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Institute
- Medizinische Klinik und Poliklinik II (45) (remove)
Objective
The current article encompasses a literature review and recommendations for radiotherapy in nodal oligorecurrent prostate cancer.
Materials and methods
A literature review focused on studies comparing metastasis-directed stereotactic ablative radiotherapy (SABR) vs. external elective nodal radiotherapy (ENRT) and studies analyzing recurrence patterns after local nodal treatment was performed. The DEGRO Prostate Cancer Expert Panel discussed the results and developed treatment recommendations.
Results
Metastasis-directed radiotherapy results in high local control (often > 90% within a follow-up of 1–2 years) and can be used to improve progression-free survival or defer androgen deprivation therapy (ADT) according to prospective randomized phase II data. Distant progression after involved-node SABR only occurs within a few months in the majority of patients. ENRT improves metastases-free survival rates with increased toxicity in comparison to SABR according to retrospective comparative studies. The majority of nodal recurrences after initial local treatment of pelvic nodal metastasis are detected within the true pelvis and common iliac vessels.
Conclusion
ENRT with or without a boost should be preferred to SABR in pelvic nodal recurrences. In oligometastatic prostate cancer with distant (extrapelvic) nodal recurrences, SABR alone can be performed in selected cases. Application of additional systemic treatments should be based on current guidelines, with ADT as first-line treatment for hormone-sensitive prostate cancer. Only in carefully selected patients can radiotherapy be initially used without additional ADT outside of the current standard recommendations. Results of (randomized) prospective studies are needed for definitive recommendations.
Background: Chimeric antigen receptor (CAR) T-cells are changing the therapeutic landscape of hematologic malignancies. Severe side effects include cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), but prolonged cytopenia has also been reported. The underlying mechanism for prolonged cytopenia is poorly understood so far. Cases: Severe pancytopenia with grade 2-3 anemia was marked 2–3 months after treatment. Laboratory evaluation revealed undetectable levels of haptoglobin with increased reticulocyte counts. Coomb's tests were negative, no schistocytes were detected on blood smear, and infectious causes were ruled out. Increased erythropoiesis without lymphoma infiltration was noted on bone marrow biopsy. A spontaneous increase in haptoglobin and hemoglobin levels was observed after several weeks. For one patient, peripheral CAR-T levels were monitored over time. We observed a decline at the same time as hemoglobin levels began to rise, implying a potential causality. Conclusion: To our knowledge, we describe the first two cases of Coombs-negative hemolytic anemia after CAR-T treatment for B-cell lymphoma. We encourage routine monitoring for hemolytic anemia after CAR-T treatment and also encourage further investigations on the underlying mechanism.
Limiting bone resorption and regenerating bone tissue are treatment goals in myeloma bone disease (MMBD). Physical stimuli such as mechanical loading prevent bone destruction and enhance bone mass in the MOPC315.BM.Luc model of MMBD. It is unknown whether treatment with the Bruton's tyrosine kinase inhibitor CC-292 (spebrutinib), which regulates osteoclast differentiation and function, augments the anabolic effect of mechanical loading. CC-292 was administered alone and in combination with axial compressive tibial loading in the MOPC315.BM.Luc model for three weeks. However, neither CC-292 alone nor its use in combination with mechanical loading was more effective in reducing osteolytic bone disease or rescuing bone mass than mechanical stimuli alone, as evidenced by microcomputed tomography (microCT) and histomorphometric analysis. Further studies are needed to investigate novel anti-myeloma and anti-resorptive strategies in combination with physical stimuli to improve treatment of MMBD.
Chronic hepatitis C can be treated very effectively with direct-acting antivirals (DAA) with only minor side effects compared to an interferon-containing treatment regimen. The significance of metabolic comorbidities after HCV cure is not well defined. This study aims to investigate short- and long-term weight change of patients receiving interferon-free antiviral treatment for chronic hepatitis C. The German Hepatitis C-registry (DHC-R) is a national multicenter real-world cohort. A total of 5111 patients were followed prospectively after DAA treatment for up to 3 years. Weight change compared to baseline was analyzed at end of treatment and at years 1, 2, and 3 after completion of antiviral therapy. Regression analysis was performed to identify baseline predictors for weight change. While there was no relevant mean weight change (−0.2 kg, SD 4.3 kg) at the end of antiviral treatment, weight started to increase during long-term follow-up reaching +1.7 kg (SD 8.0 kg, p < 0.001) compared to baseline at 3 years (follow-up year 3, FU3) after completion of antiviral therapy. 48%, 31%, and 22% of patients had a weight gain greater than 1, 3, and 5 kg at FU3, respectively. During follow-up, a body mass index (BMI) <30 proved to be the only consistent predictor for weight gain. DAA treatment is followed by a substantial weight gain (+3 kg or more) in one-third of the patients during long-term follow-up. Non-obese patients seemed to be most vulnerable to weight gain. The body compartment involved in weight gain as well as the mechanism of weight gain remain to be elucidated.
Occupational mold exposure can lead to Aspergillus-associated allergic diseases including asthma and hypersensitivity pneumonitis. Elevated IL-17 levels or disbalanced T-helper (Th) cell expansion were previously linked to Aspergillus-associated allergic diseases, whereas alterations to the Th cell repertoire in healthy occupationally exposed subjects are scarcely studied. Therefore, we employed functional immunoassays to compare Th cell responses to A. fumigatus antigens in organic farmers, a cohort frequently exposed to environmental molds, and non-occupationally exposed controls. Organic farmers harbored significantly higher A. fumigatus-specific Th-cell frequencies than controls, with comparable expansion of Th1- and Th2-cell frequencies but only slightly elevated Th17-cell frequencies. Accordingly, Aspergillus antigen-induced Th1 and Th2 cytokine levels were strongly elevated, whereas induction of IL-17A was minimal. Additionally, increased levels of some innate immune cell-derived cytokines were found in samples from organic farmers. Antigen-induced cytokine release combined with Aspergillus-specific Th-cell frequencies resulted in high classification accuracy between organic farmers and controls. Aspf22, CatB, and CipC elicited the strongest differences in Th1 and Th2 responses between the two cohorts, suggesting these antigens as potential candidates for future bio-effect monitoring approaches. Overall, we found that occupationally exposed agricultural workers display a largely balanced co-expansion of Th1 and Th2 immunity with only minor changes in Th17 responses.
Current limitations and perspectives of chimeric antigen receptor-T-cells in acute myeloid leukemia
(2021)
Adoptive transfer of gene-engineered chimeric antigen receptor (CAR)-T-cells has emerged as a powerful immunotherapy for combating hematologic cancers. Several target antigens that are prevalently expressed on AML cells have undergone evaluation in preclinical CAR-T-cell testing. Attributes of an ‘ideal’ target antigen for CAR-T-cell therapy in AML include high-level expression on leukemic blasts and leukemic stem cells (LSCs), and absence on healthy tissues, normal hematopoietic stem and progenitor cells (HSPCs). In contrast to other blood cancer types, where CAR-T therapies are being similarly studied, only a rather small number of AML patients has received CAR-T-cell treatment in clinical trials, resulting in limited clinical experience for this therapeutic approach in AML. For curative AML treatment, abrogation of bulk blasts and LSCs is mandatory with the need for hematopoietic recovery after CAR-T administration. Herein, we provide a critical review of the current pipeline of candidate target antigens and corresponding CAR-T-cell products in AML, assess challenges for clinical translation and implementation in routine clinical practice, as well as perspectives for overcoming them.
Clinical and biological characteristics of medullary and extramedullary plasma cell dyscrasias
(2021)
Background: Extramedullary plasma cell (PC) disorders may occur as extramedullary disease in multiple myeloma (MM-EMD) or as primary extramedullary plasmocytoma (pEMP)/solitary osseous plasmocytoma (SOP). In this study, we aimed to obtain insights into the molecular mechanisms of extramedullary spread of clonal PC. Methods: Clinical and biological characteristics of 87 patients with MM-EMD (n = 49), pEMP/SOP (n = 20) and classical MM (n = 18) were analyzed by using immunohistochemistry (CXCR4, CD31, CD44 and CD81 staining) and cytoplasmic immunoglobulin staining combined with fluorescence in situ hybridization (cIg-FISH). Results: High expression of CD44, a cell-surface glycoprotein involved in cell-cell interactions, was significantly enriched in MM-EMD (90%) vs. pEMP/SOP (27%) or classical MM (33%) (p < 0.001). In addition, 1q21 amplification by clonal PC occurred at a similar frequency of MM-EMD (33%), pEMP/SOP (57%) and classical MM (44%). Conversely, del(17p13), t(4;14) and t(14;16) were completely absent in pEMP/SOP. Besides this, 1q21 amplification was identified in 64% of not paraskeletal samples from MM-EMD or pEMP compared to 9% of SOP or paraskeletal MM-EMD/pEMP and 44% of classical MM samples, respectively (p = 0.02). Conclusion: Expression of molecules involved in homing and cytogenetic aberrations differ between MM with or without EMD and pEMP/SOP.
(1) Background: The aim of our study was to identify specific risk factors for fatal outcome in critically ill COVID-19 patients. (2) Methods: Our data set consisted of 840 patients enclosed in the LEOSS registry. Using lasso regression for variable selection, a multifactorial logistic regression model was fitted to the response variable survival. Specific risk factors and their odds ratios were derived. A nomogram was developed as a graphical representation of the model. (3) Results: 14 variables were identified as independent factors contributing to the risk of death for critically ill COVID-19 patients: age (OR 1.08, CI 1.06–1.10), cardiovascular disease (OR 1.64, CI 1.06–2.55), pulmonary disease (OR 1.87, CI 1.16–3.03), baseline Statin treatment (0.54, CI 0.33–0.87), oxygen saturation (unit = 1%, OR 0.94, CI 0.92–0.96), leukocytes (unit 1000/μL, OR 1.04, CI 1.01–1.07), lymphocytes (unit 100/μL, OR 0.96, CI 0.94–0.99), platelets (unit 100,000/μL, OR 0.70, CI 0.62–0.80), procalcitonin (unit ng/mL, OR 1.11, CI 1.05–1.18), kidney failure (OR 1.68, CI 1.05–2.70), congestive heart failure (OR 2.62, CI 1.11–6.21), severe liver failure (OR 4.93, CI 1.94–12.52), and a quick SOFA score of 3 (OR 1.78, CI 1.14–2.78). The nomogram graphically displays the importance of these 14 factors for mortality. (4) Conclusions: There are risk factors that are specific to the subpopulation of critically ill COVID-19 patients.
Background: Neuralgic amyotrophy (NA) has been described as a possible extrahepatic manifestation of hepatitis E virus (HEV) infection. Usually, HEV-associated NA occurs bilaterally. The clinical characteristics determining the course of HEV-associated NA have still not been defined. Methods: In this retrospective multicentric case series, 16 patients with HEV-associated NA were studied and compared to 176 HEV patients without NA in terms of their age, sex, and ALT levels. Results: Neither gender distribution (75% vs. 67% male) nor age (47 vs. 48 years median) differed significantly between the NA patients and controls. Eight NA patients (50%) presented with bilateral involvement — seven of these had right-side dominance and one had left-side dominance. Thirteen cases (81%) were hospitalized. Eight of these patients stayed in hospital for five to seven days, and five patients stayed for up to two weeks. The time from the onset of NA to the HEV diagnosis, as well as the diagnostic and therapeutic proceedings, showed a large variability. In total, 13 (81%) patients received treatment: 1/13 (8%) received intravenous immunoglobulins, 8/13 (62%) received glucocorticoids, 3/13 (23%) received ribavirin, and 6/13 (46%) received pregabalin/gabapentin. Patients with ages above the median (47 years) were more likely to be treated (p = 0.001). Conclusion: HEV-associated NA causes a relevant morbidity. In our case series neither the type of treatment nor the time of initiation of therapy had a significant effect on the duration of hospitalization or the course of the disease. The clinical presentation, the common diagnostic and therapeutic procedures, and the patients' characteristics showed large variability, demonstrating the necessity of standardized protocols for this rare but relevant disease.
Loss of Somatostatin Receptor 2 (SSTR2) expression and rising CXC Chemokine Receptor Type 4 (CXCR4) expression are associated with dedifferentiation in neuroendocrine tumors (NET). In NET, CXCR4 expression is associated with enhanced metastatic and invasive potential and worse prognosis but might be a theragnostic target. Likewise, activation of Wnt/β-catenin signaling may promote a more aggressive phenotype in NET. We hypothesized an interaction of the Wnt/β-catenin pathway with CXCR4 expression and function in NET. The NET cell lines BON-1, QGP-1, and MS-18 were exposed to Wnt inhibitors (5-aza-CdR, quercetin, and niclosamide) or the Wnt activator LiCl. The expressions of Wnt pathway genes and of CXCR4 were studied by qRT-PCR, Western blot, and immunohistochemistry. The effects of Wnt modulators on uptake of the CXCR4 ligand [\(^{68}\)Ga] Pentixafor were measured. The Wnt activator LiCl induced upregulation of CXCR4 and Wnt target gene expression. Treatment with the Wnt inhibitors had opposite effects. LiCl significantly increased [\(^{68}\)Ga] Pentixafor uptake, while treatment with Wnt inhibitors decreased radiopeptide uptake. Wnt pathway modulation influences CXCR4 expression and function in NET cell lines. Wnt modulation might be a tool to enhance the efficacy of CXCR4-directed therapies in NET or to inhibit CXCR4-dependent proliferative signaling. The underlying mechanisms for the interaction of the Wnt pathway with CXCR4 expression and function have yet to be clarified.
Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8\(^+\) counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.
Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens.
Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation (alloSCT). Sera were tested prospectively twice weekly for Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-β-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% (95% confidential interval (CI): 28–99%) and 55% (95% CI: 32–77%) for BDG, 40% (95% CI: 5–85%) and 100% (95% CI: 83–100%) for GM, and 60% (95% CI: 15–95%) and 95% (95% CI: 75–100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56–94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68–99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay.
Purpose
Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects.
Methods
262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed.
Results
Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05).
Conclusion
Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.
Extramedullary disease (EMD) represents a high-risk state of multiple myeloma (MM) associated with poor prognosis. While most anti-myeloma therapeutics demonstrate limited efficacy in this setting, some studies exploring the utility of chimeric antigen receptor (CAR)-modified T cells reported promising results. We have recently designed SLAMF7-directed CAR T cells for the treatment of MM. SLAMF7 is a transmembrane receptor expressed on myeloma cells that plays a role in myeloma cell homing to the bone marrow. Currently, the only approved anti-SLAMF7 therapeutic is the monoclonal antibody elotuzumab, but its efficacy in EMD has not been investigated thoroughly. Thus, we retrospectively analyzed the efficacy of elotuzumab-based combination therapy in a cohort of 15 patients with EMD. Moreover, since the presence of the target antigen is an indispensable prerequisite for effective targeted therapy, we investigated the SLAMF7 expression on extramedullary located tumor cells before and after treatment. We observed limited efficacy of elotuzumab-based combination therapies, with an overall response rate of 40% and a progression-free and overall survival of 3.8 and 12.9 months, respectively. Before treatment initiation, all available EMD tissue specimens (n = 3) demonstrated a strong and consistent SLAMF7 surface expression by immunohistochemistry. Furthermore, to investigate a potential antigen reduction under therapeutic selection pressure, we analyzed samples of de novo EMD (n = 3) outgrown during elotuzumab treatment. Again, immunohistochemistry documented strong and consistent SLAMF7 expression in all samples. In aggregate, our data point towards a retained expression of SLAMF7 in EMD and encourage the development of more potent SLAMF7-directed immunotherapies, such as CAR T cells.
Acute myeloid leukemia (AML) is characterized by recurrent genetic events. The BCL6 corepressor (BCOR) and its homolog, the BCL6 corepressor-like 1 (BCORL1), have been reported to be rare but recurrent mutations in AML. Previously, smaller studies have reported conflicting results regarding impacts on outcomes. Here, we retrospectively analyzed a large cohort of 1529 patients with newly diagnosed and intensively treated AML. BCOR and BCORL1 mutations were found in 71 (4.6%) and 53 patients (3.5%), respectively. Frequently co-mutated genes were DNTM3A, TET2 and RUNX1. Mutated BCORL1 and loss-of-function mutations of BCOR were significantly more common in the ELN2017 intermediate-risk group. Patients harboring loss-of-function mutations of BCOR had a significantly reduced median event-free survival (HR = 1.464 (95%-Confidence Interval (CI): 1.005–2.134), p = 0.047), relapse-free survival (HR = 1.904 (95%-CI: 1.163–3.117), p = 0.01), and trend for reduced overall survival (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056) in multivariable analysis. Our study establishes a novel role for loss-of-function mutations of BCOR regarding risk stratification in AML, which may influence treatment allocation.
Background: Preservation of kidney function in newly diagnosed (ND) multiple myeloma (MM) helps to prevent excess toxicity. Patients (pts) from two prospective trials were analyzed, provided postinduction (PInd) restaging was performed. Pts received three cycles with bortezomib (btz), cyclophosphamide, and dexamethasone (dex; VCD) or btz, lenalidomide (len), and dex (VRd) or len, adriamycin, and dex (RAD). The minimum required estimated glomerular filtration rate (eGFR) was >30 mL/min. We analyzed the percent change of the renal function using the International Myeloma Working Group (IMWG) criteria and Kidney Disease: Improving Global Outcomes (KDIGO)-defined categories. Results: Seven hundred and seventy-two patients were eligible. Three hundred and fifty-six received VCD, 214 VRd, and 202 RAD. VCD patients had the best baseline eGFR. The proportion of pts with eGFR <45 mL/min decreased from 7.3% at baseline to 1.9% PInd (p < 0.0001). Thirty-seven point one percent of VCD versus 49% of VRd patients had a decrease of GFR (p = 0.0872). IMWG-defined “renal complete response (CRrenal)” was achieved in 17/25 (68%) pts after VCD, 12/19 (63%) after RAD, and 14/27 (52%) after VRd (p = 0.4747). Conclusions: Analyzing a large and representative newly diagnosed myeloma (NDMM) group, we found no difference in CRrenal that occurred independently from the myeloma response across the three regimens. A trend towards deterioration of the renal function with VRd versus VCD may be explained by a better pretreatment “renal fitness” in the latter group.
Background: Clinical practice guidelines for patients with primary biliary cholangitis (PBC) have been recently revised and implemented for well-established response criteria to standard first-line ursodeoxycholic acid (UDCA) therapy at 12 months after treatment initiation for the early identification of high-risk patients with inadequate treatment responses who may require treatment modification. However, there are only very limited data concerning the real-world clinical management of patients with PBC in Germany. Objective: The aim of this retrospective multicenter study was to evaluate response rates to standard first-line UDCA therapy and subsequent Second-line treatment regimens in a large cohort of well-characterized patients with PBC from 10 independent hepatological referral centers in Germany prior to the introduction of obeticholic acid as a licensed second-line treatment option. Methods: Diagnostic confirmation of PBC, standard first-line UDCA treatment regimens and response rates at 12 months according to Paris-I, Paris-II, and Barcelona criteria, the follow-up cut-off alkaline phosphatase (ALP) ≤ 1.67 × upper limit of normal (ULN) and the normalization of bilirubin (bilirubin ≤ 1 × ULN) were retrospectively examined between June 1986 and March 2017. The management and hitherto applied second-line treatment regimens in patients with an inadequate response to UDCA and subsequent response rates at 12 months were also evaluated. Results: Overall, 480 PBC patients were included in this study. The median UDCA dosage was 13.2 mg UDCA/kg bodyweight (BW)/d. Adequate UDCA treatment response rates according to Paris-I, Paris-II, and Barcelona criteria were observed in 91, 71.3, and 61.3% of patients, respectively. In 83.8% of patients, ALP ≤ 1.67 × ULN were achieved. A total of 116 patients (24.2%) showed an inadequate response to UDCA according to at least one criterion. The diverse second-line treatment regimens applied led to significantly higher response rates according to Paris-II (35 vs. 60%, p = 0.005), Barcelona (13 vs. 34%, p = 0.0005), ALP ≤ 1.67 × ULN and bilirubin ≤ 1 × ULN (52.1 vs. 75%, p = 0.002). The addition of bezafibrates appeared to induce the strongest beneficial effect in this cohort (Paris II: 24 vs. 74%, p = 0.004; Barcelona: 50 vs. 84%, p = 0.046; ALP < 1.67 × ULN and bilirubin ≤ 1 × ULN: 33 vs. 86%, p = 0.001). Conclusion: Our large retrospective multicenter study confirms high response rates following UDCA first-line standard treatment in patients with PBC and highlights the need for close monitoring and early treatment modification in high-risk patients with an insufficient response to UDCA since early treatment modification significantly increases subsequent response rates of these patients.
Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells' lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.
Axicabtagene ciloleucel (axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for relapsed or refractory large B-cell lymphoma (R/R LBCL). To reduce axi-cel–related toxicity, several exploratory safety management cohorts were added to ZUMA-1 (NCT02348216), the pivotal phase 1/2 study of axi-cel in refractory LBCL. Cohort 4 evaluated the rates and severity of cytokine release syndrome (CRS) and neurologic events (NEs) with earlier corticosteroid and tocilizumab use. Primary endpoints were incidence and severity of CRS and NEs. Patients received 2 × 106 anti-CD19 CAR T cells/kg after conditioning chemotherapy. Forty-one patients received axi-cel. Incidences of any-grade CRS and NEs were 93% and 61%, respectively (grade ≥ 3, 2% and 17%). There was no grade 4 or 5 CRS or NE. Despite earlier dosing, the cumulative cortisone-equivalent corticosteroid dose in patients requiring corticosteroid therapy was lower than that reported in the pivotal ZUMA-1 cohorts. With a median follow-up of 14·8 months, objective and complete response rates were 73% and 51%, respectively, and 51% of treated patients were in ongoing response. Earlier and measured use of corticosteroids and/or tocilizumab has the potential to reduce the incidence of grade ≥ 3 CRS and NEs in patients with R/R LBCL receiving axi-cel.
Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia
(2021)
The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD+AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD+AML. Garitano-Trojaola et al. used a combination of human acute myeloid leukemia (AML) cell lines and primary samples to show that RAC1-dependent actin cytoskeleton remodeling through BCL2 family plays a key role in resistance to the FLT3 inhibitor, Midostaurin in AML. They showed that by targeting RAC1 and BCL2, Midostaurin resistance was diminished, which potentially paves the way for an innovate treatment approach for FLT3 mutant AML.
Psoriasis is an immune-mediated systemic inflammatory disease that is not limited to the skin but may be associated with arthritis, cardiovascular diseases, metabolic syndrome including diabetes and obesity and, as identified more recently, non-alcoholic fatty liver disease (NAFLD) that occurs in approximately 50 % of all patients with psoriasis. NAFLD is characterized by accumulation of fat in hepatocytes in the absence of excessive alcohol consumption. Over the last two decades, NAFLD has developed to the most common chronic liver disease with an estimated prevalence of 25 % in the Western population. NAFLD ranges from non-inflammatory or bland hepatic steatosis to inflammation of hepatic tissue (non-alcoholic steatohepatitis, NASH) and consecutive liver fibrosis. It is controversial whether the underlying systemic inflammation of psoriasis is contributing to development of NAFLD or if comorbid diseases such as obesity enhance NAFLD development. Recent findings indicate that cytokine-mediated inflammation through TNFα, interleukin (IL)-6 and IL-17 might be the common link between psoriasis and NAFLD. Considering the shared inflammatory pathways, IL-17 pharmacological blockade, which is already well-established for psoriasis, may be a promising strategy to treat both psoriasis and NAFLD. Therefore, early detection of NAFLD and a better understanding of its pathophysiology in the context of the systemic inflammation in psoriasis is important with regard to individualized treatment approaches.
Background
Genital human papillomavirus (HPV)-infections are common in the general population and are responsible for relevant numbers of epithelial malignancies. Much data on the HPV-prevalence is available for secondary immunodeficiencies, especially for patients with human immunodeficiency virus (HIV)-infection. Little is known about the genital HPV-prevalence in patients with primary immunodeficiencies (PIDs).
Methods
We performed a cross-sectional study of patients with PIDs and took genital swabs from male and female patients, which were analyzed with polymerase chain reaction for the presence of HPV-DNA. Clinical and laboratory data was collected to identify risk factors.
Results
28 PID patients were included in this study. 10 of 28 (35.7%) had HPV-DNA in their genital swabs. 6 patients had high-risk HPV-types (21.4%). Most patients had asymptomatic HPV-infections, as genital warts were rare (2 of 28 patients) and HPV-associated malignancy was absent. Differences in the HPV-positivity regarding clinical PID-diagnosis, duration of PID, age, sex, immunosuppression, immunoglobulin replacement, or circumcision in males were not present. HPV-positive PID patients had higher numbers of T cells (CD3\(^+\)), of cytotoxic T cells (CD3\(^+\)/CD8\(^+\)), of transitional B cells (CD19\(^+\)/CD38\(^{++}\)/CD10\(^+\)/IgD\(^+\)), and of plasmablasts (CD19\(^+\)/CD38\(^+\)/CD27\(^{++}\)/IgD\(^-\)) compared to HPV-negative.
Conclusion
PID patients exhibit a high rate of genital HPV-infections with a high rate of high-risk HPV-types. Regular screening for symptomatic genital HPV-infection and HPV-associated malignancy in PID patients seems recommendable.
Autologous hematopoietic stem cell transplantation (aHSCT) represents an effective treatment for systemic sclerosis (SSc), but it also can cause immunological adverse events (iAEs). Therefore, we aimed to determine the frequency of iAEs [engraftment syndrome (ES) and secondary autoimmune disorder (sAD)] and to identify potential risk factors for their development in a retrospective analysis on 22 patients similarly transplanted due to SSc. While nine patients (41%) suffered from ESs, seven sADs occurred in six patients (27%). Patients who developed ES were older in our cohort (52.45 vs. 42.58 years, p = .0433, Cohen’s d = 0.86), and cardiac involvement by SSc was associated with development of ES (OR = 40.11, p = .0017). Patients with manifestation of sAD had a higher modified Rodnan skin score (mRSS) reduction after aHSCT (90.50% vs. 60.00%, p = .0064, r = .65). Thus, IAEs are common after aHSCT for SSc and can occur in different stages during and after aHSCT with characteristic clinical manifestations. Good cutaneous response after aHSCT might be considered as a risk factor for sAD, and higher age at aHSCT and cardiac involvement might be considered as risk factors for the development of ES.
Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.
Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9\(^+\) T Cells
(2021)
Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3\(^+\) T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9\(^+\)CD3\(^+\) T cells, CD4\(^+\) and CD8\(^+\) conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9\(^+\)CD3\(^+\) T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.
IL-9-producing Th9 cells display a group of helper T cells with similarities to Th17 and Th2 T cells and have been shown to be involved in synovial inflammation in rheumatoid arthritis (RA) patients. So far, it is unclear which parameters drive Th9 differentiation in lymphocytes derived from RA patients compared to immunologically healthy individuals and whether autocrine mechanisms are able to enhance Th9 polarization. Further, parallel pathways of induction of IL-17-producing cells with Th9 phenotype have to be distinguished from exclusively Th9-inductive mechanisms. Thus, the present study aimed to determine the parameters of Th9 induction by simulation in a standardized inflammatory cytokine milieu.Peripheral naive and non-naive T cells of RA patients and healthy donors (HD) were cultured under Th9 and Th17-driving conditions and phenotypically analyzed by flow cytometry and molecular analysis.Our findings indicate a similar differentiation pathway of Th9 and Th17 cells and similar distributions of IL-9+ T cells in RA and HD regardless of Th9- or Th17-promoting cytokine milieus. Whereas the magnitude and direction of Th9- or Th17-polarization was about the same in RA and HD, IL-17+ CD4+ T cells were significantly stimulated by Th17-inducing conditions in HD. In conclusion, the results indicate that Th9- and Th17-inducing cytokine conditions mimicking autoimmune inflammation in RA may have similar stimulatory effects regarding polarization of peripheral naive and non-naive T cells into Th9 or Th17 cells. The results suggest that the differentiation of Th9 cells may be also induced by Th17-driving conditions.
Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti-Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4\(^+\) and CD8\(^+\) T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA.
Background: Renal cell carcinoma (RCC) is divided into three major histopathologic groups—clear cell (ccRCC), papillary (pRCC) and chromophobe RCC (chRCC). We performed a comprehensive re-analysis of publicly available RCC datasets from the TCGA (The Cancer Genome Atlas) database, thereby combining samples from all three subgroups, for an exploratory transcriptome profiling of RCC subgroups.
Materials and Methods: We used FPKM (fragments per kilobase per million) files derived from the ccRCC, pRCC and chRCC cohorts of the TCGA database, representing transcriptomic data of 891 patients. Using principal component analysis, we visualized datasets as t-SNE plot for cluster detection. Clusters were characterized by machine learning, resulting gene signatures were validated by correlation analyses in the TCGA dataset and three external datasets (ICGC RECA-EU, CPTAC-3-Kidney, and GSE157256).
Results: Many RCC samples co-clustered according to histopathology. However, a substantial number of samples clustered independently from histopathologic origin (mixed subgroup)—demonstrating divergence between histopathology and transcriptomic data. Further analyses of mixed subgroup via machine learning revealed a predominant mitochondrial gene signature—a trait previously known for chRCC—across all histopathologic subgroups. Additionally, ccRCC samples from mixed subgroup presented an inverse correlation of mitochondrial and angiogenesis-related genes in the TCGA and in three external validation cohorts. Moreover, mixed subgroup affiliation was associated with a highly significant shorter overall survival for patients with ccRCC—and a highly significant longer overall survival for chRCC patients.
Conclusions: Pan-RCC clustering according to RNA-sequencing data revealed a distinct histology-independent subgroup characterized by strengthened mitochondrial and weakened angiogenesis-related gene signatures. Moreover, affiliation to mixed subgroup went along with a significantly shorter overall survival for ccRCC and a longer overall survival for chRCC patients. Further research could offer a therapy stratification by specifically addressing the mitochondrial metabolism of such tumors and its microenvironment.
Whole-Body [\(^{18}\)F]FDG PET/CT Can Alter Diagnosis in Patients with Suspected Rheumatic Disease
(2021)
The 2-deoxy-d-[\(^{18}\)F]fluoro-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) is widely utilized to assess the vascular and articular inflammatory burden of patients with a suspected diagnosis of rheumatic disease. We aimed to elucidate the impact of [\(^{18}\)F]FDG PET/CT on change in initially suspected diagnosis in patients at the time of the scan. Thirty-four patients, who had undergone [\(^{18}\)F]FDG PET/CT, were enrolled and the initially suspected diagnosis prior to [18F]FDG PET/CT was compared to the final diagnosis. In addition, a semi-quantitative analysis including vessel wall-to-liver (VLR) and joint-to-liver (JLR) ratios was also conducted. Prior to [\(^{18}\)F]FDG PET/CT, 22/34 (64.7%) of patients did not have an established diagnosis, whereas in 7/34 (20.6%), polymyalgia rheumatica (PMR) was suspected, and in 5/34 (14.7%), giant cell arteritis (GCA) was suspected by the referring rheumatologists. After [\(^{18}\)F]FDG PET/CT, the diagnosis was GCA in 19/34 (55.9%), combined GCA and PMR (GCA + PMR) in 9/34 (26.5%) and PMR in the remaining 6/34 (17.6%). As such, [\(^{18}\)F]FDG PET/CT altered suspected diagnosis in 28/34 (82.4%), including in all unclear cases. VLR of patients whose final diagnosis was GCA tended to be significantly higher when compared to VLR in PMR (GCA, 1.01 ± 0.08 (95%CI, 0.95–1.1) vs. PMR, 0.92 ± 0.1 (95%CI, 0.85–0.99), p = 0.07), but not when compared to PMR + GCA (1.04 ± 0.14 (95%CI, 0.95–1.13), p = 1). JLR of individuals finally diagnosed with PMR (0.94 ± 0.16, (95%CI, 0.83–1.06)), however, was significantly increased relative to JLR in GCA (0.58 ± 0.04 (95%CI, 0.55–0.61)) and GCA + PMR (0.64 ± 0.09 (95%CI, 0.57–0.71); p < 0.0001, respectively). In individuals with a suspected diagnosis of rheumatic disease, an inflammatory-directed [\(^{18}\)F]FDG PET/CT can alter diagnosis in the majority of the cases, particularly in subjects who were referred because of diagnostic uncertainty. Semi-quantitative assessment may be helpful in establishing a final diagnosis of PMR, supporting the notion that a quantitative whole-body read-out may be useful in unclear cases.
Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
Despite the increasing incidence and prevalence of Crohn’s Disease (CD), no curative options exist and treatment remains complex. While therapy has mainly focused on medical approaches in the past, growing evidence reveals that in cases of limited inflammation, surgery can suffice as an alternative primary treatment. We retrospectively assessed the disease course and outcomes of 103 patients with terminal Ileitis who underwent primary surgery (n = 29) or received primary medical treatment followed by surgery (n = 74). Primary endpoint was the need for immunosuppressive medication after surgical treatment (ileocecal resection, ICR) during a two-years follow-up. Rates for laparoscopic ICR were enhanced in case of early surgery, but no differences were seen for postoperative complications. In case of immunosuppressive medication, patients with ICR at an early state of disease needed significantly less anti-inflammatory medication during the two-year postoperative follow-up compared to patients who were primarily treated medically. Furthermore, in a subgroup analysis for patients with localized ileocecal disease manifestation, early surgery consistently resulted in a decreased amount of medical therapy postoperatively. In conclusion primary ICR is safe and effective in patients with limited CD, and the need for immunosuppressive medication during the postoperative follow-up is low compared to patients receiving surgery at a later stage of disease.
The liver‐derived, circulating transport protein transthyretin (TTR) is the cause of systemic hereditary (ATTRv) and wild‐type (ATTRwt) amyloidosis. TTR stabilization and knockdown are approved therapies to mitigate the otherwise lethal disease course. To date, the variety in phenotypic penetrance is not fully understood. This systematic review summarizes the current literature on TTR pathophysiology with its therapeutic implications. Tetramer dissociation is the rate‐limiting step of amyloidogenesis. Besides destabilizing TTR mutations, other genetic (RBP4, APCS, AR, ATX2, C1q, C3) and external (extracellular matrix, Schwann cell interaction) factors influence the type of onset and organ tropism. The approved small molecule tafamidis stabilizes the tetramer and significantly decelerates the clinical course. By sequence‐specific mRNA knockdown, the approved small interfering RNA (siRNA) patisiran and antisense oligonucleotide (ASO) inotersen both significantly reduce plasma TTR levels and improve neuropathy and quality of life compared to placebo. With enhanced hepatic targeting capabilities, GalNac‐conjugated siRNA and ASOs have recently entered phase III clinical trials. Bivalent TTR stabilizers occupy both binding groves in vitro, but have not been tested in trials so far. Tolcapone is another stabilizer with the potential to cross the blood–brain barrier, but its half‐life is short and liver failure a potential side effect. Amyloid‐directed antibodies and substances like doxycycline aim at reducing the amyloid load, however, none of the yet developed antibodies has successfully passed clinical trials. ATTR‐amyloidosis has become a model disease for pathophysiology‐based treatment. Further understanding of disease mechanisms will help to overcome the remaining limitations, including application burden, side effects, and blood–brain barrier permeability.
Background: With the availability of T-cell-directed therapy and next-generation compounds of established classes of drugs, the treatment of relapsed/refractory (r/r) myeloma is getting more complex. However, treatment options in practice are limited by availability, approval, and patient comorbidity. The aim of this article is to provide a practical approach toward the choice of treatment for r/r myeloma patients. Summary: Regarding market authorization and current guidelines, at least in Germany, most patients nowadays will have received a doublet or triplet combination as first-line therapy containing a proteasome inhibitor and an immunomodulatory drug, mostly lenalidomide. We focus on the treatment options for patients that are ineligible for (another) stem cell transplantation. We will review treatment options for relapse after first- or second-line therapy and beyond third-line. Key Messages: There is promising data supporting the efficacy and safety of triplet combinations containing anti-CD38-monoclonal antibodies (anti-CD38 mAbs) at first or second relapse in combination with next-generation compounds. For the treatment beyond third-line, comparative studies are scarce but some promising compounds are available via conditional authorization, and there is more to come in the future. We will present some early phase trials featuring promising results.
Background
The anti-SLAMF7 monoclonal antibody, elotuzumab (elo), plus lenalidomide (len) and dexamethasone (dex) is approved for relapsed/refractory MM in the U.S. and Europe. Recently, a small phase 2 study demonstrated an advantage in progression-free survival (PFS) for elo plus pomalidomide (pom)/dex compared to pom/dex alone and resulted in licensing of this novel triplet combination, but clinical experience is still limited.
Purpose
To analyze the efficacy and safety of elo/pom/dex in a “real world” cohort of patients with advanced MM, we queried the databases of the university hospitals of Würzburg and Vienna.
Findings
We identified 22 patients with a median number of five prior lines of therapy who received elo/pom/dex prior to licensing within an early access program. Patients received a median number of 5 four-week treatment cycles. Median PFS was 6.4 months with 12-month and 18-month PFS rates of 35% and 28%, respectively. The overall response rate was 50% and 64% of responding patients who achieved a longer PFS with elo/pom/dex compared to their most recent line of therapy. Objective responses were also seen in five patients who had been pretreated with pomalidomide. Low tumor burden was associated with improved PFS (13.5 months for patients with ISS stage I/II at study entry v 6.4 months for ISS III), although this difference did not reach statistical significance. No infusion-related reactions were reported. The most frequent grade 3/4 adverse events were neutropenia and pneumonia.
Conclusion
Elo/pom/dex is an active and well-tolerated regimen in highly advanced MM even after pretreatment with pomalidomide.
Purpose
In cancer of unknown primary (CUP), positron emission tomography/computed tomography (PET/CT) with the glucose analog [\(^{18}\)F]FDG represents the standard imaging approach for localization of the malignant primary. Frequently, however, [\(^{18}\)F]FDG PET/CT cannot precisely distinguish between small occult tumors and chronic inflammation, especially in Waldeyer’s tonsillar ring. To improve the accuracy for detecting primary tumors in the Waldeyer’s tonsillar ring, the novel PET tracer [\(^{68}\)Ga]Ga-FAPI-4 for specific imaging of fibroblast activation protein (FAP) expression was used as a more specific target for cancer imaging.
Methods
Eight patients with suspicion of a malignant tumor in Waldeyer’s tonsillar ring or a CUP syndrome were examined. PET/CT scans with [\(^{18}\)F]-FDG and [\(^{68}\)Ga]Ga-FAPI-4 were performed for pre-operative tumor localization. After surgical resection, histopathological and immunohistochemical results were compared to PET/CT findings.
Results
Histopathology revealed a palatine or lingual tonsil carcinoma in all patients. In case of lymph node metastases smaller than 7 mm in size, the [\(^{18}\)F]FDG PET/CT detection rate of cervical lymph node metastases was higher than that of [\(^{68}\)Ga]FAPI PET/CT, while both tracers identified the primary tumors in all eight cases. The size of the primary and the lymph node metastases was directly correlated to the respective FAP expression, as detected by immunohistochemistry. The mean SUVmax for the primary tumors was 21.29 ± 7.97 for \(^{18}\)F-FDG and 16.06 ± 6.29 for \(^{68}\)Ga-FAPI, respectively (p = 0.2). The mean SUVmax for the healthy contralateral tonsils was 8.38 ± 2.45 for [\(^{18}\)F]FDG and 3.55 ± 0.47 for [\(^{68}\)Ga]FAPI (p < 0.001). The SUVmax ratio of [68Ga]FAPI was significantly different from [\(^{18}\)F] FDG (p = 0.03). Mean TBRmax for the [\(^{68}\)Ga]Ga-FAPI-4 tracer was markedly higher in comparison to [\(^{18}\)F]FDG (10.90 vs. 4.11).
Conclusion
Non-invasive imaging of FAP expression by [\(^{68}\)Ga]FAPI PET/CT resulted in a better visual detection of the malignant primary in CUP, as compared to [\(^{18}\)F]FDG imaging. However, the detection rate of lymph node metastases was inferior, presumably due to low FAP expression in small metastases. Nevertheless, by offering a detection method for primary tumors with the potential of lower false positive rates and thus avoiding biopsies, patients with CUP syndrome may benefit from [\(^{68}\)Ga]FAPI PET/CT imaging.
Background
International consensus criteria (ICC) have redefined borderline resectability for pancreatic ductal adenocarcinoma (PDAC) according to three dimensions: anatomical (BR-A), biological (BR-B), and conditional (BR-C). The present definition acknowledges that resectability is not just about the anatomic relationship between the tumour and vessels but that biological and conditional dimensions also are important.
Methods
Patients’ tumours were retrospectively defined borderline resectable according to ICC. The study cohort was grouped into either BR-A or BR-B and compared with patients considered primarily resectable (R). Differences in postoperative complications, pathological reports, overall (OS), and disease-free survival were assessed.
Results
A total of 345 patients underwent resection for PDAC. By applying ICC in routine preoperative assessment, 30 patients were classified as stage BR-A and 62 patients as stage BR-B. In total, 253 patients were considered R. The cohort did not contain BR-C patients. No differences in postoperative complications were detected. Median OS was significantly shorter in BR-A (15 months) and BR-B (12 months) compared with R (20 months) patients (BR-A vs. R: p = 0.09 and BR-B vs. R: p < 0.001). CA19-9, as the determining factor of BR-B patients, turned out to be an independent prognostic risk factor for OS.
Conclusions
Preoperative staging defining surgical resectability in PDAC according to ICC is crucial for patient survival. Patients with PDAC BR-B should be considered for multimodal neoadjuvant therapy even if considered anatomically resectable.
Purpose
Local treatment of small well-differentiated rectal neuroendocrine tumors (NETs) is recommended by current guidelines. However, although several endoscopic methods have been established, the highest R0 rate is achieved by transanal endoscopic microsurgery (TEM). Since a recently published study about endoscopic full thickness resection (eFTR) showed a R0 resection rate of 100%, the aim of this study was to evaluate both methods (eFTR vs. TEM).
Methods
We retrospectively analyzed all patients with rectal NET treated either by TEM (1999–2018) or eFTR (2016–2019) in two tertiary centers (University Hospital Wuerzburg and Ulm). We analyzed clinical, procedural, and histopathological outcomes in both groups.
Results
Twenty-eight patients with rectal NET received local treatment (TEM: 13; eFTR: 15). Most tumors were at stage T1a and grade G1 or G2 (in the TEM group two G3 NETs were staged T2 after neoadjuvant chemotherapy). In both groups, similar outcomes for en bloc resection rate, R0 resection rate, tumor size, or specimen size were found. No procedural adverse events were noted. Mean procedure time in the TEM group was 48.9 min and 19.2 min in the eFTR group.
Conclusion
eFTR is a convincing method for local treatment of small rectal NETs combining high safety and efficacy with short interventional time.
The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens.
This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly.
Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells.
CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus.
A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib,cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for theapplication in daily clinical routine has been developed and validated according to the US Food and Drug Administration andEuropean Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples withacetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5μm(2.1×50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A andmethanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stableisotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min perrun. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2–500 ng/mLfor afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6–1500 ng/mL for cabozantinib, dabrafenib, nilotinib,and osimertinib (coefficients of correlation≥0.99). Validation assays for accuracy and precision, matrix effect, recovery,carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughputand was successfully applied to monitor concentrations of kinase inhibitors in patients.
Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8\(^+\) T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.
Myeloid-derived suppressor cells (MDSCs) represent a major population controlling T cell immune responses. However, little is known about their molecular requirements for homing and T cell interaction to mediate suppression. Here, we investigated the functional role of the homing and collagen IV receptor VLA-1 (α1β1-integrin) on in vitro GM-CSF generated murine MDSCs from wild-type (WT) and CD49a/α1-integrin (Itga1\(^{−/−}\)) gene-deficient mice. Here, we found that effector (Teff) but not naive (Tn) CD4\(^+\) T cells express VLA-1 and monocytes further up-regulated their expression after culture in GM-CSF when they differentiated into the monocytic subset of resting MDSCs (R-MDSCs). Subsequent activation of R-MDSCs by LPS+IFN-γ (A-MDSCs) showed increased in vitro suppressor potential, which was independent of VLA-1. Surprisingly, VLA-1 deficiency did not influence A-MDSC motility or migration on collagen IV in vitro. However, interaction times of Itga1\(^{−/−}\) A-MDSCs with Teff were shorter than with WT A-MDSCs on collagen IV but not on fibronectin substrate in vitro. After injection, A-MDSCs homed to the splenic red pulp where they co-localized with Teff and showed immediate suppression already after 6 h as shown by inhibition of T cell proliferation and induction of apoptosis. Injection of A-MDSCs from Itga1\(^{−/−}\) mice showed equivalent homing into the spleen but a reduced suppressive effect. Interaction studies of A-MDSCs with Teff in the subcapsular red pulp with intravital two-photon microscopy revealed also here that MDSC motility and migration parameters were not altered by VLA-1 deficiency, but the interaction times with Teff were reduced. Together, our data point to a new role of VLA-1 adhesion to collagen IV as a prerequisite for extended contact times with Teff required for suppression.