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.

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.

Leukemic stem cells and therapy resistance in acute myeloid leukemia.

A major obstacle in the treatment of acute myeloid leukemia (AML) is refractory disease or relapse after achieving remission. The latter arises from a few therapy-resistant cells within minimal residual disease (MRD). Resistant cells with long-term self-renewal capacity that drive clonal outgrowth are referred to as leukemic stem cells (LSC). The cancer stem cell concept considers LSC as relapse-initiating cells residing at the top of each genetically defined AML subclone forming epigenetically controlled downstream hierarchies. LSC display significant phenotypic and epigenetic plasticity, particularly in response to therapy stress, which results in various mechanisms mediating treatment resistance. Given the inherent chemotherapy resistance of LSC, targeted strategies must be incorporated into first-line regimens to prevent LSC-mediated AML relapse. The combination of venetoclax and azacitidine is a promising current strategy for the treatment of AML LSC. Nevertheless, the selection of patients who would benefit either from standard chemotherapy or venetoclax + azacitidine treatment in first-line therapy has yet to be established and the mechanisms of resistance still need to be discovered and overcome. Clinical trials are currently underway that investigate LSC susceptibility to first-line therapies. The era of single-cell multi-omics has begun to uncover the complex clonal and cellular architectures and associated biological networks. This should lead to a better understanding of the highly heterogeneous AML at the inter- and intra-patient level and identify resistance mechanisms by longitudinal analysis of patients' samples. This review discusses LSC biology and associated resistance mechanisms, potential therapeutic LSC vulnerabilities and current clinical trial activities.

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.

Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the ß-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the ß-subunit. The examined promoter region of the human FTase ß-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Alternative splicing of the TNFSF13B (BAFF) pre-mRNA and expression of the BAFFX1 isoform in human immune cells.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.

Blinatumomab or Inotuzumab Ozogamicin as Bridge to Allogeneic Stem Cell Transplantation for Relapsed or Refractory B-lineage Acute Lymphoblastic Leukemia: A Retrospective Single-Center Analysis.

BACKGROUND: Blinatumomab and inotuzumab ozogamicin are now widely used to treat relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). PATIENTS AND METHODS: We have reported the clinical course of 34 adult patients with r/r B-ALL receiving blinatumomab or inotuzumab ozogamicin at our institution from 2009 to 2019. RESULTS: Blinatumomab-based salvage therapy was applied for overt r/r B-ALL (n = 13) or minimal residual disease (MRD) positivity (n = 5). Of the 13 patients with r/r B-ALL, 9 (69%; 95% confidence interval [CI], 39%-91%) achieved complete remission (CR), with 78% of CR patients (95% CI, 40%-97%) reaching MRD negativity. MRD negativity was also achieved in all 5 patients treated for MRD positivity. The 1-year overall survival of patients receiving blinatumomab for r/r B-ALL and MRD positivity was 54% (n = 13; 95% CI, 26%-81%) and 80% (n = 5; 95% CI, 44-100), respectively. In the inotuzumab ozogamicin group, all 16 patients were treated for overt r/r B-ALL. The rate of CR was 94% (95% CI, 70%-100%), with 67% (95% CI, 38%-88%) of CR patients reaching MRD negativity. The 1-year OS after the first application of inotuzumab ozogamicin was 46% (95% CI, 18%-74%). Of those patients receiving blinatumomab and inotuzumab ozogamicin as a bridge-to-transplant strategy, 79% and 80%, respectively, proceeded to allogeneic stem cell transplantation. The most frequent drug-specific adverse events were similar to those previously reported, including cytokine release syndrome, capillary leak syndrome, and neurotoxicity for blinatumomab and transplant-associated veno-occlusive disease of the liver for inotuzumab ozogamicin. CONCLUSION: Together with previous observations from phase III clinical trials, these data suggest that blinatumomab and inotuzumab ozogamicin are highly effective salvage regimens in r/r B-ALL.

Monitoring minimal residual/relapsing disease after allogeneic haematopoietic stem cell transplantation in adult patients with acute lymphoblastic leukaemia.

Relapse after allogeneic haematopoietic stem cell transplantation (SCT) is a major cause of death in patients with acute lymphoblastic leukaemia (ALL). Here, we retrospectively analysed the contributions of lineage-sorted donor cell chimerism (sDCC) and quantitative PCR (qPCR) targeting disease-specific genetic rearrangements to detect minimal residual/relapsing disease (MRD) and predict impending relapse in 94 adult ALL patients after SCT. With a median follow-up of surviving patients (n = 61) of 3.3 years, qPCR and/or sDCC measurements turned positive in 38 patients (40%). Of these, 22 patients relapsed and 16 remained in complete remission. At 3 years, qPCR and/or sDCC positive patients showed an increased incidence of relapse (50% vs. 4%, p < 0.0001), decreased relapse-free survival (RFS, 40% vs. 85%, p < 0.0001), and decreased overall survival (OS, 47% vs. 87%, p 0.004). Both, qPCR and sDCC pre-detected 11 of 21 relapses occurring within the first two years after SCT and, overall, complemented for each other method in four of the relapsing and four of the non-relapsing cases. Patients receiving pre-emptive MRD-driven interventions (n = 11) or not (n = 10) showed comparable median times until relapse, RFS, and OS. In our single centre cohort, qPCR and sDCC were similarly effective and complementary helpful to indicate haematological relapse of ALL after SCT.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines.

BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.

Relationship between GNAS1 T393C polymorphism and aseptic loosening after total hip arthroplasty.

BACKGROUND: Aseptic loosening is a main cause for revision surgery after total hip arthroplasty (THA) and there is no reliable marker for the early detection of patients at high risk. This study has been performed to validate association of the T393C polymorphism (rs7121) in the GNAS1 gene, encoding for the alpha-subunit of heterotrimeric G-protein Gs, with risk for and time to aseptic loosening after THA, which has been demonstrated in our previous study. METHODS: 231 patients with primary THA and 234 patients suffering from aseptic loosening were genotyped for dependency on GNAS1 genotypes and analyzed. RESULTS: Genotyping revealed almost similar minor allele frequencies of 0.49 and 0.46, respectively. Consistently, genotype distributions of both groups were not significantly different (p = 0.572). Neither gender nor GNAS1 genotype showed a statistically significant association with time to loosening (p = 0.501 and p = 0.840). Stratification by gender, as performed in our previous study, was not able to show a significant genotype-dependent difference in time (female p = 0.313; male p = 0.584) as well as median time to aseptic loosening (female p = 0.353; male p = 0.868). CONCLUSION: This study was not able to confirm the results of our preliminary study. An association of the GNAS1 T393C polymorphisms with risk for and time to aseptic loosening after THA is unlikely.

Impact of BCL2 polymorphisms on survival in transitional cell carcinoma of the bladder.

PURPOSE: To evaluate the impact of three BCL2 single-nucleotide polymorphisms, i.e., c.-938C>A (rs2279115), c.21G>A (rs1801018), and c.*2203A>G (rs4987853) on survival in patients with transitional cell carcinoma of the bladder. METHODS: We analyzed 179 patients who underwent surgical treatment for bladder cancer at the Clinic of Urology, University Hospital Essen, Germany. Genomic DNA was extracted and genotyped for the polymorphisms. For all polymorphisms, linkage analysis was performed. Kaplan-Meier and Cox regression analyses were used to determine the putative impact of the three polymorphisms on outcome. RESULTS: c.-938C>A and c.21G>A, but not c.*2203A>G, are in strong linkage disequilibrium (D' 0.96). We found a significant association between c.-938C>A and relapse-free survival (p = 0.024) with an allele dose effect. In the same way, c.21G>A had a significant impact on both relapse-free survival (p = 0.009) and progression-free survival (p = 0.012), as well as a pronounced allele dose effect. Regression analysis proved c.21G>A and c.-938C>A, to be an independent risk factor in univariate and multivariable analyses. CONCLUSIONS: In our cohort, both c.-938C>A and c.21G>A showed a significant impact on outcome with TCC of the bladder. Due to the linkage disequilibrium of both SNPs, maybe, only one of them could mediate this effect. In multivariable analysis, however, both proved to be independently associated with overall survival. Contrary to other findings which found the c.-938C>A mainly influencing outcome, our data may suggest that the main effect on TCC could be due to the c.21G>A polymorphism.

Association of a Common Oxytocin Receptor Gene Polymorphism with Self-Reported 'Empathic Concern' in a Large Population of Healthy Volunteers.

BACKGROUND: Previous research has linked genomic variations of the oxytocin receptor (OXTR) gene with individual differences in empathy. The impact of these variations on specific cognitive and emotional aspects of empathy, however, remains to be clarified. METHODS: We analysed associations of a common OXTR polymorphism (rs53576) with trait empathy in a sample of 421 blood donors (231 M, 190 F; age 18-74) using the Interpersonal Reactivity Index (IRI) as an established multidimensional self-report measure of empathy. RESULTS: Female sex was significantly associated with higher empathy scores in all IRI scales (p<0.001) with the exception of the cognitive perspective taking scale (p = 0.09). The overall trait empathy score was significantly associated with rs53576 (p = 0.01), with mean scores increasing from AA to GG genotypes. An analysis of the IRI subscores revealed that the polymorphism was especially associated with the emotional empathic concern scale (p = 0.02). Separate analysis of the male and female subgroup revealed a significant association of the polymorphism with female (p = 0.04), but not with male (p = 0.20) empathic concern. A comparison of effect sizes between the groups showed greater effects for women compared to men although effect size differences did not become significant in our sample. CONCLUSIONS: Our findings suggest a significant association of the rs53576 OXTR gene polymorphism with trait empathy and especially with emotional aspects of empathy. This association is possibly weaker or absent in men compared to women.

The BCL2 -938C>A Promoter Polymorphism Is Associated with Risk for and Time to Aseptic Loosening of Total Hip Arthroplasty.

Aseptic loosening is a major cause of revision surgery of total hip arthroplasty (THA). Only few host factors affecting aseptic loosening have been identified until now, although they are urgently needed to identify and possibly treat those patients at higher risk for aseptic loosening. To determine whether the functional single nucleotide polymorphism (SNP) c.-938C>A (rs2279115), located in the promoter region of the BCL2 gene has an impact on aseptic loosening of THA we genotyped and analyzed 234 patients suffering from aseptic loosening and 231 patients after primary THA. The polymorphism is associated with risk for aseptic loosening with the CC genotype at highest risk for aseptic loosening, Odds Ratio CC vs. AA 1.93, 95%CI 1.15-3.25, p = 0.013. In contrast, low risk AA genotype carriers that still developed aseptic loosening showed a significantly shorter time to aseptic loosening than patients carrying the C allele (p = 0.004). These results indicate that the BCL2 -938C>A polymorphism influences the occurrence and course of aseptic loosening and suggests this polymorphism as an interesting candidate for prospective studies and analyses in THA registers.