The tamoxifen-regulated, long non-coding RNA LINC00992 affects proliferation, migration, and expression of tamoxifen resistance-associated genes in MCF-7 breast cancer cells.

Introduction: Tamoxifen-adapted MCF-7 breast cancer cells (MCF-7-TAM-R) are a model for acquired tamoxifen resistance in oestrogen receptor-positive breast cancer. In this system, the expression of long-non-coding RNA LINC00992 is decreased. LINC00992 might therefore contribute to tamoxifen adaption and associated gene expres-sion changes. Here, we investigated whether a modulation of LINC00992 modifies gene expression, proliferation, and migration. Material and methods: Up- and down-- regulation of LINC00992 was performed using plasmid vectors and siRNA. Gene expression was measured via nCounter® and quantitative real-time polymerase chain reaction. Database analysis was performed using GEPIA2 and cBioportal. Furthermore, we performed scratch assays, colony-forming assays, and proliferation assays with MCF-7 and MCF-7-TAM-R after up-regulation of LINC00992. Results: Up- and down-regulation of LINC00992 caused gene expression changes in 4 of the 42 tamoxifen-regulated genes tested. Especially ubiquitin D, single-minded homologue 1 (SIM1) carcinoembryonic antigen-related cell adhesion molecule 5 and the G-protein coupled oestrogen receptor 1 were affected. In tamoxifen-adapted MCF-7-TAM-R cells, LINC00992 overexpression resulted in augmented viability and proliferation and enhanced migration. Database analyses revealed that luminal breast cancers have increased expression of LINC00992 compared to Her2-type/neu- or basal type. Furthermore, higher expression of LINC00992 was associated with poor prognosis in luminal-A carcinomas. Conclusions: Changes in the expression of tamoxifen-regulated genes could be induced by manipulating LINC00992's abundance, suggesting that it is at least partially involved in the establishment of the tamoxifen-induced gene expression pattern. LINC00992 may also serve as a prognostic biomarker and may indicate the development of tamoxifen resistance.

Allogeneic stem cell transplantation for mantle cell lymphoma-update of the prospective trials of the East German Study Group Hematology/Oncology (OSHO#60 and #74).

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

The impact of G protein-coupled oestrogen receptor 1 on male breast cancer: a retrospective analysis.

INTRODUCTION: The G protein-coupled oestrogen receptor 1 (GPER-1) is a potential prognostic marker in breast cancer. However, its role in male breast cancer (MBC) is still unknown. This study evaluates the expression of GPER-1 in MBC samples and correlates these data with clinical and pathological parameters including patients' survival. MATERIAL AND METHODS: For this retrospective analysis of a prospectively maintained cohort of patients with MBC, we examined 161 specimens for GPER-1 expression using immunohistochemistry. An immunoreactive score (IRS) was calculated based on staining intensity and the percentage of positive tumour cells. Then, we correlated GPER-1 IRS with clinical and pathological parameters, and overall and relapse-free survival. RESULTS: About 40% of MBC samples were positive for GPER-1 expression (IRS ≥ 4). There was no significant correlation with clinicopathological parameters, such as hormone receptor status or grading. However, a statistical trend was observed for tumour size (≥ 2 cm, p = 0.093). Kaplan-Meier survival analysis revealed no significant correlation with relapse-free survival. However, there was a significant correlation with overall survival, but when we adjusted the log-rank p-value to compensate for the cut-off point optimization method, it rose above 0.1. Additionally, GPER-1-positive patients were older at diagnosis. When adjusted for age by multivariable Cox regression analysis, the significance of GPER-1 status for survival was further reduced. CONCLUSIONS: We found no significant prognostic value of GPER-1 in this MBC cohort as anticipated from studies on female BC. Future studies with higher sample size are needed to further verify a potential sex-specific role of GPER-1.

Pretreatment CD34+/CD38- Cell Burden as Prognostic Factor in Myelodysplastic Syndrome Patients Receiving Allogeneic Stem Cell Transplantation.

Myelodysplastic syndrome (MDS) is a highly heterogeneous clonal hematopoietic disorder. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment and is of particular interest in patients at high risk for progression to acute myeloid leukemia (AML). In MDS, CD34+/CD38- cells possess MDS stem cell potential, and secondary AML (sAML) clones originate from the MDS disease stage. However, the prognostic impact of the pretreatment stem cell population burden in MDS remains unknown. We retrospectively analyzed the prognostic impact of the pretreatment CD34+/CD38- cell burden in 124 MDS patients who received allogeneic HSCT at our institution. A high pretreatment bone marrow CD34+/CD38- cell burden (≥1%) was associated with worse genetic risk and a higher incidence of blast excess. Patients with a high CD34+/CD38- cell burden had a significantly higher cumulative incidence of MDS relapse, a higher cumulative incidence of secondary AML, and a trend for shorter overall survival after allogeneic HSCT. In multivariable analyses this prognostic impact was shown to be independent of other clinical and cytogenetic risk factors in MDS. Patients suffering MDS relapse or progression to AML also had a higher pre-treatment CD34+/CD38- cell burden as a continuous variable. The observed prognostic impact is likely mediated by MDS stem cells within the CD34+/CD38- cell population initiating MDS relapse or progression to AML. New therapeutic strategies targeting MDS stem cells might improve outcomes.

Comparison of non-myeloablative and reduced-intensity allogeneic stem cell transplantation in older patients with myelodysplastic syndromes.

Allogeneic stem cell transplantation (HSCT) remains the only curative treatment for myelodysplastic syndromes (MDS) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. The introduction of reduced intensity (RIC) and non-myeloablative (NMA) conditioning enabled HSCT in older or comorbid individuals representing the majority of patients. Studies comparing RIC and NMA conditioning are limited. We retrospectively analyzed 151 MDS or MDS/MPN patients older than 50 years who received NMA- or RIC-HSCT. Patients younger or older than 65 years at HSCT were analyzed separately. Patients receiving RIC-HSCT or NMA-HSCT were balanced in factors reflecting disease aggressiveness and the HCT-CI comorbidity score. The NMA conditioned patients had a higher incidence of graft rejection and chronic graft-vs-host disease. Cumulative incidence of relapse (CIR), non-relapse mortality (NRM) and overall survival (OS), did not differ significantly with regard to the conditioning regime in the whole cohort. In patients <65 years at HSCT, NMA conditioning associated with higher NRM and shorter OS by trend, while CIR was similar in both groups. In multivariable analyzes, the conditioning regimen remained a prognostic factor for NRM and OS in patients <65 years at HSCT. In MDS patients NMA and RIC conditioning result in similar disease control, but especially patients <65 years may benefit from RIC-HSCT.

Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients.

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable ("minimal") residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers-also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients' outcomes.

Primary prophylaxis of invasive fungal infections in patients with haematological malignancies: 2017 update of the recommendations of the Infectious Diseases Working Party (AGIHO) of the German Society for Haematology and Medical Oncology (DGHO).

Immunocompromised patients are at high risk of invasive fungal infections (IFI), in particular those with haematological malignancies undergoing remission-induction chemotherapy for acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS) and recipients of allogeneic haematopoietic stem cell transplants (HSCT). Despite the development of new treatment options in the past decades, IFI remains a concern due to substantial morbidity and mortality in these patient populations. In addition, the increasing use of new immune modulating drugs in cancer therapy has opened an entirely new spectrum of at risk periods. Since the last edition of antifungal prophylaxis recommendations of the German Society for Haematology and Medical Oncology in 2014, seven clinical trials regarding antifungal prophylaxis in patients with haematological malignancies have been published, comprising 1227 patients. This update assesses the impact of this additional evidence and effective revisions. Our key recommendations are the following: prophylaxis should be performed with posaconazole delayed release tablets during remission induction chemotherapy for AML and MDS (AI). Posaconazole iv can be used when the oral route is contraindicated or not feasible. Intravenous liposomal amphotericin B did not significantly decrease IFI rates in acute lymphoblastic leukaemia (ALL) patients during induction chemotherapy, and there is poor evidence to recommend it for prophylaxis in these patients (CI). Despite substantial risk of IFI, we cannot provide a stronger recommendation for these patients. There is poor evidence regarding voriconazole prophylaxis in patients with neutropenia (CII). Therapeutic drug monitoring TDM should be performed within 2 to 5 days of initiating voriconazole prophylaxis and should be repeated in case of suspicious adverse events or of dose changes of interacting drugs (BIItu). General TDM during posaconazole prophylaxis is not recommended (CIItu), but may be helpful in cases of clinical failure such as breakthrough IFI for verification of compliance or absorption.

Diagnosis of invasive fungal diseases in haematology and oncology: 2018 update of the recommendations of the infectious diseases working party of the German society for hematology and medical oncology (AGIHO).

Invasive fungal diseases (IFD) are a primary cause of morbidity and mortality in patients with haematological malignancies. These infections are mostly life-threatening and an early diagnosis and initiation of appropriate antifungal therapy are essential for the clinical outcome. Most commonly, Aspergillus and Candida species are involved. However, other Non-Aspergillus moulds are increasingly identified in case of documented IFD. For definite diagnosis of IFD, a combination of diagnostic tools have to be applied, including conventional mycological culture and non-conventional microbiological tests such as antibody/antigen and molecular tests, as well as histopathology and radiology. Although varying widely in cancer patients, the risk of invasive fungal infection is highest in those with allogeneic stem cell transplantation and those with acute leukaemia and markedly lower in patients with solid cancer. Since the last edition of Diagnosis of Invasive Fungal Diseases recommendations of the German Society for Hematology and Oncology in 2012, integrated care pathways have been proposed for the management and therapy of IFDs with either a diagnostic driven strategy as opposed to a clinical or empirical driven strategy. This update discusses the impact of this additional evidence and effective revisions.

Prognostic impact of the CD34+/CD38- cell burden in patients with acute myeloid leukemia receiving allogeneic stem cell transplantation.

In acute myeloid leukemia (AML), leukemia-initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Percentage of bone marrow CD34+/CD38- cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival which may be mediated by residual leukemia-initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia-initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.

Transcription factor C/EBPα-induced microRNA-30c inactivates Notch1 during granulopoiesis and is downregulated in acute myeloid leukemia.

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBPα exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBPα during granulopoiesis. Thus, wild-type C/EBPα-p42 directly upregulates miR-30c expression, whereas C/EBPα-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBPα-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBPα, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Prevention of graft-versus-host-disease with preserved graft-versus-leukemia-effect by ex vivo and in vivo modulation of CD4(+) T-cells.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation.

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3ß), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3ß are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3ß negatively regulates G-CSFR expression and its downstream signaling.

Allogeneic stem cell transplantation for mantle cell lymphoma--final report from the prospective trials of the East German Study Group Haematology/Oncology (OSHO).

This study was conducted in order to evaluate allogeneic stem cell transplantation (alloSCT) as consolidation for patients with mantle cell lymphoma (MCL). Patients with MCL were included into two prospective trials OSHO #060 (refractory/relapsed) and #074 (de novo). Induction was rituximab and chemotherapy. Responding patients proceeded to alloSCT. Minimal residual disease was monitored by quantitative RT-PCR detecting either t(11;14) or clonospecific CDR-III regions. In case of circulating lymphoma cells, immunomodulation (cyclosporine A withdrawal, rituximab, donor lymphocyte infusion) was initiated. Thirty-three of 39 patients underwent alloSCT after myeloablative (n = 7) or toxicity-reduced (n = 26) conditioning. Leukocytes engrafted at day +16 (median, range 0-101) and platelets at day +14 (0-142). Acute graft-versus-host disease stages I-II occurred in 42 % and stages III-IV in 15 %. Five patients have relapsed after SCT. The overall mortality after SCT was 24 % (n = 8). Median follow-up after SCT was 2.8 years (range 0.0-10.9). Five-year progression-free survival was 67 %, and overall survival 73 % after SCT. The results were comparable for primary MCL and refractory/relapsed disease as well as for related vs. unrelated SCT. Younger patients had a significantly better outcome than the elderly. AlloSCT is a feasible and promising consolidation therapy for relapsed and refractory disease and an attractive option for young patients with de novo MCL of high risk.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells.

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.

Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes.

Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5-7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone.

MicroRNA Expression Patterns Reveal a Role of the TGF-ß Family Signaling in AML Chemo-Resistance.

Resistance to chemotherapy is ultimately responsible for the majority of AML-related deaths, making the identification of resistance pathways a high priority. Transcriptomics approaches can be used to identify genes regulated at the level of transcription or mRNA stability but miss microRNA-mediated changes in translation, which are known to play a role in chemo-resistance. To address this, we compared miRNA profiles in paired chemo-sensitive and chemo-resistant subclones of HL60 cells and used a bioinformatics approach to predict affected pathways. From a total of 38 KEGG pathways implicated, TGF-ß/activin family signaling was selected for further study. Chemo-resistant HL60 cells showed an increased TGF-ß response but were not rendered chemo-sensitive by specific inhibitors. Differential pathway expression in primary AML samples was then investigated at the RNA level using publically available gene expression data in the TGCA database and by longitudinal analysis of pre- and post-resistance samples available from a limited number of patients. This confirmed differential expression and activity of the TGF-ß family signaling pathway upon relapse and revealed that the expression of TGF-ß and activin signaling genes at diagnosis was associated with overall survival. Our focus on a matched pair of cytarabine sensitive and resistant sublines to identify miRNAs that are associated specifically with resistance, coupled with the use of pathway analysis to rank predicted targets, has thus identified the activin/TGF-ß signaling cascade as a potential target for overcoming resistance in AML.

Characterization of post-vaccination SARS-CoV-2 T cell subtypes in patients with different hematologic malignancies and treatments.

Background: To evaluate the benefits of SARS-CoV-2 vaccination in cancer patients it is relevant to understand the adaptive immune response elicited after vaccination. Patients affected by hematologic malignancies are frequently immune-compromised and show a decreased seroconversion rate compared to other cancer patients or controls. Therefore, vaccine-induced cellular immune responses in these patients might have an important protective role and need a detailed evaluation. Methods: Certain T cell subtypes (CD4, CD8, Tfh, γδT), including cell functionality as indicated by cytokine secretion (IFN, TNF) and expression of activation markers (CD69, CD154) were assessed via multi-parameter flow cytometry in hematologic malignancy patients (N=12) and healthy controls (N=12) after a second SARS-CoV-2 vaccine dose. The PBMC of post-vaccination samples were stimulated with a spike-peptide pool (S-Peptides) of SARS-CoV-2, with CD3/CD28, with a pool of peptides from the cytomegalovirus, Epstein-Barr virus and influenza A virus (CEF-Peptides) or left unstimulated. Furthermore, the concentration of spike-specific antibodies has been analyzed in patients. Results: Our results indicate that hematologic malignancy patients developed a robust cellular immune response to SARS-CoV-2 vaccination comparable to that of healthy controls, and for certain T cell subtypes even higher. The most reactive T cells to SARS-CoV-2 spike peptides belonged to the CD4 and Tfh cell compartment, being median (IQR), 3.39 (1.41-5.92) and 2.12 (0.55-4.14) as a percentage of IFN- and TNF-producing Tfh cells in patients. In this regard, the immunomodulatory treatment of patients before the vaccination period seems important as it was strongly associated with a higher percentage of activated CD4 and Tfh cells. SARS-CoV-2- and CEF-specific T cell responses significantly correlated with each other. Compared to lymphoma patients, myeloma patients had an increased percentage of SARS-CoV-2-specific Tfh cells. T-SNE analysis revealed higher frequencies of γδT cells in patients compared to controls, especially in myeloma patients. In general, after vaccination, SARS-CoV-2-specific T cells were also detectable in patients without seroconversion. Conclusion: Hematologic malignancy patients are capable of developing a SARS-CoV-2-specific CD4 and Tfh cellular immune response after vaccination, and certain immunomodulatory therapies in the period before vaccination might increase the antigen-specific immune response. A proper response to recall antigens (e.g., CEF-Peptides) reflects immune cellular functionality and might be predictive for generating a newly induced antigen-specific immune response as is expected after SARS-CoV-2 vaccination.

Monoclonal Antibody Therapy for COVID-19: A Retrospective Observational Study at a Regional Hospital.

BACKGROUND: Monoclonal antibodies represent one option for treatment of COVID-19 early after infection. Although large clinical trials have been successfully conducted, real world data are needed to obtain a realistic assessment of the assumed effect on hospitalization rates. METHODS: For this retrospective, observational study, clinical data were collected in 2021 from outpatients (402) as well as hospitalized patients (350) receiving monoclonal antibodies Bamlanivimab, Casirivimab/Imdevimab or Etesevimab/Bamlanivimab. These data were compared with data from a control group of patients not receiving antibodies because admission to the hospital was too late for this therapy. RESULTS: Both groups showed a comparable spectrum of risk factors. Due to the late hospitalization of control patients, a higher frequency of severe symptoms, such as fever, dyspnea, syncope and lower viral load, were observed. CRP and leukocytes counts were also higher in the untreated group. Most importantly, hospitalization time was significantly shorter and the number of deaths was also lower in the treated group. CONCLUSIONS: Apparently, the application of anti-SARS-CoV-2 antibodies reduced the work load of our hospital as shown by the shorter hospitalization time and lower number of COVID-19-related deaths.