Therapeutic benefit of larotrectinib over the historical standard of care in patients with locally advanced or metastatic infantile fibrosarcoma (EPI VITRAKVI study).

BACKGROUND: Patients with infantile fibrosarcoma (IFS) have shown strong and long-lasting responses to larotrectinib, a tropomyosin receptor kinase inhibitor (TRKi), in single-arm clinical trials. Conventional chemotherapy has also shown important efficacy. But, until now, no comparative data exist. This study aims to assess the therapeutic benefit of larotrectinib over the current standard of care (SOC) of chemotherapy in paediatric patients with locally advanced or metastatic IFS. PATIENTS AND METHODS: EPI VITRAKVI is a retrospective, observational, externally controlled study (NCT05236257). Data of patients aged ≤21 years with locally advanced or metastatic IFS treated with larotrectinib in the phase I/II SCOUT trial (NCT02637687) were compared with those of an external historical control group (data of Institut Curie and Cooperative Weichteilsarkom Studiengruppe) treated with a chemotherapy-based regimen. Between-group differences were assessed after balancing groups using inverse probability of treatment weighting (IPTW). RESULTS: In total, 93 patients were compared, 51 in the larotrectinib arm and 42 in the external control arm. After therapy, 4 patients (7.8%) in the larotrectinib group had a medical treatment failure event [start of new systemic treatment (2 cases), mutilating surgery (2 cases)] versus 15 (35.7%) in the external control group [start of new systemic treatment (6 cases), mutilating surgery (5 cases), radiation therapy (2 cases), and death (2 cases)]. Larotrectinib was associated with an 80% reduced likelihood of encountering a medical treatment failure event, when compared to the external control group (weighted and stratified hazard ratio 0.20, 95% confidence interval 0.06-0.63, P = 0.0060). These results were confirmed by sensitivity analyses, including exact matching, and subgroup analyses for number of lines of treatment. CONCLUSIONS: Treatment with larotrectinib reduced the need of subsequent therapies compared to SOC with chemotherapy in children with locally advanced or metastatic IFS, regardless of the line of treatment.

Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196.

The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.

[Critical aspects of the transmandibular approach to the oral cavity and oropharynx]. / Kritische Aspekte des transmandibulären Zugangswegs zu Mundhöhle und Oropharynx.

BACKGROUND: In the context of tumor surgery, the median mandibulotomy as an access route to the oral cavity and oropharynx provides an excellent overview of the surgical site. However, it is not regarded entirely unproblematic with regard to early and later complications that may arise. OBJECTIVE: The results and complications of the median mandibulotomy will be presented based on data collected from our own patient collective. MATERIALS AND METHODS: A total of 21 patients who had undergone a median mandibulotomy as part of tumor surgery at the Department of Otorhinolaryngology of the SRH Zentralklinikum Suhl were examined over a period from 01 January 2010 to 31 December 2020. The patient files were retrospectively evaluated. RESULTS: A stair-step median mandibulotomy was performed in all 21 patients. Reconstruction was performed using a 2.8 mm thick angled mandibular plate and bicortical locking screws. The mean follow-up period was 29.8 months. In all, 7 patients (33%) had a recurrence at the time of surgery; 5 patients (24%) had already undergone pre-radiation. Furthermore, 18 patients (85.7%) received adjuvant radiotherapy. We found plate loosening or extrusion in 0 of 21 cases. A salivary fistula (4.7%) was observed in 1 patient. Trismus was found in 4 (19%) cases during follow-up. Osteoradionecrosis was found in 2 (9.5%) of 21 cases. Cosmetic deficits were not observed. CONCLUSION: Our results show that the stair-step median mandibulotomy in combination with a 2.8 mm thick reconstruction plate and bicortical locking screws leads to a stable and safe reconstruction even with pre-irradiated situations. Plate loosening or extrusion did not occur.

More Retrieval Attempts are Associated with Poorer Functional Outcome After Unsuccessful Thrombectomy.

PURPOSE: In mechanical thrombectomy, it has been hypothesized that multiple retrieval attempts might the improve reperfusion rate but not the clinical outcome. In order to assess a potential harmful effect of a mechanical thrombectomy on patient outcome, the number of retrieval attempts was analyzed. Only patients with a thrombolysis in cerebral infarction (TICI) score of 0 were reviewed to exclude the impact of eventual successful reperfusion on the mechanical hazardousness of repeated retrievals. METHODS: In this study 6635 patients who underwent endovascular thrombectomy (EVT) for acute large vessel occlusion (LVO) from the prospectively administered multicenter German Stroke Registry were screened. Insufficient reperfusion was defined as no reperfusion (TICI score of 0), whereas a primary outcome was defined as functional independence (modified Rankin scale [mRS] 0-2 at day 90). Propensity score matching and multivariable logistic regressions were then performed to adjust for confounders. RESULTS: A total of 377 patients (7.8%) had a final TICI score of 0 and were included in the study. After propensity score matching functional independence was found to be significantly more frequent in patients who underwent ≤ 2 retrieval attempts (14%), compared to patients with > 2 retrieval attempts (3.9%, OR 0.29, 95% CI 0.07-0.73, p = 0.009). After adjusting for age, sex, admission NIHSS score, and location of occlusion, more than two retrieval attempts remained significantly associated with lower odds of functional independence at 90 days (OR 0.2, 95% CI 0.07-0.52, p = 0.002). CONCLUSION: In patients with failure of reperfusion, more than two retrieval attempts were associated with a worse clinical outcome, therefore indicating a possible harmful effect of multiple retrieval attempts.

Efficient conversion of hemicellulose sugars from spent sulfite liquor into optically pure L-lactic acid by Enterococcus mundtii.

Spent sulfite liquor (SSL), a waste stream from wood pulp production, has great potential as carbon source for future industrial fermentations. In the present study, SSL was separated into a hemicellulose derived sugar syrup (HDSS) and a lignosulfonic fraction by simulated moving bed chromatography. The recovery of SSL sugars in the HDSS was 89% and the fermentation inhibitors furfural, 5-hydroxymethylfurfural and acetic acid were removed by 98.7%, 60.5% and 75.5%, respectively. The obtained sugars have been converted to L-lactic acid, a building block for bioplastics, by fermentation with the lactic acid bacterium Enterococcus mundtii DSM4838. Batch fermentations on HDSS produced up to 56.3 g/L L-lactic acid. Simultaneous conversion of pentose and hexose sugars during fed-batch fermentation of wildtype E. mundtii led to 87.9 g/L optically pure (>99%) L-lactic acid, with maximum productivities of 3.25 g/L.h and yields approaching 1.00 g/g during feeding phase from HDSS as carbon source.

Inequalities in diagnosis and registration of pediatric very rare tumors: a European study on pleuropulmonary blastoma.

Very rare tumors (VRTs) account for up to 11% of childhood cancers. Dedicated national groups and registries only exist in some European countries. Pleuropulmonary blastoma (PPB) is a very rare intrathoracic pediatric tumor with a potentially severe prognosis. Due to its rarity, it sometimes goes unrecognized. We investigated PPB diagnostic capability and possible correlations between diagnostic performance and VRT-dedicated activities. The number of cases of PPB registered between 2000 and 2014 at pediatric oncology centers in Europe was compared with the number of expected cases. Data sources included VRT registries, population-based cancer registries, and hospital registries. Data were obtained for 25 countries, grouped into 4 geographical regions. The expected cases were 111, and the observed cases were 129. The observed-to-expected ratio was 1.86 for Northern Europe, 1.33 for Southern Europe, 1.22 for Central Europe, and 0.65 for Eastern Europe. More cases than expected were registered in all countries with an official VRT registry.Conclusion: The number of cases observed is consistent with expectations, but disparities exist across Europe. Difficulties in diagnosing PPB emerged in most Eastern countries. The incidence rate of PPB may be underestimated. The creation of VRT-dedicated groups and a European Registry for VRTs could help to reduce inequalities.What is Known:• Very rare pediatric tumors are often not recognized, despite representing almost 11% of childhood cancers .• Pleuropulmonary blastoma is a rare pediatric tumor with a poor prognosis.What is New:• The ability to diagnose and register pleuropulmonary blastoma varies in Europe.Registries dedicated to very rare pediatric tumors improve the diagnostic rates.• The incidence rate of pleuropulmonary blastoma may currently be underestimated.

Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine.

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.

Development of a Novel Human Cell-Derived Tissue-Engineered Heart Valve for Transcatheter Aortic Valve Replacement: an In Vitro and In Vivo Feasibility Study.

Transcatheter aortic valve replacement (TAVR) is being extended to younger patients. However, TAVR-compatible bioprostheses are based on xenogeneic materials with limited durability. Off-the-shelf tissue-engineered heart valves (TEHVs) with remodeling capacity may overcome the shortcomings of current TAVR devices. Here, we develop for the first time a TEHV for TAVR, based on human cell-derived extracellular matrix and integrated into a state-of-the-art stent for TAVR. The TEHVs, characterized by a dense acellular collagenous matrix, demonstrated in vitro functionality under aortic pressure conditions (n = 4). Next, transapical TAVR feasibility and in vivo TEHV functionality were assessed in acute studies (n = 5) in sheep. The valves successfully coped with the aortic environment, showing normal leaflet motion, free coronary flow, and absence of stenosis or paravalvular leak. At explantation, TEHVs presented full structural integrity and initial cell infiltration. Its long-term performance proven, such TEHV could fulfill the need for next-generation lifelong TAVR prostheses.

Compensating human subjects providing oocytes for stem cell research: 9-year experience and outcomes.

PURPOSE: Human oocytes are arguably one of the most important cell types in humans, yet they are one of the least investigated cells. Because oocytes are limited in number, the use of high-quality oocytes is almost entirely in reproduction. Furthermore, regulatory hurdles for research on gametes and regulations on funding related to research on gametes present significant obstacles to research and the advancement of reproductive treatments. Here we report the outcomes of the largest compensated oocyte donation program for research in the USA to date, and probably worldwide. METHODS: Women who participated in oocyte donation for research between 2008 and 2017 were contacted in a phone interview and completed a standardized questionnaire. RESULTS: Of 114 participants, 98 oocyte donors completed donation, donating 1787 mature MII oocytes and a total of 86 skin biopsies. Complication rate, including minor complications, of oocyte donation was 8/98, or 8.1%, for which two involved follow-up. Fifty-seven donors answered questions about their experience. Participants were incentivized primarily by money and a desire to help others and reported an overall favorable experience. Most, but not all, human subjects recalled that they had donated for research, and approximately half recalled that their oocytes were being used specifically for stem cell research. CONCLUSIONS: Compensated oocyte donation provides a reliable path to obtaining high-quality oocytes for research and is reviewed favorably by oocyte donors. The continuation of programs that offer compensation for oocyte donation is invaluable to continued progress and advancements in stem cell research and human embryology, and for the advancement of novel reproductive treatments.

[Influence of rocuronium dose on the effectiveness of mask ventilation : A prospective, randomized clinical trial]. / Der Einfluss der Rocuroniumdosis auf die Effektivität der Maskenbeatmung : Eine prospektive, randomisierte, klinische Studie.

BACKGROUND: Mask ventilation could improve after administration of muscle relaxants if there is a functional obstruction of the airway, such as laryngospasm, vocal cord closure or opioid-induced muscle rigidity. On the other hand, muscle relaxants could worsen mask ventilation because they induce upper airway collapse; however, clinical studies showed that rocuronium (Roc) improved mask ventilation or it remained unchanged. In most cases Roc 0.06 mg/kgBW was used. OBJECTIVE: The optimal dose of Roc has not been studied; therefore, we studied the quality of mask ventilation with three different doses of Roc (0.3, 0.6 and 0.9 mg/kgBW) and compared them with a control group receiving saline. MATERIAL AND METHODS: In this prospective clinical trial 200 patients were randomized into 4 groups: NaCl (saline), Roc03 (Roc 0.3 mg/kgBW), Roc06 (Roc 0.6 mg/kgBW) and Roc09 (Roc 0.9 mg/kgBW). Mask ventilation was performed to reach a tidal volume (TV) of >5.0 ml/kgBW; maximum ventilation peak pressure (ppeak) was limited to 25 mbar. If this TV was not reached, mask ventilation was improved by better mask position, head position or/and usage of a Guedel tube (oropharyngeal airway). During mask ventilation ppeak and TV were recorded and the quality of mask ventilation was assessed with the Han and Warters scores. The quality of mask ventilation between the four groups was compared for all patients and a subgroup analysis was performed for patients of the study groups, who had to be ventilated with a Guedel tube and for obese patients (body mass index ≥26 kg/m2). A sample size calculation revealed that at least 38 patients were necessary for each group to detect a statistically significant difference between groups; it was assumed that Roc improved the efficacy of mask ventilation by 20% compared to saline (α = 0.05, 1-ß = 0.8). RESULTS: The administration of Roc significantly improved the TV/ppeak ratio compared to saline (p = 0.04); however, this effect was irrespective of the dose. In patients who were ventilated with the Guedel tube the TV/ppeak ratio increased after Roc03 (p ≤ 0.01) and after Roc06 (p < 0.02) compared to the saline group. In obese patients who were ventilated with the Guedel tube the TV/ppeak ratio increased after Roc03 (p ≤ 0.01), after Roc06 (p = 0.03) and after Roc09 (p = 0.02) compared to the saline group. There were no significant differences between the Roc groups; however, the effect was more pronounced in the Roc03 patients compared to the other Roc groups. The Han and Warters scores were not significantly different between the Roc groups and the saline group. CONCLUSION: The efficacy of mask ventilation was equal or improved after administration of Roc but did not become worse. Patients who were ventilated with a Guedel tube had higher TV/ppeak ratios after Roc03 and Roc06 compared to saline. Higher dosages (>Roc06), however, had no advantages concerning quality of mask ventilation. In obese patients who had to be ventilated with a Guedel tube, Roc also improved the efficacy of mask ventilation. We conclude that administration of Roc is effective in improving mask ventilation and this effect was seen after 30-60 s even after Roc03.

Advocacy, support and survivorship in prostate cancer.

Across Australia, prostate cancer support groups (PCSG) have emerged to fill a gap in psychosocial care for men and their families. However, an understanding of the triggers and influencers of the PCSG movement is absent. We interviewed 21 SG leaders (19 PC survivors, two partners), of whom six also attended a focus group, about motivations, experiences, past and future challenges in founding and leading PCSGs. Thematic analysis identified four global themes: illness experience; enacting a supportive response; forming a national collective and challenges. Leaders described men's feelings of isolation and neglect by the health system as the impetus for PCSGs to form and give/receive mutual help. Negotiating health care systems was an early challenge. National affiliation enabled leaders to build a united voice in the health system and establish a group identity and collective voice. Affiliation was supported by a symbiotic relationship with tensions between independence, affiliation and governance. Future challenges were group sustainability and inclusiveness. Study findings describe how a grassroots PCSG movement arose consistent with an embodied health movement perspective. Health care organisations who seek to leverage these community resources need to be cognisant of SG values and purpose if they are to negotiate effective partnerships that maximise mutual benefit.

Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD.

Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10-6 and 10-9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M-1s-1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.

Incorporation studies of clickable ceramides in Jurkat cell plasma membranes.

The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.

Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells.

Tumor surveillance of natural killer (NK) cells is mediated by the cytotoxicity receptor natural-killer group 2 member D (NKG2D). Ligands for NKG2D are generally not expressed on healthy cells, but induced on the surface of malignant cells. To date, NKG2D ligand (NKG2D-L) induction was mainly described to depend on the activation of the DNA damage response, although the molecular mechanisms that regulate NKG2D-L expression remain largely unknown. Here, we show that the acetyltransferases CBP (CREB-binding protein) and p300 play a crucial role in the regulation of NKG2D-L on tumor cells. Loss of CBP/p300 decreased the basal cell surface expression of human ligands and reduced the upregulation of MICA/B and ULBP2 in response to histone deacetylase inhibitors or DNA damage. Furthermore, CBP/P300 deficiency abrogated the sensitivity of stressed cells to NK cell-mediated killing. CBP/p300 were also identified as major regulators of mouse NKG2D ligand RAE-1 in vitro and in vivo using the Eµ-Myc lymphoma model. Mechanistically, we observed an enhanced activation of the CBP/p300 binding transcription factor CREB (cAMP response element-binding protein) correlating to the NKG2D-L upregulation. Moreover, increased binding of CREB and CBP/p300 to NKG2D-L promoters and elevated histone acetylation were detectable. This study provides strong evidence for a major role of CBP and p300 in orchestrating NKG2D-L induction and consequently immunosurveillance of tumors in mice and humans. These findings might help to develop novel immunotherapeutic approaches against cancer.

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.