HER3 (ERBB3) amplification in liposarcoma - a putative new therapeutic target?

BACKGROUND: Liposarcomas are among the most common mesenchymal malignancies. However, the therapeutic options are still very limited and so far, targeted therapies had not yet been established. Immunotherapy, which has been a breakthrough in other oncological entities, seems to have no efficacy in liposarcoma. Complicating matters further, classification remains difficult due to the diversity of morphologies and nonspecific or absent markers in immunohistochemistry, leaving molecular pathology using FISH or sequencing as best options. Many liposarcomas harbor MDM2 gene amplifications. In close relation to the gene locus of MDM2, HER3 (ERBB3) gene is present and co-amplification could occur. Since the group of HER/EGFR receptor tyrosine kinases and its inhibitors/antibodies play a role in a broad spectrum of oncological diseases and treatments, and some HER3 inhibitors/antibodies are already under clinical investigation, we hypothesized that in case of HER3 co-amplifications a tumor might bear a further potential therapeutic target. METHODS: We performed FISH analysis (MDM2, DDIT3, HER3) in 56 archived cases and subsequently performed reclassification to confirm the diagnosis of liposarcoma. RESULTS: Next to 16 out of 56 cases needed to be re-classified, in 20 out of 54 cases, a cluster-amplification of HER3 could be detected, significantly correlating with MDM2 amplification. Our study shows that the entity of liposarcomas show specific molecular characteristics leading to reclassify archived cases by modern, established methodologies. Additionally, in 57.1% of these cases, HER3 was cluster-amplified profusely, presenting a putative therapeutic target for targeted therapy. CONCLUSION: Our study serves as the initial basis for further investigation of the HER3 gene as a putative therapeutic target in liposarcoma.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

γ-Secretase Modulator BPN15606 Reduced Aß42 and Aß40 and Countered Alzheimer-Related Pathologies in a Mouse Model of Down Syndrome.

OBJECTIVES: Due to increased gene dose for the amyloid precursor protein (APP), elderly adults with Down syndrome (DS) are at a markedly increased risk of Alzheimer's disease (AD), known as DS-AD. How the increased APP gene dose acts and which APP products are responsible for DS-AD is not well understood, thus limiting strategies to target pathogenesis. As one approach to address this question, we used a novel class of γ-secretase modulators that promote γ-site cleavages by the γ-secretase complex, resulting in lower levels of the Aß42 and Aß40 peptides. METHODS: Ts65Dn mice, which serve as a model of DS, were treated via oral gavage with 10 mg/kg/weekday of BPN15606 (a potent and novel pyridazine-containing γ-secretase modulators). Treatment started at 3 months-of-age and lasted for 4 months. RESULTS: Demonstrating successful target engagement, treatment with BPN15606 significantly decreased levels of Aß40 and Aß42 in the cortex and hippocampus; it had no effect on full-length APP or its C-terminal fragments in either 2 N or Ts65Dn mice. Importantly, the levels of total amyloid-ß were not impacted, pointing to BPN15606-mediated enhancement of processivity of γ-secretase. Additionally, BPN15606 rescued hyperactivation of Rab5, a protein responsible for regulating endosome function, and normalized neurotrophin signaling deficits. BPN15606 treatment also normalized the levels of synaptic proteins and tau phosphorylation, while reducing astrocytosis and microgliosis, and countering cognitive deficits. INTERPRETATION: Our findings point to the involvement of increased levels of Aß42 and/or Aß40 in contributing to several molecular and cognitive traits associated with DS-AD. They speak to increased dosage of the APP gene acting through heightened levels of Aß42 and/or Aß40 as supporting pathogenesis. These findings further the interest in the potential use of γ-secretase modulators for treating and possibly preventing AD in individuals with DS. ANN NEUROL 2024.

Liquid Biopsies as Non-Invasive Tools for Mutation Profiling in Multiple Myeloma: Application Potential, Challenges, and Opportunities.

Over the last decades, the survival of multiple myeloma (MM) patients has considerably improved. However, despite the availability of new treatments, most patients still relapse and become therapy-resistant at some point in the disease evolution. The mutation profile has an impact on MM patients' outcome, while typically evolving over time. Because of the patchy bone marrow (BM) infiltration pattern, the analysis of a single bone marrow sample can lead to an underestimation of the known genetic heterogeneity in MM. As a result, interest is shifting towards blood-derived liquid biopsies, which allow for a more comprehensive and non-invasive genetic interrogation without the discomfort of repeated BM aspirations. In this review, we compare the application potential for mutation profiling in MM of circulating-tumor-cell-derived DNA, cell-free DNA and extracellular-vesicle-derived DNA, while also addressing the challenges associated with their use.

Listeria monocytogenes Infections in Hematopoietic Cell Transplantation Recipients: Clinical Manifestations and Risk Factors. A Multinational Retrospective Case-Control Study from the Infectious Diseases Working Party of the European Society for Blood and Marrow Transplantation.

Listeriosis is rare after hematopoietic stem cell transplantation (HCT). Little is known about listeriosis in this population. In this retrospective international case-control study, we evaluated 41 listeriosis episodes occurring between 2000 and 2021 in HCT recipients (111 transplant centers in 30 countries) and assessed risk factors for listeriosis by comparisons with matched controls. The 41 listeriosis episodes (all due to Listeria monocytogenes [LM]) occurred in 30 allogeneic (allo)-HCT recipients and 11 autologous (auto)-HCT recipients at a median of 6.2 months (interquartile range [IQR], 1.6 to 19.3 months) post-HCT. The estimated incidence was 49.8/100,000 allo-HCT recipients and 13.7/100,000 auto-HCT recipients. The most common manifestations in our cohort were fever (n = 39; 95%), headache (n = 9; 22%), diarrhea, and impaired consciousness (n = 8 each; 20%). Four patients (10%) presented with septic shock, and 19 of 38 (50%) were severely lymphocytopenic. Thirty-seven patients (90%) had LM bacteremia. Eleven patients (27%) had neurolisteriosis, of whom 4 presented with nonspecific signs and 5 had normal brain imaging findings. Cerebrospinal fluid analysis revealed high protein and pleocytosis (mainly neutrophilic). Three-month mortality was 17% overall (n = 7), including 27% (n = 3 of 11) in patients with neurolisteriosis and 13% (n = 4 of 30) in those without neurolisteriosis. In the multivariate analysis comparing cases with 74 controls, non-first HCT (odds ratio [OR], 5.84; 95% confidence interval [CI], 1.10 to 30.82; P = .038); and lymphocytopenia <500 cells/mm3 (OR, 7.54; 95% CI, 1.50 to 37.83; P = .014) were significantly associated with listeriosis. There were no statistically significant differences in background characteristics, immunosuppression, and cotrimoxazole prophylaxis between cases and controls. HCT recipients are at increased risk for listeriosis compared to the general population. Listeriosis cause severe disease with septic shock and mortality. Neurolisteriosis can present with nonspecific signs and normal imaging. Lymphocytopenia and non-first HCT are associated with an increased risk of listeriosis, and cotrimoxazole was not protective.

Targeting S100A9 protein affects mTOR-ER stress signaling and increases venetoclax sensitivity in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with limited treatment options and a high demand for novel targeted therapies. Since myeloid-related protein S100A9 is abundantly expressed in AML, we aimed to unravel the therapeutic impact and underlying mechanisms of targeting both intracellular and extracellular S100A9 protein in AML cell lines and primary patient samples. S100A9 silencing in AML cell lines resulted in increased apoptosis and reduced AML cell viability and proliferation. These therapeutic effects were associated with a decrease in mTOR and endoplasmic reticulum stress signaling. Comparable results on AML cell proliferation and mTOR signaling could be observed using the clinically available S100A9 inhibitor tasquinimod. Interestingly, while siRNA-mediated targeting of S100A9 affected both extracellular acidification and mitochondrial metabolism, tasquinimod only affected the mitochondrial function of AML cells. Finally, we found that S100A9-targeting approaches could significantly increase venetoclax sensitivity in AML cells, which was associated with a downregulation of BCL-2 and c-MYC in the combination group compared to single agent therapy. This study identifies S100A9 as a novel molecular target to treat AML and supports the therapeutic evaluation of tasquinimod in venetoclax-based regimens for AML patients.

Correction: Analysis of epidemiological association patterns of serum thyrotropin by combining random forests and Bayesian networks.

[This corrects the article DOI: 10.1371/journal.pone.0271610.].

Alzheimer's disease linked Aß42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid ß (Aß) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aß42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aß42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We show that human Aß42 peptides, but neither murine Aß42 nor human Aß17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, Aß42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aß42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aß toxicity in the context of γ-secretase-dependent homeostatic signaling.

Modeling Alzheimer's disease related phenotypes in the Ts65Dn mouse: impact of age on Aß, Tau, pTau, NfL, and behavior.

Introduction: People with DS are highly predisposed to Alzheimer's disease (AD) and demonstrate very similar clinical and pathological features. Ts65Dn mice are widely used and serve as the best-characterized animal model of DS. Methods: We undertook studies to characterize age-related changes for AD-relevant markers linked to Aß, Tau, and phospho-Tau, axonal structure, inflammation, and behavior. Results: We found age related changes in both Ts65Dn and 2N mice. Relative to 2N mice, Ts65Dn mice showed consistent increases in Aß40, insoluble phospho-Tau, and neurofilament light protein. These changes were correlated with deficits in learning and memory. Discussion: These data have implications for planning future experiments aimed at preventing disease-related phenotypes and biomarkers. Interventions should be planned to address specific manifestations using treatments and treatment durations adequate to engage targets to prevent the emergence of phenotypes.

Disease Perception Is Correlated with Health-Related Quality of Life in Patients Suffering from Myelodysplastic Syndromes: Results of the Belgian Be-QUALMS Study.

Patients with myelodysplastic syndromes suffer from an impaired quality of life that is only partially explained by physical symptoms. In an observational study, we aimed to investigate the impact of current MDS treatments and the influence of disease perception on quality of life. Serial measurement of health-related quality of life was performed by 'the QUALMS', a validated MDS-specific patient reported outcome tool. Disease perception was evaluated by means of the Brief Illness Perception Questionnaire (B-IPQ). We prospectively collected data on 75 patients that started on a new treatment and could not demonstrate a significant change in QUALMS score or B-IPQ score during treatment. Six out of eight items evaluated in the B-IPQ correlated significantly with QUALMS score. In this small sample, no significant difference in QUALMS score was found between lower vs. higher risk MDS patients or other studied variables, e.g., targeted hemoglobin showed no correlation with QUALMS score. In daily practice attention must be paid to initial formation of disease perception as it correlates independently with health-related quality of life and does not change during treatment (clinicaltrials.gov identifier: NCT04053933).

Parameter-based patient-specific restoration of physiological knee morphology for optimized implant design and matching.

Total knee arthroplasty (TKA) patients may present with genetic deformities, such as trochlear dysplasia, or deformities related to osteoarthritis. This pathologic morphology should be corrected by TKA to compensate for related functional deficiencies. Hence, a reconstruction of an equivalent physiological knee morphology would be favorable for detailed preoperative planning and the patient-specific implant selection or design process. A parametric database of 673 knees, each described by 36 femoral parameter values, was used. Each knee was classified as pathological or physiological based on cut-off values from literature. A clinical and a mathematical classification approach were developed to distinguish between affected and unaffected parameters. Three different prediction methods were used for the restoration of physiological parameter values: regression, nearest neighbor search and artificial neural networks. Several variants of the respective prediction model were considered, such as different network architectures. Regarding all methods, the model variant chosen resulted in a prediction error below the parameters' standard deviation, while the regression yielded the lowest errors. Future analyses should consider other deformities, also of tibia and patella. Furthermore, the functional consequences of the parameter changes should be analyzed.

Retromer Proteins Reduced in Down Syndrome and the Dp16 Model: Impact of APP Dose and Preclinical Studies of a γ-Secretase Modulator.

OBJECTIVE: The retromer complex plays an essential role in intracellular endosomal sorting. Deficits in the retromer complex are linked to enhanced Aß production. The levels of the components of the retromer complex are reported to be downregulated in Alzheimer disease (AD). Down syndrome (DS) shares neuropathological features with AD. Recent evidence points to dysregulation of the retromer complex in DS. The mechanisms underlying retromer deficits in DS and AD are poorly understood. METHODS: We measured the levels of retromer components in the frontal cortex of cases of DS-AD (AD in DS) as well as DS; the frontal cortex of a person partially trisomic (PT-DS) for human chromosome 21 (HSA21), whose genome had only the normal 2 copies of the APP gene, was also examined. We also analyzed these proteins in the Dp16 mouse model of DS. To further explore the molecular mechanism for changes in the retromer complex, we treated Dp16 mice with a γ-secretase modulator (GSM; 776890), a treatment that reduces the levels of Aß42 and Aß40. RESULTS: We found VPS26A, VPS26B, and VPS29, but not VPS35, were significantly reduced in both DS and DS-AD, but not in PT-DS. Downregulation of VPS26A, VPS26B, and VPS29 was recapitulated in the brains of old Dp16 mice (at 16 months of age) and required increased App gene dose. Significantly, GSM treatment completely prevented reductions of the retromer complex. INTERPRETATION: Our studies point to increased APP gene dose as a compromising retromer function in DS and suggest a causal role for Aß42 and Aß40. ANN NEUROL 2023;94:245-258.

Outcome of SARS-CoV2 infection in hematopoietic stem cell transplant recipients for autoimmune diseases.

Hematopoietic stem cell transplant (HSCT) recipients may be at high risk of mortality from coronavirus disease 2019 (COVID-19). However, specific data on COVID-19 after treatment with HSCT in patients affected by autoimmune diseases (ADs) are still lacking. In this multicenter observational study of the European Society for Blood and Marrow Transplantation (EBMT), clinical data on COVID-19 in 11 patients affected by severe ADs treated with HSCT (n = 3 allogeneic transplant; n = 8 autologous transplant) are reported. All patients were symptomatic during the initial phase of the SARS-CoV-2 infection. At screening, 5 patients reported upper respiratory symptoms, 3 patients had cough without oxygen requirement, and 6 patients exhibited extra-pulmonary symptoms. Four cases developed a lower respiratory tract disease (LRTD). Hospitalization was required in 6 cases, without necessity of intensive care unit (ICU) admission and/or ventilation/supplemental oxygen. Different interventions were adopted: remdesivir (n = 1), nirmatrelvir/ritonavir (n = 1), sotrovimab (n = 1), immunoglobulins (n = 1). At last follow-up, all patients are alive and had resolution of the infection. The current analysis describing the mild-moderate course of COVID-19 in transplant recipients affected by ADs, similar to the course observed in ADs under standard treatments, provides useful information to support the delivery of HSCT programs in this field. Vaccination and new treatments available for SARS-CoV-2 may be useful to further minimize the risk of infection.

Multipotent mesenchymal stromal cells as treatment for poor graft function after allogeneic hematopoietic cell transplantation: A multicenter prospective analysis.

Introduction: Poor graft function (PGF) is a rare but serious complication of allogeneic hematopoietic cell transplantation (alloHCT). Due to their hematopoietic supporting properties and immune regulatory effects, multipotent mesenchymal stromal cells (MSC) could be considered a good candidate to help to restore bone marrow (BM) niches homeostasis and facilitate hematopoiesis after alloHCT. Methods: We prospectively assessed the efficacy and safety of ex-vivo expanded BM-derived MSC from third-party donor in a series of 30 patients with prolonged severe cytopenia and PGF after alloHCT. This multicenter trial was registered at www.clinicaltrials.gov (#NTC00603330). Results: Within 90 days post-MSC infusion, 53% (95% CI, 35 - 71%) of patients improved at least one cytopenia (overall response, OR) and 37% (95% CI, 19 - 54%) achieved a complete hematological response (CR: absolute neutrophil count, ANC >0.5 x 109/L, Hb > 80g/L and platelet count > 20 x 109/L with transfusion independence). Corresponding response rates increased to 67% (95% CI, 50 - 84%) OR and 53% (95% CI, 35 - 71%) CR within 180 days after MSC infusion. A significant decrease in red blood cells and platelets transfusion requirement was observed after MSC (median of 30-days transfusion requirement of 0.5 and 0 from d90-120 post-MSC versus 5 and 6.5 before MSC, respectively, p ≤0.001). An increase in ANC was also noted by day +90 and +180, with 3/5 patients with severe neutropenia having recovered an ANC > 1 x 109/L within the 90-120 days after MSC infusion. Overall survival at 1 year post-MSC was 70% (95% CI, 55.4 - 88.5), with all but one of the patients who achieved CR being alive. A single infusion of third-party MSC appeared to be safe, with the exception of one deep vein thrombotic event possibly related to the intervention. Discussion: In conclusion, a single i.v. infusion of BM-derived MSC from third party donor seemed to improve hematological function after alloHCT, although spontaneous amelioration cannot be excluded. Comparative studies are warranted to confirm these encouraging results.

Reduced synaptic proteins and SNARE complexes in Down syndrome with Alzheimer's disease and the Dp16 mouse Down syndrome model: Impact of APP gene dose.

INTRODUCTION: Synaptic failure, a hallmark of Alzheimer's disease (AD), is correlated with reduced levels of synaptic proteins. Though people with Down syndrome (DS) are at markedly increased risk for AD (AD-DS), few studies have addressed synapse dysfunction. METHODS: Synaptic proteins were measured in the frontal cortex of DS, AD-DS, sporadic AD cases, and controls. The same proteins were examined in the Dp16 model of DS. RESULTS: A common subset of synaptic proteins were reduced in AD and AD-DS, but not in DS or a case of partial trisomy 21 lacking triplication of APP gene. Pointing to compromised synaptic function, the reductions in AD and AD-DS were correlated with reduced SNARE complexes. In Dp16 mice reductions in syntaxin 1A, SNAP25 and the SNARE complex recapitulated findings in AD-DS; reductions were impacted by both age and increased App gene dose. DISCUSSION: Synaptic phenotypes shared between AD-DS and AD point to shared pathogenetic mechanisms.

Veno-venous extra-corporeal membrane oxygenation in a COVID-19 patient with cold-agglutinin haemolytic anaemia: A case report.

OVERVIEW: The use of extra-corporeal membrane oxygenation (ECMO) therapy to treat severe COVID-19 patients with acute respiratory failure is increasing worldwide. We reported herein the use of veno-venous ECMO in a patient with cold agglutinin haemolytic anaemia (CAHA) who suffered from severe COVID-19 infection. DESCRIPTION: A 64-year-old man presented to the emergency department (ED) with incremental complaints of dyspnoea and cough since one week. His history consisted of CAHA, which responded well to corticosteroid treatment. Because of severe hypoxemia, urgent intubation and mechanical ventilation were necessary. Despite deep sedation, muscle paralysis and prone ventilation, P/F ratio remained low. Though his history of CAHA, he still was considered for VV-ECMO. As lab results pointed to recurrence of CAHA, corticosteroids and rituximab were started. The VV-ECMO run was short and rather uncomplicated. Although, despite treatment, CAHA persisted and caused important complications of intestinal ischemia, which needed multiple surgical interventions. Finally, the patient suffered from progressive liver failure, thought to be secondary to ischemic cholangitis. One month after admission, therapy was stopped and patient passed away. CONCLUSION: Our case report shows that CAHA is no contraindication for VV-ECMO, even when both titre and thermal amplitude are high. Although, the aetiology of CAHA and its response to therapy will determine the final outcome of those patients.

Genetic profiling of human bone marrow mesenchymal stromal cells after in vitro expansion in clinical grade human platelet lysate.

Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

Liquid Biopsy-Derived DNA Sources as Tools for Comprehensive Mutation Profiling in Multiple Myeloma: A Comparative Study.

The analysis of bone marrow (BM) samples in multiple myeloma (MM) patients can lead to the underestimation of the genetic heterogeneity within the tumor. Blood-derived liquid biopsies may provide a more comprehensive approach to genetic characterization. However, no thorough comparison between the currently available circulating biomarkers as tools for mutation profiling in MM has been published yet and the use of extracellular vesicle-derived DNA for this purpose in MM has not yet been investigated. Therefore, we collected BM aspirates and blood samples in 30 patients with active MM to isolate five different DNA types, i.e., cfDNA, EV-DNA, BM-DNA and DNA isolated from peripheral blood mononucleated cells (PBMNCs-DNA) and circulating tumor cells (CTC-DNA). DNA was analyzed for genetic variants with targeted gene sequencing using a 165-gene panel. After data filtering, 87 somatic and 39 germline variants were detected among the 149 DNA samples used for sequencing. cfDNA showed the highest concordance with the mutation profile observed in BM-DNA and outperformed EV-DNA, CTC-DNA and PBMNCs-DNA. Of note, 16% of all the somatic variants were only detectable in circulating biomarkers. Based on our analysis, cfDNA is the preferable circulating biomarker for genetic characterization in MM and its combined use with BM-DNA allows for comprehensive mutation profiling in MM.

Cellular and Humoral Immune Response to a Third Dose of BNT162b2 COVID-19 Vaccine - A Prospective Observational Study.

Background: Since the introduction of various vaccines against SARS-CoV-2 at the end of 2020, infection rates have continued to climb worldwide. This led to the establishment of a third dose vaccination in several countries, known as a booster. To date, there has been little real-world data about the immunological effect of this strategy. Methods: We compared the humoral- and cellular immune response before and after the third dose of BioNTech/Pfizer vaccine BNT162b2, following different prime-boost regimen in a prospective observational study. Humoral immunity was assessed by determining anti-SARS-CoV-2 binding antibodies using a standardized quantitative assay. In addition, neutralizing antibodies were measured using a commercial surrogate ELISA-assay. Interferon-gamma release was measured after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay to evaluate the cellular immune response. Results: We included 243 health-care workers who provided blood samples and questionnaires pre- and post- third vaccination. The median antibody level increased significantly after the third vaccination dose to 2663.1 BAU/ml vs. 101.4 BAU/ml (p < 0.001) before administration of the booster dose. This was also detected for neutralizing antibodies with a binding inhibition of 99.68% ± 0.36% vs. 69.06% ± 19.88% after the second dose (p < 0.001). 96.3% of the participants showed a detectable T-cell-response after the booster dose with a mean interferon-gamma level of 2207.07 mIU/ml ± 1905 mIU/ml. Conclusion: This study detected a BMI-dependent antibody increase after the third dose of BNT162b2 following different vaccination protocols. All participants showed a significant increase in their immune response. This, in combination with the low rate of post-vaccination-symptoms underlines the potential beneficial effect of a BNT162b2-booster dose.