Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.

Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.

Treatment with the apoptosis inhibitor Asunercept reduces clone sizes in patients with lower risk Myelodysplastic Neoplasms.

In low-risk Myelodysplastic Neoplasms (MDS), increased activity of apoptosis-promoting factors such as tumor necrosis factor (TNFα) and pro-apoptotic Fas ligand (CD95L) have been described as possible pathomechanisms leading to impaired erythropoiesis. Asunercept (APG101) is a novel therapeutic fusion protein blocking CD95, which has previously shown partial efficacy in reducing transfusion requirement in a clinical phase I trial for low-risk MDS patients (NCT01736436; 2012-11-26). In the current study we aimed to evaluate the effect of Asunercept therapy on the clonal bone marrow composition to identify potential biomarkers to predict response. Bone marrow samples of n = 12 low-risk MDS patients from the above referenced clinical trial were analyzed by serial deep whole exome sequencing in a total of n = 58 time points. We could distinguish a mean of 3.5 molecularly defined subclones per patient (range 2-6). We observed a molecular response defined as reductions of dominant clone sizes by a variant allele frequency (VAF) decrease of at least 10% (mean 20%, range: 10.5-39.2%) in dependency of Asunercept treatment in 9 of 12 (75%) patients. Most of this decline in clonal populations was observed after completion of 12 weeks treatment. Particularly early and pronounced reductions of clone sizes were found in subclones driven by mutations in genes involved in regulation of methylation (n = 1 DNMT3A, n = 1 IDH2, n = 1 TET2). Our results suggest that APG101 could be efficacious in reducing clone sizes of mutated hematopoietic cells in the bone marrow of Myelodysplastic Neoplasms, which warrants further investigation.

Therapy resistance mechanisms in hematological malignancies.

Hematologic malignancies are model diseases for understanding neoplastic transformation and serve as prototypes for developing effective therapies. Indeed, the concept of systemic cancer therapy originated in hematologic malignancies and has guided the development of chemotherapy, cellular therapies, immunotherapy and modern precision oncology. Despite significant advances in the treatment of leukemias, lymphomas and multiple myelomas, treatment resistance associated with molecular and clinical relapse remains very common. Therapy of relapsed and refractory disease remains extremely difficult, and failure of disease control at this stage remains the leading cause of mortality in patients with hematologic malignancies. In recent years, many efforts have been made to identify the genetic and epigenetic mechanisms that drive the development of hematologic malignancies to the stage of full-blown disease requiring clinical intervention. In contrast, the mechanisms responsible for treatment resistance in hematologic malignancies remain poorly understood. For example, the molecular characteristics of therapy-resistant persisting cells in minimal residual disease (MRD) remain rather elusive. In this mini-review we want to discuss that cellular heterogeneity and plasticity, together with adaptive genetic and epigenetic processes, lead to reduced sensitivity to various treatment regimens such as chemotherapy and pathway inhibitors such as tyrosine kinase inhibitors. However, resistance mechanisms may be conserved across biologically distinct cancer entities. Recent technological advances have made it possible to explore the underlying mechanisms of therapy resistance with unprecedented resolution and depth. These include novel multi-omics technologies with single cell resolution combined with advanced biocomputational approaches, along with artificial intelligence (AI) and sophisticated disease models for functional validation.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Response and resistance to cladribine in patients with advanced systemic mastocytosis: a registry-based analysis.

We sought to evaluate the efficacy of the purine analogue cladribine in 79 patients with advanced systemic mastocytosis (AdvSM) using data from the 'German Registry on Disorders of Eosinophils and Mast Cells (GREM)'. The overall response rate according to modified Valent criteria (46 evaluable patients) for first- (1L) and second-line (2L) cladribine treatment was 41% (12/29) and 35% (6/17, P = 0.690), respectively, and the median overall survival (OS, all patients evaluable) was 1.9 years (n = 48) and 1.2 years (n = 31; P = 0.311). Univariate and multivariable analyses of baseline and on-treatment parameters identified diagnosis of mast cell leukemia (hazard ratio [HR] 3.5, 95% confidence interval [CI, 1.3-9.1], P = 0.012), eosinophilia ≥ 1.5 × 109/L (HR 2.9 [CI 1.4-6.2], P = 0.006) and < 3 cycles of cladribine (HR 0.4 [CI 0.2-0.8], P = 0.008) as independent adverse prognostic parameters for OS. There was no impact of other laboratory (anemia, thrombocytopenia, serum tryptase) or genetic markers (mutations in SRSF2, ASXL1 or RUNX1) on OS. In consequence, none of the recently established prognostic scoring systems (MARS, IPSM, MAPS or GPSM) was predictive for OS. Modified Valent criteria were superior to a single factor-based response assessment (HR 2.9 [CI 1.3-6.6], P = 0.026). In conclusion, cladribine is effective in 1L and 2L treatment of AdvSM. Mast cell leukemia, eosinophilia, application of < 3 cycles and a lack of response are adverse prognostic markers.

Prospective analysis of the attendance behaviour of the participants of a facilitated support group for patients after allogeneic hematopoietic cell transplantation.

PURPOSE: Support groups might help survivors of allogeneic hematopoietic cell transplantations (HCT) to cope with medical, psychological, and social challenges. The aim of this project was (1) to establish a facilitated post-HCT support group and (2) to assess the participation behaviour. METHODS: From 11/2013 until 7/2017, all adult patients who had received a HCT at our centre were invited to participate in a professionally facilitated support group. The format of the group was unstructured without any rules regarding regular attendance. The attendance was prospectively minuted by the facilitator. Reasons for non-attendance were assessed by a survey. RESULTS: During the observation period, 53 group meetings were scheduled. Nine meetings were cancelled because of low attendance. Altogether 23 different patients (F: n=10; M: n=13) and 10 spouses (F: n=9; M: n=1) participated. Median participation was 5 [range 2-11]. With respect to all HCT patients who had the theoretical opportunity to attend, the mean participation rate was 7%. Thirteen patients and four spouses attended more than one meeting. The median count of participations among those participants was 8 [2-32]. The median interval from the first until the last participation was 16 months. The main reason reported for non-participation was the effort to get to the venue of the support group. CONCLUSIONS: To our knowledge, this is the first analysis on the attendance behaviour of the participants of a support group for HCT survivors. The results provide guidance for the organization of future support groups and indicate what participation rates can be expected and how they might be increased.

In Silico Pan-Cancer Analysis Reveals Prognostic Role of the Erythroferrone (ERFE) Gene in Human Malignancies.

The erythroferrone gene (ERFE), also termed CTRP15, belongs to the C1q tumor necrosis factor-related protein (CTRP) family. Despite multiple reports about the involvement of CTRPs in cancer, the role of ERFE in cancer progression is largely unknown. We previously found that ERFE was upregulated in erythroid progenitors in myelodysplastic syndromes and strongly predicted overall survival. To understand the potential molecular interactions and identify cues for further functional investigation and the prognostic impact of ERFE in other malignancies, we performed a pan-cancer in silico analysis utilizing the Cancer Genome Atlas datasets. Our analysis shows that the ERFE mRNA is significantly overexpressed in 22 tumors and affects the prognosis in 11 cancer types. In certain tumors such as breast cancer and adrenocortical carcinoma, ERFE overexpression has been associated with the presence of oncogenic mutations and a higher tumor mutational burden. The expression of ERFE is co-regulated with the factors and pathways involved in cancer progression and metastasis, including activated pathways of the cell cycle, extracellular matrix/tumor microenvironment, G protein-coupled receptor, NOTCH, WNT, and PI3 kinase-AKT. Moreover, ERFE expression influences intratumoral immune cell infiltration. Conclusively, ERFE is aberrantly expressed in pan-cancer and can potentially function as a prognostic biomarker based on its putative functions during tumorigenesis and tumor development.

Molecular and phenotypic diversity of CBL-mutated juvenile myelomonocytic leukemia.

Mutations in the gene CBL were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found this disease to be highly diverse in presentation and overall outcome. Moreover, we discovered somatically-acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish somatic CBL patients from germline CBL patients, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among germline CBL patients. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.

Definition of factors associated with negative antibody response after COVID-19 vaccination in patients with hematological diseases.

COVID-19 in patients with hematological diseases is associated with a high mortality. Moreover, preventive vaccination demonstrated reduced efficacy and the knowledge on influencing factors is limited. In this single-center study, antibody levels of the SARS-CoV-2 spike protein were measured ≥ 2 weeks after 2nd COVID-19 vaccination with a concentration ≥ 0.8 U/mL considered positive. Between July and October 2021, in a total of 373 patients (median age 64 years, 44% women) with myeloid neoplasms (n = 214, 57%), lymphoid neoplasms (n = 124, n = 33%), and other diseases (n = 35, 10%), vaccination was performed with BNT162b2 (BioNTech), mRNA-1273 (Moderna), ChADOx1 (AstraZeneca), or a combination. A total of 229 patients (61%) were on active therapy within 3 months prior vaccination and 144 patients (39%) were previously treated or treatment naïve. Vaccination-related antibody response was negative in 56/373 patients (15%): in 39/124 patients with lymphoid neoplasms, 13/214 with myeloid neoplasms, and 4/35 with other diseases. Active treatment per se was not correlated with negative response. However, rituximab and BTK inhibitor treatment were correlated significantly with a negative vaccination response, whereas younger age and chronic myeloid leukemia (CML) disease were associated with positive response. In addition, 5 of 6 patients with myeloproliferative neoplasm (MPN) and negative vaccination response were on active treatment with ruxolitinib. In conclusion, a remarkable percentage of patients with hematological diseases had no response after 2nd COVID-19 vaccination. Multivariable analysis revealed important factors associated with response to vaccination. The results may serve as a guide for better protection and surveillance in this vulnerable patient cohort.

The insertion site is the main risk factor for central venous catheter-related complications in patients with hematologic malignancies.

Central venous catheters (CVC) placed either via the internal jugular vein (IJV) or the subclavian vein (SCV) are routinely used in patients with hematologic malignancies. In this retrospective study, we systematically compared CVC-associated complications for both insertion sites, IJV and SCV. Between January 2011 and June 2013, all consecutive patients (n = 87) were included with at least one CVC (n = 153; n = 94 IJV; n = 59 SCV) at our institution due to induction/consolidation for AML/ALL or autologous hematopoietic cell transplantation (HCT). Primary study endpoints were central line-associated (CLABSI), catheter-related (CRBSI) blood stream infections and local inflammation (LI) at the insertion site. CRBSI occurred earlier and more frequently in the IJV- versus the SCV-group with an incidence rate of CRBSI at day 15 of 10% versus 0% (p = .04) and a rate of CRBSI per 1000 CVC days of 5.7 versus 1.2. In addition, CLABSI was detected more often in IJV- compared to SCV-CVC (26% vs. 8%, p = .009). Conversely, LI occurred more frequently and earlier in SCV- versus IJV-CVC (88% vs. 56%, p < .0001) with a median time to LI of 9 versus 14 days (p < .0001). The strongest risk factor for the endpoints CRBSI, CLABSI, and LI was the insertion site. However, SCV insertion was a risk factor for LI (p = .001, HR: 2.0), insertion in the IJV a risk factor for CLABSI (p = .044, HR: 2.7) and CRBSI (p = .036, HR: 5.4). These results demonstrate a differential effect of the insertion site of CVC in neutropenic patients with a significantly reduced frequency of CVC-related blood stream infections in SCV-CVC.

Low risk of contrast media-induced hypersensitivity reactions in all subtypes of systemic mastocytosis.

BACKGROUND: Patients with systemic mastocytosis (SM) are at increased risk of hypersensitivity reactions (HRs). Although Hymenoptera venoms are the predominant triggers, cases of contrast media-induced HR (CMIHR) have also been reported and prophylactic premedication is often performed. However, data from larger series are limited and differences between indolent and advanced SM have not yet been investigated. OBJECTIVE: To determine the incidence and severity of CMIHR in all subtypes of SM. METHODS: We analyzed 162 adult patients with SM (indolent systemic mastocytosis [ISM], n = 65; advanced systemic mastocytosis [advSM], n = 97). First, the cumulative incidence of CMIHR was retrospectively assessed in the patient's history. Second, at our institution, patients underwent 332 contrast media (CM)-enhanced imaging including 80 computed tomography (CT) scans with iodine-based contrast agent and 252 magnetic resonance imaging (MRI) with a gadolinium-based contrast agent, and tolerance was assessed. RESULTS: Previous CMIHRs to CT (vomiting, n = 1, erythema, n = 1, cardiovascular shock, n = 1), and MRI (dyspnea, n = 1, cardiovascular shock, n = 1) had been reported by 4 out of 162 (2.5%) patients (ISM, n = 3; advSM, n = 1). In contrast, during or after 332 CM-enhanced CT or MRI examinations at our institution, no CMIHRs were reported. Premedication was solely given to 3 patients before CT scans, including 1 with previous CMIHR, who tolerated the imaging well. CONCLUSION: We conclude that: (1) there is a substantial discrepancy between the perception and prevalence of HRs to CM in SM; (2) reactions are scarce in ISM and even rarer in advSM; and (3) in SM patients without previous history of CM hypersensitivity, prophylactic premedication before CM-enhanced CT or MRI is dispensable.

Clinical and histopathological features of myeloid neoplasms with concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V mutations.

We report on 45 patients with myeloid neoplasms and concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V (JAK2pos. /KITpos. ) mutations, which are individually identified in >60% of patients with classical myeloproliferative neoplasms (MPN) and >90% of patients with systemic mastocytosis (SM) respectively. In SM, the concurrent presence of a clonal non-mast cell neoplasm [SM with associated haematological neoplasm (SM-AHN)] usually constitutes a distinct subtype associated with poor survival. All 45 patients presented with a heterogeneous combination of clinical/morphological features typical of the individual disorders (e.g. leuco-/erythro-/thrombocytosis and elevated lactate dehydrogenase for MPN; elevated serum tryptase and alkaline phosphatase for SM). Overlapping features identified in 70% of patients included splenomegaly, cytopenia(s), bone marrow fibrosis and additional somatic mutations. Molecular dissection revealed discordant development of variant allele frequency for both mutations and absence of concurrently positive single-cell derived colonies, indicating disease evolution in two independent clones rather than monoclonal disease in >60% of patients examined. Overall survival of JAK2pos. /KITpos. patients without additional somatic high-risk mutations [HRM, e.g. in serine and arginine-rich splicing factor 2 (SRSF2), additional sex combs like-1 (ASXL1) or Runt-related transcription factor 1 (RUNX1)] at 5 years was 77%, indicating that the mutual impact of JAK2 V617F and KIT D816V on prognosis is fundamentally different from the adverse impact of additional HRM in the individual disorders.

High erythroferrone expression in CD71+ erythroid progenitors predicts superior survival in myelodysplastic syndromes.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Genome-wide DNA methylation analysis pre- and post-lenalidomide treatment in patients with myelodysplastic syndrome with isolated deletion (5q).

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets.

Adverse Prognostic Impact of the KIT D816V Transcriptional Activity in Advanced Systemic Mastocytosis.

In systemic mastocytosis (SM), qualitative and serial quantitative assessment of the KIT D816V mutation is of diagnostic and prognostic relevance. We investigated peripheral blood and bone marrow samples of 161 patients (indolent SM (ISM), n = 40; advanced SM, AdvSM, n = 121) at referral and during follow-up for the KIT D816V variant allele frequency (VAF) at the DNA-level and the KIT D816V expressed allele burden (EAB) at the RNA-level. A round robin test with four participating laboratories revealed an excellent correlation (r > 0.99, R2 > 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM (r = 0.91, coefficient of determination, R2 = 0.84) but only to a lesser extent in AdvSM (r = 0.71; R2 = 0.5). However, as compared to an EAB/VAF ratio ≤2 (cohort A, 77/121 patients, 64%) receiver operating characteristic (ROC) analysis identified an EAB/VAF ratio of >2 (cohort B, 44/121 patients, 36%) as predictive for an advanced phenotype and a significantly inferior median survival (3.3 vs. 11.7 years; p = 0.005). In terms of overall survival, Cox-regression analysis was only significant for the EAB/VAF ratio >2 (p = 0.006) but not for VAF or EAB individually. This study demonstrates for the first time that the transcriptional activity of KIT D816V may play an important role in the pathophysiology of SM.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Influence of the Insertion Site on Central Venous Catheter-Related Complications in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation.

Central venous catheters (CVCs) are extensively used in patients undergoing allogeneic hematopoietic cell transplantation (HCT). In these patients CVC are placed routinely either via the internal jugular vein (IJV) or the subclavian vein (SCV). Purpose of this study was to systematically analyze complications of CVC at different insertion sites in HCT recipients. In this retrospective analysis, all consecutive patients (n = 56) who received a CVC (n = 101) due to allogeneic HCT at our institution between January 2011 and June 2013 were included. Three-lumen standard, nontunneled CVCs were placed via either the IJV (n = 60; 59%) or the SCV (n = 41; 41%). Study endpoints were time to local inflammation at the insertion site, time to fever, time to a combined endpoint of inflammation and fever, central line-associated bloodstream infection (CLABSI), duration of catheterization, catheter lumen obstruction, deep-vein thrombosis, pneumothorax, and catheter-related death. The median duration of catheterization per CVC was almost identical for the IJV and SCV sites (18 days versus 17 days; P not significant). There were no differences in the frequency of CLABSI, deep-vein thrombosis, pneumothorax, and catheter lumen obstruction between IJV and SCV CVC insertion sites. None of the patients died due to a CVC-related cause. Local inflammation occurred less frequently (48% versus 71%; P = .025) and later (median time to local inflammation, 25 days versus 12 days; P = .01) in IJV CVCs versus SCV CVCs. There was a trend toward a median longer time to the occurrence of fever for IJV CVCs compared with SCV CVCs (20 days versus 13 days; P = .07). In the multivariate analysis, diagnosis of acute leukemia (hazard ratio [HR], 1.696; P = .036), SCV CVC (HR, 1.617; P  = .039), and neutropenic CVC-days (HR, 2.477; P = .01) were identified as risk factors for the occurrence of local inflammation or fever. In contrast to earlier studies in patients without hematologic malignancies, these data demonstrate that CVCs placed in the SCV are not superior over IJV CVCs. Moreover, local inflammation occurred earlier and more frequently in patients with an SCV CVC.