Neurologic complications (NCs), especially those of the central nervous system (CNS), represent a severe complication after allogeneic stem cell transplantation (allo-HSCT) and are associated with relevant morbidity and mortality. We aimed to characterize the potential risk factors for the development of CNS-NC, with a special focus on the role of calcineurin inhibitors (CNIs) as a predisposing factor. For this purpose, we compared cyclosporin A (CsA) versus tacrolimus (TAC) with respect to their influence on the incidence and type of CNS-NC after allo-HSCT. We retrospectively analyzed the incidence, risk factors, and impact on outcomes of CNS-NC diagnosed during the post-transplantation follow-up in patients with different high-risk hematologic malignancies who underwent allo-HSCT at our institution over a 20-year period. All patients included in the analysis received CNI (CsA or TAC) as graft-versus-host disease (GVHD) prophylaxis. We evaluated a total of 739 consecutive patients who underwent transplantation between December 1999 and April 2019. During a median follow-up of 6.8 years, we observed a CNS-NC incidence of 17%. The development of CNS-NC was associated with decreased overall survival (OS) and increased transplantation-related mortality (TRM). The most frequent CNS-NCs were infections (30%) and neurologic adverse events related to the administration of CNI, TAC, or CsA as GVHD prophylaxis (42%). In the multivariable analysis, age, total body irradiation (TBI), and severe acute GVHD and chronic GVHD were significant risk factors in the development of CNS-NCs. TAC compared with CsA emerged as an independent predisposing factor for CNS-NCs. The TAC-associated risk of CNS-NCs was related mostly to the occurrence of transplantation-associated thrombotic microangiopathy (TA-TMA) with neurologic manifestations (neuro-TA-TMA), although the general TA-TMA incidence was comparable in the 2 CNI subgroups. CNS-NCs are associated with poor prognosis after allo-HSCT, with TAC emerging as a potential yet insufficiently characterized predisposing factor.

In allogenic stem cell recipients, invasive fungal disease is a common yet dreaded complication with high mortality. Among these, fusariosis is especially complex to treat due to high intrinsic resistance and few antimycotic options, requiring close cooperation of all involved departments. We here report an instructive case of disseminated fusariosis after allogenic stem cell transplantation with fatal outcome despite maximum treatment.

Steroid-refractory acute and chronic graft-versus-host disease (SR-a/cGvHD) represents a potential life-threatening complication following allogeneic stem-cell transplantation (allo-SCT). The JAK1/2-inhibitor ruxolitinib and the extracorporeal photopheresis (ECP) have been shown to significantly improve the overall response rate (ORR) in this setting. However, about 30-40 % of high-risk patients do not respond to monotherapy and/or experience side effects. Considering the potential synergic mechanism of action of ruxolitinib and ECP and the good safety profile, we decided to investigate the role of a treatment strategy of ruxolitinib in combination with ECP in frail patients with high-risk SR-a/cGvHD. We conducted a retrospective single-center study comprising 47 patients who underwent allo-SCT from November 2018 to October 2023 and received treatment for SR-aGvHD (n=20) or SR-cGvHD (n=27) with ruxolitinib and ECP. In the SR-aGvHD group, 95 % of patients had a lower GI-tract involvement, with 80 % presenting with a grade III-IV SR-aGvHD. The ORR at day +28 was 65 %, with a 30 % CR rate. The 1-year overall survival (OS) for responders (PR and CR) was 33 % (95 % CI, 10 %-59 %). In the SR-cGvHD group, 55.6 % and 44.4 % had moderate and severe SR-cGvHD, respectively. The majority of patients (66.7 %) had a GI-involvement. The ORR at week 24 was 88 %, including 12 % CR and 76 % PR. The 1-year OS for responders was 76 % (95 % CI, 47 %-90 %). Our retrospective analysis shows that the treatment of ruxolitinib in combination with ECP has potential efficacy in patients with SR-a/cGvHD with a high-risk for transplantation-associated mortality.

Introduction. Letermovir (LTV) showed to reduce the incidence of Cytomegalovirus (CMV) reactivations in CMV-seropositive patients after allogeneic stem cell transplantation (allo-SCT). CMV reactivations are known to stimulate polyfunctional T-cell responses, which foster the development of CMV-specific immune reconstitution (IR) and contribute to the advent of the polyclonal IR after allo-SCT. Since IR plays an important role in influencing outcome of transplanted patients, we aimed at investigating the impact of LTV in the advent and timing of polyclonal IR after allo-SCT. Methods. We conducted a retrospective analysis of CMV-seropositive patients consecutively undergoing allo-SCT at our center. We defined two cohorts according to the administration of LTV: (1) the no-LTV-cohort, encompassing patients who did not receive LTV, but pre-emptive treatment (PET) in case of CMV-reactivation (historical cohort, transplanted between 2013-2018); (2) the LTV-cohort, including patients who received LTV until at least day +100 after allo-SCT (transplanted between 2019-2023). CMV-DNA monitoring was performed weekly using real-time PCR from blood samples for the first 100 days after allo-SCT. Immune monitoring was performed using a flow cytometry-based assay to identify and determine the percentages and absolute counts of T (CD3+), B (CD19+), and natural killer (NK) cells (CD16+CD56+) as well as the CD4 and CD8 subpopulations of T cells in peripheral blood. Immune monitoring was performed monthly during the first year after allo-SCT and afterwards every 3 months until IR, which was defined as the detection in 2 consecutive measurements of CD3+CD4+-T-cells > 200 cells/µl and CD19+-B-cells > 50cells/µl. Statistical analysis included univariate methods using the Mann-Whitney test for continuous variables and the chi-square and Fischer exact test for nominal ones. For multivariable analysis, Cox- and competing risks regressions were used. Results. The study included 363 CMV-seropositive transplanted patients, with 128 (35%) in the LTV cohort and 235 patients (65%) in the historical no-LTV cohort. Patient characteristics, including age at time of allo-SCT, type of disease, disease status at allo-SCT, EBMT risk score, conditioning regimen and graft versus host (GVHD) prophylaxis, mainly based on ATG, were homogenously balanced between the two groups. The cumulative incidence (CI) of IR at day+ 100 and +180 after allo-SCT showed no significant difference between the two cohorts . Multivariable analysis indicated that LTV did not affect the occurrence of IR 1 year (y) after allo-SCT. Factors such as older age, high EBMT risk score, active disease at the time of allo-SCT and a CMV-seronegative donor significantly negatively impacted the advent of polyclonal IR. We then analyzed the different cellular population in order to assess the dynamics of the shaping of the immune system in the 2 groups. We found that in the no LTV-group there was a significantly higher expansion of the CD3+CD8+-T-cell fraction during the first 6 months after transplantation as compared to the LTV-cohort: at day +100 we detected 228 CD3+CD8+-T-cells/µl in the no LTV cohort vs. 114/µl in the LTV-cohort (p=0.0016). At day +180 the difference was preserved, with 425 CD3+CD8+-T-cells/µl vs. 204/µl respectively, p=0.001. No difference was observed in the CD3+CD8+-T-cells expansion at 1 y after allo-SCT. We also found a significantly higher peak of expansion of NK-cells at day +100 in the LTV-cohort (243 NK-cells/µl vs. 206 NK-cells/µl in the no LTV, p=0.008), but also in this setting no relevant difference was observed in the long-term follow-up (day+180 and +365). No significant differences were observed between the two cohorts considering the dynamic of expansion of CD3+CD4+-T-cells and CD19+-B-cells during the follow-up post allo-SCT. Conclusions. This is one of the largest retrospective studies evaluating occurrence and dynamic of IR in patients receiving LTV after allo-SCT. We confirmed that LTV could potentially (but transitorily) decrease T-cells (especially in the CD3+CD8+-T-cell fraction), with a peak of expansion of NK-cells in our study occurring at day +100. Despite these differences in the dynamic of polyclonal IR during the early phase after allo-SCT, in the long-term follow-up LTV does not appear to influence the advent, the qualitative composition and the incidence of IR.

BackgroundLow aerobic capacity is associated with an increased mortality risk in allogenic stem-cell transplantation (alloSCT) patients, but currently used risk scores in the pre-transplantation workup are still underestimating physical activity as a prognostic factor.AimTo examine the physical condition, muscle function, blood inflammation and training adherence of alloSCT patients during inpatient time to identify potential biomarkers associated with development of myopathy and sarcopenia.MethodsPatients undergoing alloSCT were examined at four time points (T0: before alloSCT; Tha: hospital admission; T1: engraftment; T2: inpatient discharge). T0 included cardiopulmonary performance, body composition, grip and knee strength, motor skill tests (One-leg stand/Tinetti/Chair-rising), blood sampling (blood cell profiling and inflammation targets (Kynurenin/high sensitivity C-reactive Protein (hsCRP)/Tumor necrosis factor alpha (TNF-alpha)/Musclin/Galectin-3) and quality of life, state of health, fatigue, muscle weakness and physical activity by questionnaires (IPAQ/BSA/SARC-F/Fatigue). At T1 and T2, blood samples, grip strength and motor skill tests were repeated. Glucocorticoid dose and daily physical activity were documented during inpatient stay.Results26 of 35 included patients (4 females; age 55.58 ± 12.32 years; BMI 24.70 ± 3.27 kg/m2; VO2peak 16.55 ± 4.06 ml/min/kg) could proceed to alloSCT. Grip strength and Tinetti decreased from T0 until T2, no difference in Chair-rising test, One-leg and Tandem stand. All patients engrafted after 24.9 days ± 3.9 days. HsCRP and Kynurenine increased from T0 to T1, decreased at T2. TNF-alpha (T0vsT2/T1vsT2) and Musclin (T0vsT1) decreased. At T2, Galectin-3 was higher compared to T0/T1. Correlation analysis of grip strength and inflammatory markers revealed a positive correlation with TNF-alpha at T2. 50% of patients documented physical activity and questionnaire and reported a 50%-reduction of daily endurance and strength training between T1 to T2.ConclusionAllogeneic stem-cell transplantation is associated with immune system vulnerability due to conditioning, increased inflammation and fatigue, and loss of muscle strength and function. In addition to hsCRP, Kynurenine seems to be a reliable biomarker to monitor acute and regenerative inflammation status of alloSCT patients, while Musclin and Galectin-3 may be added to physiological assessment regarding myopathy and sarcopenia. Grip strength and daily activity level should be documented by professionals to identify risk patients early and support them with optimal (exercise) therapy.

Introduction Allogeneic hematopoietic stem cell transplantation (alloHCT) is the only potential curative treatment for patients (pts) with high-risk myeloid malignancies. Despite advances in the supportive care over the past decades, alloHCT is still associated with significant toxicity, especially in the elderly population. The choice of an adequate conditioning regimen has become essential in order to prevent treatment-related morbidity and mortality. Since myeloablative conditioning (MAC) is mostly restricted to the younger population due to the high toxicity, reduced-intensity conditioning (RIC) provides an alternative solution. However, especially for pts with high-risk disease, a potential increase in the incidence of relapse must be taken into account. The advent of reduced-toxicity conditioning regimens (RTC), mostly based on treosulfan in combination with fludarabine, offers a balance between a favorable toxicity profile and a strong anti-neoplastic effect. Considering the increasing rates of alloHCT among older pts in the past decades, finding the optimal conditioning regimen is still an unmet need for this collective. The objective of this study is to analyze the outcome of pts aged ≥60 years who underwent alloHCT for myelodysplastic or myeloproliferative disease and received a treosulfan-based conditioning. In order to maximize the anti-leukemic effect of the conditioning in this population we chose the myeloablative dose of treosulfan of 14g/m2 daily. Materials and methods We retrospectively analyzed the outcome of adult pts ≥60 years with myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) or myelofibrosis (MF) who underwent their first allo-HCT from a matched or mismatched donor over a period of 10 years at the adult Bone Marrow Transplantation Unit of our institution. The conditioning consisted of a combination of treosulfan 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -2, alone or combined with 2 Gy TBI (day 0) for MDS and treosulfan, 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -3 for CMML and MF. Endpoints of this analysis were engraftment rate (together with time to engraftment), relapse-free survival (RFS), overall survival (OS), and non-relapse mortality (NRM). Continuous variables were illustrated as median values. NRM, RFS and OS for the whole cohort were analyzed with the Kaplan-Meier method and presented as survival functions. Results Between 01/2012 and 12/2022 91 consecutive elderly pts were eligible for this analysis. Patient and transplant characteristics are listed in Table 1. The median follow-up after allo-HCT was 3.12 years (4 months-10.5 years). Median age was 65 years, 76% (n = 69) of patients had an EBMT (European Society for Blood and Marrow Transplantation) risk score of ≥ 4. In 20 of 91 pts (22%) a 9/10 matched unrelated donor was selected, due to the lack of a more suitable donor. The standard graft versus host disease (GVHD) prophylaxis was a combination of calcineurin inhibitors (CNI) +/- mycophenolate mofetil and Anti-T-lymphocyte globulin (ATG) (n= 85, 94%). Neutrophil engraftment was achieved in 98% (n= 89) of pts. The median time to engraftment was 13 days (12, 15). Fourteen of 91 pts (15%) developed severe (grade III-IV) acute GVHD, and 44% (n= 40) had chronic GVHD of any grade. Considering early toxicity, 2 patients of 91 (2%) died before day +30 after alloHCT due to transplant-related toxicity, overall 6 of 91 (7%) in the first 100 days after alloHCT. The 2-year OS was 55% (95% CI, 44.7-.66.2) (Figure 1) and RFS was 48.5% (95% CI, 38.9-60.5). The 2-year NRM was 37% (95% CI, 33.5-39.5). No significant differences have been observed in the outcome of the different disease entities. Main causes of death were infections (n= 15, 32%), GVHD (n=13, 27.7%) and disease relapse (n= 13, 27.7%). Conclusions Our retrospective analysis suggests that a treosulfan-based RTC with myeloablative potential in an elderly and frail population of pts with high-risk myeloid diseases is feasible, with an excellent engraftment rate and a limited early toxicity, and promotes disease control. We report a 2-year NRM of 37%, which was partially due to severe GvHD in a cohort of pts with a relatively high-proportion of mismatched donors (20%). The role of post-transplant cyclophosphamide as backbone of the GvHD prophylaxis of RTC regimes should be further evaluated, in prospective trials.

Introduction. Persistent thrombocytopenia (PT) due to delayed primary platelet recovery or secondary graft failure with prolonged thrombocytopenia is a relevant and relatively common complication after allogeneic stem cell transplantation (alloSCT). It can result in transfusion dependence and hemorrhagic events, leading to increase of morbidity and mortality. Concomitant causes of PT after alloSCT are several and can include viral infections, graft-versus-host disease (GvHD), drug toxicity, thrombotic microangiopathy (TMA) and immune thrombocytopenia (ITP). Together with the treatment of the primary cause and the support with platelet transfusion, the use of thrombopoietin receptor agonists (TPO-RAs), which proved to be effective in several scenarios (e.g. ITP or aplastic anemia), is gaining a fundamental role also in the post alloSCT setting. To date, the use of TPO-RAs after transplant has been investigated in small studies or case reports. In this study, we evaluated the use of romiplostim in a larger patient cohort to determine its safety, efficacy and potential predictors of response. Materials and methods. We conducted a retrospective study on consecutive patients undergoing alloSCT and receiving romiplostim for the treatment of PT at the bone marrow transplantation unit of the University Hospital of Ulm. PT was defined as a platelet count < 20 Giga/l for 7 consecutive days after engraftment, the need for continuous transfusion during the post-transplant follow-up or a not rapidly reversible decrease of >50% of the platelet count not due to relapse of underlying hematological disease. Response to treatment was defined as a platelet count >50 Giga/l for at least 7 consecutive days without transfusion. Patients with residual bone marrow involvement of primary hematological disease were excluded. Univariate analysis was conducted using Fisher's exact test or Mann-Whitney-U-test. Results. Between 01/2019 and 12/2021 we identified a total of 119 out of 255 transplanted patients (46,7%) with PT receiving, as a part of the treatment, romiplostim during the post-transplant follow-up. Patient characteristics are listed in Table 1. The median platelet count at baseline was 32 Giga/l (r = 0 - 88). We started with romiplostim 250μg on a weekly basis, in 46,2% of cases the dose was escalated to 500 µg during treatment. Seventy-three percent showed a clinical response to treatment with a median time to response of 40 days (r = 7 - 565). We observed adverse events like thrombosis, pain syndrome, dizziness in only 8,4% of patients. The median duration of treatment was 168 days (r = 18 - 761). In patients with response, the median platelet count was 91 Giga/l (r = 53 - 212). In univariate analysis, we evaluated potential risk factors associated with poor response to romiplostim. While the presence of a viral reactivation (like CMV, EBV, BKV, HSV1) requiring systemic treatment was associated with a significant lower response rate (47.6% versus 79.6%, respectively for patients with or without reactivation, p= 0.005), the presence of active GvHD (also in case of steroid-refractory (SR) forms), of TMA and the number of CD34-positive stem cells of the graft did not play a role in determining the efficacy of romiplostim. We also evaluated the influence of graft cryopreservation, which was performed increasingly during the SARS-CoV-2 pandemic, on the rate of efficacy of romiplostim in PT treatment. We could not identify a significant difference as compared to the non-cryopreserved grafts. Conclusion. Our results indicate that romiplostim is overall well tolerated and represents a reasonable and effective treatment for PT after alloSCT. We observed that viral infections requiring systemic antiviral treatment could represent a predictor of worse response, which could potentially be explained by the myelotoxicity induced not only by the virus itself but also as a consequence of antiviral substances. Of note, especially considering that SR GvHD or TMA did not seem to impair the efficacy of romiplostim, the stimulation of TPO receptor could play a role in reducing the rate of morbidity and mortality associated with such severe complications of the post transplant follow-up. Also, cryopreservation did not impact on the rate of success of TPO-RAs based treatment strategy. These preliminary results need to be further validated in larger and prospective cohorts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The standard of care in medically and physically fit patients is intensive induction therapy. The majority of these intensively treated patients achieve a complete remission. However, a high number of these patients will experience relapse. In patients older than 60 years, the results are even worse. Therefore, new therapeutic approaches are desperately needed. One promising approach in high-risk leukemia to prevent relapse is the induction of the immune system simultaneously or after reduction of the initial tumor burden. Different immunotherapeutic approaches such as allogenic stem cell transplantation or donor lymphocyte infusions are already standard therapies, but other options for AML treatment are in the pipeline. Moreover, the therapeutic landscape in AML is rapidly changing, and in the last years, a number of immunogenic targets structures eligible for specific therapy, risk assessment or evaluation of disease course were determined. For example, leukemia-associated antigens (LAA) showed to be critical as biomarkers of disease state and survival, as well as markers of minimal residual disease (MRD). Yet many mechanisms and properties are still insufficiently understood, which also represents a great potential for this form of therapy. Therefore, targeted therapy as immunotherapy could turn into an efficient tool to clear residual disease, improve the outcome of AML patients and reduce the relapse risk. In this review, established but also emerging immunotherapeutic approaches for AML patients will be discussed.

Introduction Chimeric antigen receptor (CAR)-T cell therapy is a promising immunotherapeutic strategy approved for the treatment of relapsed or refractory (r/r) B-cell lymphomas (e.g. diffuse large B cell lymphoma (DLBCL), mantel cell lymphoma (MCL), follicular lymphoma (FL)) after two or more treatment courses. In this setting CAR-T cell therapy can induce complete remission (CR) in a substantial proportion of patients (30-40%) with poor prognosis. Nevertheless, disease relapse still represents one of the major issues. Many efforts are being made to detect factors accounting for sustained remission after cell therapy. CAR-Ts expansion and persistence are thought to represent crucial factors influencing outcome. The identification of a reliable detection strategy of CAR-T cells is thus fundamental to assess/anticipate disease relapse and to tailor post CAR-T cell therapy strategy. Materials and Methods In order to assess the presence of CAR-T cells in blood samples during follow-up of lymphoma patients undergoing CAR-T cell therapy with tisagenlecleucel (tisa-cel) or axicabtagene-ciloleucel (axi-cel), we developed a Taqman assay specific for the genomic sequence of the single-chain variable fragment (scFv), which encodes the antigen recognition site of the CAR construct of the two different CAR-T cell constructs. A semi-quantitative real-time PCR protocol was established in order to compare the amount of CAR-T DNA with reference genomic DNA sequences (KELL, IGF1, ASMA). The results were reported as CAR-T cell copies/1000 cells. CAR-T DNA was assessed from samples of peripheral blood at fixed time-points after infusion: day +7 and day +30 were the main reference points in order to detect/define expansion and persistence, respectively. Results Between 05/2019 and 04/2022 24 patients with r/r DLBCL or transformed FL were treated with CAR-T cell therapy (tisa-cel, n=16, or axi-cel, n=8) at the University Hospital of Ulm. Patients' characteristics are listed in table 1. At three months after infusion, we documented an overall response rate of 46%, with a CR rate of 33%. With our PCR assay we detected the peak of expansion with a median of 56 CAR-T cells/1000 cells (range, 0-3215) and documented the persistence with a median of 59.5 CAR-T cells/1000 cells (range: 0-284). Comparing responders vs. non-responders, we noticed no significant differences in the median cell peak at the time of expansion (p=0.2), but a significant longer persistence in responders (99 vs. 23 CAR-T cells/1000 cells, responders vs. non-responders, p=0.00798). We also compared expansion and persistence according to the type of CAR-T cell product, knowing that axi-cel presents a greater expansion peak and that tisa-cel with the 4-1bb costimulatory domain is able to persist longer. We confirmed that axi-cel has a higher expansion as compared to tisa-cel (1251 vs. 49 CAR-T cells/1000 cells at day+ 7, respectively, p=0.003). There was no significant difference considering the persistence at day +30 between the two products (62 vs. 59,5 CAR-T cells/1000 cells, respectively for tisa-cel and axi-cel, p=0.432). Furthermore, in the tisa-cel subgroup CAR-T cell persistence at day +30 proved to be associated with clinical response in univariate analysis (99 vs. 21 CAR-T cells/1000 cells, respectively responders vs. non-responders, p=0.040), while expansion-peak did not seem to play a role in inducing response after treatment. In the axi-cel subgroup both the entity of CAR-T cell peak at day+ 7 (1959 vs. 1033 CAR-T cells/1000 cells for responders vs. non-responders, p= 0.40) and the persistence (125 vs. 23,5 CAR-T cells/1000 cells for responders vs. non-responders, p= 0.40) were significantly associated with clinical response 3 months after infusion. Conclusions Reliable monitoring of CAR-T cell expansion and persistence may reveal patterns that correlate with clinical response to CAR-T treatment, thus representing a potential instrument able to give early information about the pharmacodynamic properties of CAR-Ts. This information could help to tailor the post-infusion strategy, also knowing that CAR-T cell persistence seems to exert a fundamental role in inducing continuous CR also across different CAR-T cell products. These results are retrospectively collected on a small and heterogeneous patient population and need to be validated in larger and prospective cohorts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In recent years, next-generation sequencing (NGS) technology has established itself as the gold standard in HLA genotyping.It allows high sample throughput and yields sequencing data with high quality.In combination with long-range PCR assays, it is partially possible to obtain fully phased sequences and thus achieve HLA genotyping with allelic resolution.But the HLA class II genes in particular, with an average size of 8-15 kb bring NGS, based on short-read alignments to its technical limits.Longread sequencing platforms, which are able to generate single reads of several hundred kilobases, offer the possibility to obtain fully phased sequences.The two currently available sequencing platforms were developed by Oxford Nanopore Technologies (ONT) and by Pacific Biosciences (PacBio) and differ fundamentally in their sequencing principle.In this project we evaluate the suitability of a long-read platform for the high-throughput routine HLA genotyping in the Stefan-Morsch-Foundation.Prior to the library preparation, we amplified the six transplantationrelevant HLA genes (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) as well as the non-classical HLA-E gene using inhouse developed long-range PCR assays.With the exception of HLA-DRB1 amplicon that only covers the gene from the end of intron 1 to the 3 0 UTR, the remaining amplicons comprise the full length of the corresponding genes (5 0 UTR to 3 0 UTR).The PCR amplicons were pooled in equimolar ratios.The corresponding library preparation and sequencing was performed applying platform-specific routine workflows.For genotyping, NGSengine software (GenDx, Utrecht) was used.Besides, the correct genotyping we also evaluated the quality metrics such as read distribution and read length, including the rate of sequencing errors.Further on we checked the impact of data pre-processing steps such as filtering and polishing on the genotyping results.With long-read platforms, continuous allelic sequences can be generated and thus could be used as an elegant method for ambiguityfree HLA genotyping.Especially, in combination with capture-probe based target enrichment sequencing of entire haplotypes or larger MHC regions is the next step towards improving genotype resolution.

Allogenic stem cell transplantation (aSCT) is the only potentially curative treatment for high-risk hematological diseases. Despite advancements in supportive measures, aSCT outcome is still affected by considerable transplant-related mortality. We implemented a new sarcopenia assessment prior to aSCT to evaluate its predictive capability for all-cause and non-relapse mortality. Therefore all patients initially scheduled for aSCT within a 25-month period were screened during pre-transplantation-routine for muscle mass, grip strength, and aerobic capacity (AC) by measuring peak oxygen uptake (VO2peak). Patients were assigned to one of five groups adapted according current sarcopenia guidelines. Primary endpoints were all-cause and non-relapse mortality within a follow up time of up to 12 months. A total of 178 patients were included and rated as normal (n = 48), impaired aerobic capacity (n = 56), pre-sarcopenic (n = 26), sarcopenic (n = 27), and severe sarcopenic (n = 22) without significant age-differences between groups. Patients presenting with sarcopenia showed a significant three-fold increase in all-cause and non-relapse mortality compared to patients with normal screening results. AC showed to be the strongest single predictor with a more than two-fold increase of mortality for low AC. We conclude that risk stratification based on combination of muscle mass, grip strength, and AC allowed identifying a subgroup with increased risk for complications in patients undergoing aSCT.

Abstract Introduction Different vaccines have been recently approved by FDA and EMA for the prevention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating a protection rate ranging from 60% to almost 95% in phase II/III trials. Immunocompromised patients, including those undergoing allogeneic stem cell transplantation (allo-SCT), were excluded from vaccine trials. While EBMT recommended the vaccination for transplanted patients, data concerning efficacy and safety in this particular setting are scarce. Since infections represent a relevant cause of transplant-related mortality and the treatment and management of SARS-CoV-2 infection in allo-SCT patients proved to be challenging and complex, we prospectively evaluated the safety and the development of protective response against SARS-CoV-2 vaccines in our allogeneic transplanted patients. Materials and Methods Starting in March 2021, allogeneic stem cell transplanted patients with different hematological diseases underwent COVID-19 vaccination. Taking into account the EBMT recommendations, we considered patients suitable for vaccination when (1) they were at least 3-6 months after allo-SCT, (2) didn't have any graft versus host (GvHD) activity, and (3) received less than 0,5 mg/kg steroids as part of the immunosuppressive treatment. There was no recommendation for a specific type of vaccine, with the only exception for life-attenuated vaccines, which are mostly contraindicated in the post allo-SCT setting. Vaccinated patients were regularly monitored for the potential development of adverse events. The anti-SARS-CoV-2 Spike protein antibodies were measured in blood samples to assess the humoral response. In case of no response with undetectable anti-Spike antibodies 2 week after the second dose of vaccine, we repeated the measurements at regular intervals until week + 6-8 after the completion of vaccination. Patients with no measurable antibodies 8 weeks after completion of the vaccination were considered as no responders. Results Between 03/2021 and 06/2021 a total of 83 patients underwent COVID-19 vaccination (including first and second dose) during the post allo-SCT follow-up. Patients' characteristics are listed in Table 1. Most patients (77%) received BNT162b2, while only a small subgroup (8%) underwent a mixed vaccination after a first dose of ChAdOx1-S. We considered the mixed vaccination mostly to maximize the response. Overall, the two vaccine doses were well tolerated, with only 5% of patients developing a reactivation of GvHD. No relevant grade 3 or 4 organ toxicities were observed. Overall, 66% of patients in our cohort showed a humoral response. The incidence of positive serology was lower in patients who underwent the vaccination within the first 18 months after allo-SCT (29% vs 83% for patients >18 months after allo-SCT, p< 0.001). In multivariate analysis other risk factors that were associated with poor or no response were lack of immune reconstitution (p< 0.001) and ongoing immunosuppressive therapy (p= 0.009). The age of patients at the time of vaccination, sex, intensity of conditioning regimen and the use of ATG did not prove to have an influence for a humoral response during the post-transplant follow up. Discussion The achievement of a protective immunity against SARS-CoV-2 represents a crucial event for a frail population, like allogeneic stem cell transplanted patients. So far and to our knowledge little is known about the safety and efficacy of the COVID-19 vaccination in this particular setting. Here, we report one of the first series of patients undergoing COVID-19 vaccination after allo-SCT. We demonstrated that a humoral response can be achieved, especially for those patients who are in the long-term follow-up, underwent immune reconstitution and are free from immunosuppressive drugs. For the other patients, who represent the frailer subgroup, in the absence of a documented immune response after 2 doses of vaccine, the option of a third dose in order to increase the probability of response should be evaluated in prospective clinical trials. Figure 1 Figure 1. Disclosures Viardot: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; University Hospital of Ulm: Current Employment; Amgen: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Döhner: Roche: Consultancy, Honoraria; Agios: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Ulm University Hospital: Current Employment; Abbvie: Consultancy, Honoraria, Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astex: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Pfizer: Research Funding; Berlin-Chemie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria. Sala: Novartis: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Jazz: Consultancy, Honoraria.

INTRODUCTION:Respiratory viral infections are a major cause of morbidity and mortality among stem cell transplant recipients. While there is a substantial amount of information on prognostic factors and response to ribavirin therapy is available for RSV infections, this information is largely lacking for hMPV.PATIENTS AND METHODS:In total, 71 patients were included in this study: 47 patients with RSV and 24 with hMPV. Forty-one patients presented as an upper respiratory tract infection (URTI) and 30 as a primary lower respiratory tract infection (LRTI). Patients were stratified as per ISI criteria into low-, moderate-, and high-risk groups. Twenty-two patients in the URTI cohort received treatment with ribavirin (mainly oral), and 19 patients received no antiviral therapy. The decision for antiviral treatment was at the discretion of the attending physician. All 30 patients with primary LRTI and 10 patients with secondary LRTI were treated with ribavirin, 95% with the intravenous formulation. 45% of these patients received additional treatment with intravenous immunoglobulins. The viral load was assessed indirectly by using the CT value of the RT-PCR.RESULTS:In the cohort, as whole 11.5% suffered a virus-associated death, 5% in the URTI group, and 20% in the LRTI group. Sixty-day mortality was significantly higher in the ISI high-risk group (log-rank P = .05). Mortality was independent of the type of virus (P = .817). Respiratory failure with an indication for mechanical ventilation developed in 11.5%, this risk was independent of the type of virus. Progression from URTI to LRTI was observed in 24% of cases with a significantly higher risk (75%) in the ISI high group (log-rank P = .001). In the ISI high-risk group, treatment with ribavirin significantly reduced the risk of progression (log-rank P < .001). Neither the type of virus nor the viral load in the nasopharyngeal swab impacted the risk of progression (P = .529 and P = .141, respectively). The detection of co-pathogens in the BAL fluid was borderline significant for mortality (P = .07).CONCLUSIONS:We could detect no differences between RSV and hMPV with respect to progression to LRTI, risk of respiratory failure or need for mechanical ventilation and virus-associated death. The ISI index is of predictive value in hMPV patients with a high ISI score and treatment with oral ribavirin has an equivalent protective effect in RSV and hMPV patients. Treatment of LRTI with intravenous ribavirin results in a similar outcome in RSV- and hMPV-infected patients. We could not detect any benefit of adjunctive treatment with immunoglobulins in both primary and secondary LRTI. No role of viral load as an independent prognostic marker could be detected either for progression to LRTI or death.

Rhizomucor miehei is a cause of bovine mycotic abortion and mastitis and has rarely been described in human disease. Here, we report the first isolation of R. miehei from native mitral valve tissue in a fatal case of endocarditis that substantiates its pathogenic potential. Apart from morphological criteria, molecular methods were a cornerstone for definite diagnosis.

Allogeneic stem cell transplantation (allo-SCT) is the preferred curative treatment for several hematological malignancies. The efficacy of allo-SCT depends on the graft-versus-leukemia (GvL) effect. However, the prognosis of patients with relapsed acute myeloid leukemia (AML) following allo-SCT is poor. Donor lymphocyte infusion (DLI) is utilized after allo-SCT in this setting to prevent relapse, to prolong progression free survival, to establish full donor chimerism and to restore the GvL effect in patients with hematological malignancies. Thus, there are different options for the administration of DLI in AML patients. DLI is currently used prophylactically and in the setting of an overt relapse. In addition, in the minimal residual disease (MRD) setting, DLI may be a possibility to improve overall survival. However, DLI might increase the risk of severe life-threatening complications such as graft-versus-host disease (GvHD) as well as severe infections. The transfusion of lymphocytes has been tested not only for the treatment of hematological malignancies but also chronic infections. In this context, manipulated DLI in a prophylactic or therapeutic approach are an option, e.g., virus-specific DLI using different selection methods or antigen-specific DLI such as peptide-specific CD8+ cytotoxic T lymphocytes (CTLs). In addition, T cells are also genetically engineered, using both chimeric antigen receptor (CAR) genetically modified T cells and T cell receptor (TCR) genetically modified T cells. T cell therapies in general have the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease after allo-SCT. The focus of this review is to discuss the different strategies to use donor lymphocytes after allo-SCT. Our objective is to give an insight into the functional effects of DLI on immunogenic antigen recognition for a better understanding of the mechanisms of DLI. To ultimately increase the GvL potency without raising the risk of GvHD at the same time.