Treatment of chronic COVID-19 with convalescent/postvaccination plasma in patients with hematologic malignancies.

Immunocompromised patients are at high risk to fail clearance of SARS-CoV-2. Prolonged COVID-19 constitutes a health risk and a management problem as cancer treatments often have to be disrupted. As SARS-CoV-2 evolves, new variants of concern have emerged that evade available monoclonal antibodies. Moreover, antiviral therapy promotes SARS-CoV-2 escape mutations, particularly in immunocompromised patients. These patients frequently suffer from prolonged infection. No successful treatment has been established for persistent COVID-19 infection. Here, we report on a series of 21 immunocompromised patients with COVID-19-most of them hematologic malignancies-treated with plasma obtained from recently convalescent or vaccinated donors or a combination thereof. Repeated dosing of SARS-CoV-2-antibody-containing plasma could clear SARS-CoV-2 infection in 16 out of 21 immunocompromised patients even if COVID-19-specific treatments failed to induce sustained viral clearance or to improve clinical course of SARS-CoV-2 infection. Ten patients were major responders defined as an increase delta(d)Ct of > = 5 after the first administration of convalescent and/or vaccinated plasma (C/VP). On average, SARS-CoV-2 PCR Ct values increased from a median value of 22.55 (IQR = 19.10-24.25) to a median value of 29.57 (IQR = 27.55-34.63; p = <.0001) in the major response subgroup. Furthermore, when treated a second time with C/VP, even 4 out of 5 of the initial nonresponders showed an increase in Ct-values from a median value of 23.13 (IQR = 17.75-28.05) to a median value of 32.79 (IQR = 31.75-33.75; p = .013). Our results suggest that C/VP could be a feasible treatment of COVID-19 infection in patients with hematologic malignancies who did not respond to antiviral treatment.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

Clonal hematopoiesis with DNMT3A and PPM1D mutations impairs regeneration in autologous stem cell transplant recipients.

Clonal hematopoiesis (CH) is an age-related condition driven by stem and progenitor cells harboring recurrent mutations linked to myeloid neoplasms. Currently, potential effects on hematopoiesis, stem cell function and regenerative potential under stress conditions are unknown. We performed targeted DNA sequencing of 457 hematopoietic stem cell grafts collected for autologous stem cell transplantation (ASCT) in myeloma patients and correlated our findings with high-dimensional longitudinal clinical and laboratory data (26,510 data points for blood cell counts/serum values in 25 days around transplantation). We detected CHrelated mutations in 152 patients (33.3%). Since many patients (n=54) harbored multiple CH mutations in one or more genes, we applied a non-negative matrix factorization (NMF) clustering algorithm to identify genes that are commonly co-mutated in an unbiased approach. Patients with CH were assigned to one of three clusters (C1-C3) and compared to patients without CH (C0) in a gene specific manner. To study the dynamics of blood cell regeneration following ASCT, we developed a time-dependent linear mixed effect model to validate differences in blood cell count trajectories amongst different clusters. The results demonstrated that C2, composed of patients with DNMT3A and PPM1D single and co-mutated CH, correlated with reduced stem cell yields and delayed platelet count recovery following ASCT. Also, the benefit of maintenance therapy was particularly strong in C2 patients. Taken together, these data indicate an impaired regenerative potential of hematopoietic stem cell grafts harboring CH with DNMT3A and PPM1D mutations.

Aspirin use and bleeding events during thrombocytopenia after autologous stem-cell transplantation for multiple myeloma.

Background: In patients with cardiovascular (CV) comorbidities that necessitate antiplatelet therapy (APT), its optimal management during chemotherapy-induced thrombocytopenia remains elusive, as the risk of bleeding has to be balanced against the risk of CV events. The purpose of this study was to assess the risk for bleeding with APT during thrombocytopenia in patients with multiple myeloma undergoing high-dose chemotherapy and subsequent autologous stem-cell transplantation (ASCT) with and without acetylsalicylic acid (ASA) as comedication. Methods: We assessed patients who underwent ASCT at the Heidelberg University Hospital between 2011 and 2020 for bleeding events, management strategies for ASA intake during thrombocytopenia, transfusion requirements, and the occurrence of CV events. Results: There were 57/1,113 patients who continued ASA until at least 1 day after ASCT; thus, a continuous platelet inhibition during thrombocytopenia was assumed. Most of the patients (41/57) continued ASA until they had a platelet count of 20-50/nl. This range reflects the kinetics of thrombocytopenia and nondaily measurements of platelets during ASCT. A tendency toward a higher risk for bleeding events in the ASA group was demonstrated (1.9% (control group) vs. 5.3% (ASA), p = 0.082). The risk factors for bleeding in multivariate analysis were the duration of thrombocytopenia < 50/nl, a history of gastrointestinal bleeding, and diarrhea. The factors predicting the duration of thrombocytopenia were age >60 years, a hematopoietic stem-cell transplantation comorbidity index ≥3, and an impaired bone marrow reserve at admission. CV events occurred in three patients; none of them took ASA or had an indication for APT. Conclusions: The intake of ASA until thrombocytopenia with a platelet count of 20-50/nl appears safe, although an elevated risk cannot be excluded. If ASA is indicated for the secondary prevention of CV events, the evaluation of risk factors for bleeding and a prolonged time of thrombocytopenia before conditioning is crucial to adapt the strategy for ASA intake during thrombocytopenia.

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia.

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML.

Combinatorial BCL2 Family Expression in Acute Myeloid Leukemia Stem Cells Predicts Clinical Response to Azacitidine/Venetoclax.

The BCL2 inhibitor venetoclax (VEN) in combination with azacitidine (5-AZA) is currently transforming acute myeloid leukemia (AML) therapy. However, there is a lack of clinically relevant biomarkers that predict response to 5-AZA/VEN. Here, we integrated transcriptomic, proteomic, functional, and clinical data to identify predictors of 5-AZA/VEN response. Although cultured monocytic AML cells displayed upfront resistance, monocytic differentiation was not clinically predictive in our patient cohort. We identified leukemic stem cells (LSC) as primary targets of 5-AZA/VEN whose elimination determined the therapy outcome. LSCs of 5-AZA/VEN-refractory patients displayed perturbed apoptotic dependencies. We developed and validated a flow cytometry-based "Mediators of apoptosis combinatorial score" (MAC-Score) linking the ratio of protein expression of BCL2, BCL-xL, and MCL1 in LSCs. MAC scoring predicts initial response with a positive predictive value of more than 97% associated with increased event-free survival. In summary, combinatorial levels of BCL2 family members in AML-LSCs are a key denominator of response, and MAC scoring reliably predicts patient response to 5-AZA/VEN. SIGNIFICANCE: Venetoclax/azacitidine treatment has become an alternative to standard chemotherapy for patients with AML. However, prediction of response to treatment is hampered by the lack of clinically useful biomarkers. Here, we present easy-to-implement MAC scoring in LSCs as a novel strategy to predict treatment response and facilitate clinical decision-making. This article is highlighted in the In This Issue feature, p. 1275.

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N = 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished in vitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid in vitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

Anti-SARS-CoV-2 antibody-containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19: a randomized clinical trial.

Patients with cancer are at high risk of severe coronavirus disease 2019 (COVID-19), with high morbidity and mortality. Furthermore, impaired humoral response renders severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a randomized, open-label, multicenter trial ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE ) in hospitalized patients with severe COVID-19 (n = 134) within four risk groups ((1) cancer (n = 56); (2) immunosuppression (n = 16); (3) laboratory-based risk factors (n = 36); and (4) advanced age (n = 26)) randomized to standard of care (control arm) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (plasma arm). No serious adverse events were observed related to the plasma treatment. Clinical improvement as the primary outcome was assessed using a seven-point ordinal scale. Secondary outcomes were time to discharge and overall survival. For the four groups combined, those receiving plasma did not improve clinically compared with those in the control arm (hazard ratio (HR) = 1.29; P = 0.205). However, patients with cancer experienced a shortened median time to improvement (HR = 2.50; P = 0.003) and superior survival with plasma treatment versus the control arm (HR = 0.28; P = 0.042). Neutralizing antibody activity increased in the plasma cohort but not in the control cohort of patients with cancer (P = 0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcomes in patients with cancer who are unable to intrinsically generate an adequate immune response.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML.

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

Humoral Responses and Chronic GVHD Exacerbation after COVID-19 Vaccination Post Allogeneic Stem Cell Transplantation.

The COVID-19 pandemic threatens patients with a compromised immune and endothelial system, including patients who underwent allogeneic stem cell transplantation (alloSCT). Thus, there is an unmet need for optimizing vaccination management in this high-risk cohort. Here, we monitored antibodies against SARS-CoV-2 spike protein (anti-S1) in 167 vaccinated alloSCT patients. Humoral immune responses were detectable in 81% of patients after two vaccinations with either mRNA-, vector-based, or heterologous regimens. Age, B-cell counts, time interval from vaccination, and the type of vaccine determined antibody titres in patients without systemic immunosuppression (sIS). Similar to a healthy control cohort, mRNA vaccine-based regimens induced higher titres than vector-based vaccines. Patients on two or more immunosuppressants rarely developed immunity. In contrast, 62% and 45% of patients without or on only one immunosuppressant, respectively, showed a strong humoral vaccination response (titre > 100). Exacerbation of cGVHD upon vaccination was observed in 6% of all patients and in 22% of patients receiving immunosuppression for cGVHD. cGVHD exacerbation and low antibody titres were both associated with higher angiopoietin-2 (ANG2) serum levels. In conclusion, mRNA-based vaccines elicit strong humoral responses in alloSCT patients in the absence of double sIS. Biomarkers such as ANG2 might help with weighing cGVHD risk versus beneficial responses.

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Validation of a proxy-reported SARC-F questionnaire for current and retrospective screening of sarcopenia-related functional impairments.

BACKGROUND: The strength, assistance walking, rise from a chair, climb stairs, and falls (SARC-F) questionnaire is a well-established instrument for screening of sarcopenia and sarcopenia-related functional impairments. As it is based on self-reporting, its use precludes patients who are unable to answer the questionnaire as a consequence of severe acute diseases or cognitive impairment. Therefore, we aimed to validate a proxy-reported version of the SARC-F for both ad-hoc as well as retrospective screening for severe sarcopenia-related functional impairments. METHODS: Patients aged ≥60 years completed the SARC-F and performed the short physical performance battery (SPPB) at baseline (T1). Proxies in Cohort A gave a simultaneous assessment of the patients' functional status with the proxy-reported SARC-F at T1 and again, retrospectively, after 3 months (T2). Proxies in Cohort B only completed the SARC-F retrospectively at T2. The questionnaires' performances were assessed through sensitivity/specificity analyses and receiver operating characteristic (ROC) curves. For non-inferiority analyses, results of both the patient-reported and proxy-reported SARC-F were correlated with the SPPB total score as well as the results of the chair-rise test subcategory; the respective correlation coefficients were tested against each other. RESULTS: One hundred and four patients and 135 proxies participated. Using a SPPB score < 9 points as the reference standard, the proxy-reported SARC-F identified patients at high risk for sarcopenia-related functional impairment with a sensitivity of 0.81 (ad-hoc), 0.88 (retrospective Cohort A), and 0.87 (retrospective Cohort B) as well as a specificity of 0.89 (ad-hoc), 0.78 (retrospective Cohort A), and 0.64 (retrospective Cohort B). Areas under the ROC curves were ≥ 0.9 for the ad-hoc proxy-reported SARC-F and the retrospective proxy-reported SARC-F in both cohorts. The proxy-reported SARC-F showed a non-inferior correlation with the SPPB compared with the patient-reported SARC-F for ad-hoc (P = <0.001) as well as retrospective screening for severe sarcopenia-related functional impairment in both Cohorts A (P = 0.007) and B (P = 0.026). CONCLUSIONS: Proxy-reported SARC-F is a valid instrument for both ad-hoc as well as retrospective screening for sarcopenia-related functional impairment and could become the standard tool for evaluating this risk in older adults with severe acute disease, for example, in patients with quickly evolving haematological conditions.

Hotspot DNMT3A mutations in clonal hematopoiesis and acute myeloid leukemia sensitize cells to azacytidine via viral mimicry response.

Somatic mutations in DNA methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and acute myeloid leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3AR882). Here, we demonstrate that DNMT3AR882H-dependent CH and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in individuals with DNMT3AR882 mutations, suggesting its potential as a predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3AR882H-expressing cells. Hematopoietic stem cells (HSCs) and progenitor cells expressing DNMT3AR882H exhibited cell autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3AR882H-expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.

A Randomized Open label Phase-II Clinical Trial with or without Infusion of Plasma from Subjects after Convalescence of SARS-CoV-2 Infection in High-Risk Patients with Confirmed Severe SARS-CoV-2 Disease (RECOVER): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Primary objectives • To assess the time from randomisation until an improvement within 84 days defined as two points on a seven point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of SARS-CoV-2 infection or standard of care. Secondary objectives • To assess overall survival, and the overall survival rate at 28 56 and 84 days. • To assess SARS-CoV-2 viral clearance and load as well as antibody titres. • To assess the percentage of patients that required mechanical ventilation. • To assess time from randomisation until discharge. TRIAL DESIGN: Randomised, open-label, multicenter phase II trial, designed to assess the clinical outcome of SARS-CoV-2 disease in high-risk patients (group 1 to group 4) following treatment with anti-SARS-CoV-2 convalescent plasma or standard of care. PARTICIPANTS: High-risk patients >18 years of age hospitalized with SARS-CoV-2 infection in 10-15 university medical centres will be included. High-risk is defined as SARS-CoV-2 positive infection with Oxygen saturation at ≤ 94% at ambient air with additional risk features as categorised in 4 groups: • Group 1, pre-existing or concurrent hematological malignancy and/or active cancer therapy (incl. chemotherapy, radiotherapy, surgery) within the last 24 months or less. • Group 2, chronic immunosuppression not meeting the criteria of group 1. • Group 3, age ≥ 50 - 75 years meeting neither the criteria of group 1 nor group 2 and at least one of these criteria: Lymphopenia < 0.8 x G/l and/or D-dimer > 1µg/mL. • Group 4, age ≥ 75 years meeting neither the criteria of group 1 nor group 2. Observation time for all patients is expected to be at least 3 months after entry into the study. Patients receive convalescent plasma for two days (day 1 and day 2) or standard of care. For patients in the standard arm, cross over is allowed from day 10 in case of not improving or worsening clinical condition. Nose/throat swabs for determination of viral load are collected at day 0 and day 1 (before first CP administration) and subsequently at day 2, 3, 5, 7, 10, 14, 28 or until discharge. Serum for SARS-Cov-2 diagnostic is collected at baseline and subsequently at day 3, 7, 14 and once during the follow-up period (between day 35 and day 84). There is a regular follow-up of 3 months. All discharged patients are followed by regular phone calls. All visits, time points and study assessments are summarized in the Trial Schedule (see full protocol Table 1). All participating trial sites will be supplied with study specific visit worksheets that list all assessments and procedures to be completed at each visit. All findings including clinical and laboratory data are documented by the investigator or an authorized member of the study team in the patient's medical record and in the electronic case report forms (eCRFs). INTERVENTION AND COMPARATOR: This trial will analyze the effects of convalescent plasma from recovered subjects with SARS-CoV-2 antibodies in high-risk patients with SARS-CoV-2 infection. Patients at high risk for a poor outcome due to underlying disease, age or condition as listed above are eligible for enrollment. In addition, eligible patients have a confirmed SARS-CoV-2 infection and O2 saturation ≤ 94% while breathing ambient air. Patients are randomised to receive (experimental arm) or not receive (standard arm) convalescent plasma in two bags (238 - 337 ml plasma each) from different donors (day 1, day 2). A cross over from the standard arm into the experimental arm is possible after day 10 in case of not improving or worsening clinical condition. MAIN OUTCOMES: Primary endpoints: The main purpose of the study is to assess the time from randomisation until an improvement within 84 days defined as two points on a seven-point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of a SARS-CoV-2 infection or standard of care. Secondary endpoints: • Overall survival, defined as the time from randomisation until death from any cause 28-day, 56-day and 84-day overall survival rates. • SARS-CoV-2 viral clearance and load as well as antibody titres. • Requirement mechanical ventilation at any time during hospital stay (yes/no). • Time until discharge from randomisation. • Viral load, changes in antibody titers and cytokine profiles are analysed in an exploratory manner using paired non-parametric tests (before - after treatment). RANDOMISATION: Upon confirmation of eligibility (patients must meet all inclusion criteria and must not meet exclusion criteria described in section 5.3 and 5.4 of the full protocol), the clinical site must contact a centralized internet randomization system ( https://randomizer.at/ ). Patients are randomized using block randomisation to one of the two arms, experimental arm or standard arm, in a 1:1 ratio considering a stratification according to the 4 risk groups (see Participants). BLINDING (MASKING): The study is open-label, no blinding will be performed. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total number of 174 patients is required for the entire trial, n=87 per group. TRIAL STATUS: Protocol version 1.2 dated 09/07/2020. A recruitment period of approximately 9 months and an overall study duration of approximately 12 months is anticipated. Recruitment of patients starts in the third quarter of 2020. The study duration of an individual patient is planned to be 3 months. After finishing all study-relevant procedures, therapy, and follow-up period, the patient is followed in terms of routine care and treated if necessary. Total trial duration: 18 months Duration of the clinical phase: 12 months First patient first visit (FPFV): 3rd Quarter 2020 Last patient first visit (LPFV): 2nd Quarter 2021 Last patient last visit (LPLV): 3rd Quarter 2021 Trial Report completed: 4th Quarter 2021 TRIAL REGISTRATION: EudraCT Number: 2020-001632-10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE , registered on 04/04/2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). The eCRF is attached (Additional file 3).

Donor derived HLA-G polymorphisms have a significant impact on acute rejection in kidney transplantation.

Human leucocyte antigen G (HLA-G) is a non-classical HLA-class I antigen that exerts immunoregulatory functions. The polymorphisms 14-base pair (bp) insertion/deletion (ins/del) (rs1704) and +3142C > G (rs1063320) could modify the expression level of HLA-G. We genotyped 175 kidney recipients (41 with acute rejection and 134 without rejection) and additionally the corresponding donors for both polymorphisms in order to assess their impact on acute rejections one year after transplantation. In addition, we analyzed soluble HLA-G (sHLA-G) levels in sera of 32 living kidney donors and compared the sHLA-G levels in terms of the present genotype. In kidney transplant recipients we did not observe an impact of the 14-bp ins/ins and the +3142GG genotypes on acute rejection. In contrast, we found a higher frequency of these genotypes in the donors of the no-rejection collective compared to the rejection collective (4.9% vs. 24.6%; p = 0.010; 9.8% vs. 31.3%; p = 0.006). Soluble HLA-G levels were highest in healthy kidney donors homozygous for the 14-bp insertion. We conclude that the HLA-G polymorphisms of the donor are of importance for susceptibility of acute rejection in kidney transplantation. We suggest that the 14-bp ins/ins and the +3142GG genotypes are protective against kidney transplant rejection.

14-bp ins/del polymorphism and +3142C>G SNP of the HLA-G gene have a significant impact on acute rejection after liver transplantation.

Expression of human leukocyte antigen G (HLA-G) has been associated with increased graft survival and decreased rejection episodes. It has been described that the HLA-G 14-base pair (bp) insertion/deletion (ins/del) (rs66554220) and +3142C>G (rs1063320) gene polymorphisms modify the expression level of HLA-G. The aim of the study was to investigate whether these HLA-G polymorphisms have an impact on acute rejection after liver transplantation. In total, 146 liver transplant recipients (57 with acute rejection and 89 without acute rejection) and 99 corresponding liver donors were genotyped for both polymorphisms. In liver transplantation the 14-bp ins/ins and the +3142GG genotypes are more frequent in recipients without rejection compared to recipients with rejection (3.5% vs. 31.5%, p=<0.001; 12.3% vs. 41.6%, p=<0.001) demonstrating an association with protection from acute rejection. In contrast, in liver donors we could not reveal an association. We conclude that 14-bp ins/ins and +3142GG genotypes of HLA-G in liver transplant recipients are of importance for prediction of acute rejection after liver transplantation. Thus genotyping of liver recipients for both polymorphisms might be useful to stratify liver transplant recipients according to the risk of acute liver transplant rejection.