquerdo e o acesso para introdução do portal foram as etapas que apresentaram maior tempo de execução. A OVH videoassistida empregando o novo dispositivo multiportal é factível, não demonstrando complicações. Acredita-se, portanto, que a técnica proposta torna-se uma alternativa para realização minimamente invasiva de ovariohisterectomia em felinos.

Correlation between positron emission tomography and Cerenkov luminescence imaging in vivo and ex vivo using 64Cu-labeled antibodies in a neuroblastoma mouse model.

Antibody-based therapies gain momentum in clinical therapy, thus the need for accurate imaging modalities with respect to target identification and therapy monitoring are of increasing relevance. Cerenkov luminescence imaging (CLI) are a novel method detecting charged particles emitted during radioactive decay with optical imaging. Here, we compare Position Emission Tomography (PET) with CLI in a multimodal imaging study aiming at the fast and efficient screening of monoclonal antibodies (mAb) designated for targeting of the neuroblastoma-characteristic epitope disialoganglioside GD2. Neuroblastoma-bearing SHO mice were injected with a 64Cu-labeled GD2-specific mAb. The tumor uptake was imaged 3 h, 24 h and 48 h after tracer injection with both, PET and CLI, and was compared to the accumulation in GD2-negative control tumors (human embryonic kidney, HEK-293). In addition to an in vivo PET/CLI-correlation over time, we also demonstrate linear correlations of CLI- and γ-counter-based biodistribution analysis. CLI with its comparably short acquisition time can thus be used as an attractive one-stop-shop modality for the longitudinal monitoring of antibody-based tumor targeting and ex vivo biodistribution.These findings suggest CLI as a reliable alternative for PET and biodistribution studies with respect to fast and high-throughput screenings in subcutaneous tumors traced with radiolabeled antibodies. However, in contrast to PET, CLI is not limited to positron-emitting isotopes and can therefore also be used for the visualization of mAb labeled with therapeutic isotopes like electron emitters.

Improved selectivity of mIBG uptake into neuroblastoma cells in vitro and in vivo by inhibition of organic cation transporter 3 uptake using clinically approved corticosteroids.

INTRODUCTION: Radiolabeled meta-iodobenzylguanidine (mIBG) is used for imaging and therapy of neuroblastoma as well as pheochromocytoma. However, non-tumorous tissues also incorporate mIBG mainly by organic cation transporters (OCTs). In this study, we tested different clinically approved corticosteroids as potential inhibitors of the OCT3-mediated uptake in vitro and in vivo, to achieve a more selective mIBG tumor uptake. METHODS: The in vitro incorporation of [(3)H]norepinephrine ([(3)H]NE), [(3)H]dopamine ([(3)H]DA) and [(123)I]mIBG in neuroblastoma cells (SK-N-SH, Kelly, IMR-32) and in HEK-293 cells transfected with human OCT3 was measured with and without supplemental corticosteroids (hydrocortisone, prednisolone, dexamethasone, corticosterone). The in vivo biodistribution of [(123)I]mIBG in absence and presence of corticosteroids was studied in non-tumor bearing NOD scid gamma mice. Retrospectively, we selected patients with and without corticosteroid treatment prior to [(123)I]mIBG scintigraphy. RESULTS: A concentration-dependent inhibitory effect of different corticosteroids on the [(3)H]NE and [(3)H]DA uptake via OCT3 was illustrated in vitro. The highest OCT3 inhibition was observed for corticosterone, but clinically used corticosteroids, showed also promising inhibitory effects. In contrast, the uptake in neuroblastoma cells was reduced only moderately. Hydrocortisone or prednisolone had only minor effects on [(123)I]mIBG uptake of both neuroblastoma cells, but reduced uptake in OCT3 expressing cells significantly. In mice tissues, [(123)I]mIBG uptake was inhibited by corticosteroids especially in the small intestine and kidney. Finally, in one patient with hydrocortisone treatment performed prior to [(123)I]mIBG scan, heart and liver uptake was reduced compared to untreated patients. CONCLUSIONS: The OCT3 is widely spread in many organs and responsible for non-targeted uptake of radiolabeled mIBG. In our study, clinically approved corticosteroids inhibited mIBG uptake in OCT3 expressing cells effectively, whereas tracer accumulation in NT (norepinephrine transporter) expressing neuroblastoma cells showed consistency. We conclude, that a single dose of hydrocortisone or prednisolone prior to [(123)I]mIBG scintigraphy may improve specificity and reduce radiation dose to non-target organs.

Enhanced binding of necrosis-targeting immunocytokine NHS-IL12 after local tumour irradiation in murine xenograft models.

PURPOSE: NHS-IL12 is an immunocytokine targeting necrotic tumour areas. IL12 shows anti-tumour activity. As local irradiation might induce additional necrosis in solid tumours, we aimed to evaluate the increase in intratumoural accumulation of NHS-IL12 after irradiation and correlate the findings with diffusion-weighted MRI studies in two xenograft models. METHODS: Human rhabdomyosarcoma (A204) and prostate cancer (PC3) cells were studied in vitro and as subcutaneous xenografts. Radiation sensitivity of the cell lines was assessed in vitro by colony formation assays. In vivo tumour necrosis was assessed based on apparent diffusion coefficients (ADC). Biodistribution of NHS-IL12 was evaluated with and without tumour irradiation using in vivo small-animal PET and ex vivo biodistribution. RESULTS: A204 and PC3 differed in their intrinsic radiation sensitivity. Accordingly, radiation-induced tumour necrosis was found only in A204 xenografts. In comparison with control, ADC was significantly increased after irradiation of A204 tumours with 1 × 8.0 Gy and 5 × 2.0 Gy, whereas no change in ADC was observed in PC3 xenografts in all irradiation regimes. ADC correlated with histology. An enhanced uptake of radiolabelled NHS-IL12 in A204 tumours was detected by PET and ex vivo biodistribution after tumour irradiation. In PC3 tumours, no increase in NHS-IL12 uptake was observed. CONCLUSIONS: In dependence of the tumour model, tumour irradiation enhanced tumour necrosis measured in MRI and histology. In vivo PET and ex vivo biodistribution showed enhanced binding of NHS-IL12 in rhabdomyosarcoma xenografts. Thus, enhanced binding of necrosis-targeting immunocytokines might be a novel mechanism of additive effects in combination with irradiation.

Preclinical evaluation of the anti-tumor effects of the natural isoflavone genistein in two xenograft mouse models monitored by [18F]FDG, [18F]FLT, and [64Cu]NODAGA-cetuximab small animal PET.

UNLABELLED: The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. PROCEDURES: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [18F]FDG, [18F]FLT and [64Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. RESULTS: Genistein reduced tumor growth significantly in both xenografts. [18F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [18F]FDG and [64Cu]NODAGA-cetuximab were not suitable for therapy monitoring. CONCLUSIONS: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [18F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation.

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.