Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

N-terminal pro-brain natriuretic peptide is a prognostic marker for response to intensive chemotherapy, early death, and overall survival in acute myeloid leukemia.

Patient-related factors are of prognostic importance in acute myeloid leukemia (AML). Likewise, cardiac disorders may limit the tolerance of intensive therapy. Little is known about the prognostic value of N-terminal pro-brain natriuretic peptide (NT-proBNP). We analyzed NT-proBNP levels at diagnosis in 312 AML patients (median age: 61 years; range 17-89 years) treated with 3 + 7-based induction-chemotherapy and consolidation with up to four cycles of intermediate or high-dose ARA-C. NT-proBNP levels were elevated in 199 patients (63.8%), normal (0-125 pg/ml) in 113 (36.2%), and highly elevated (>2000 pg/ml) in 20 patients (6.4%). Median NT-proBNP levels differed significantly among patients with complete remission (153.3 pg/ml), no remission (225.9 pg/ml), or early death (735.5 pg/ml) (p = .002). In multivariate analysis, NT-proBNP, age, and the 2009 European LeukemiaNet (ELN-2009) classification were independent predictors of outcome after induction chemotherapy. Overall survival (OS) differed significantly between patients with normal, moderately elevated, and highly elevated NT-proBNP (p < .001). These differences were observed in all patients and in patients <60 years but not in those ≥60 years. In multivariate analysis, NT-proBNP, age, and ELN-2009 remained independent prognostic variables for OS (p < .01). Together, NT-proBNP is an independent prognostic factor indicating the risk of induction failure, early death, and reduced OS in patients with AML.

The TGF-b/SOX4 axis and ROS-driven autophagy co-mediate CD39 expression in regulatory T-cells.

The ectonucleotidase CD39 on human regulatory T-cells (Treg) is an important immune regulator which is dysregulated in autoimmune diseases and cancer immunosuppression. We here define that CD39 expression on Treg is independent of the Treg-specific transcription factors FOXP3 and HELIOS and promoted by canonical TGF-b- and mTOR-signaling. Furthermore, the TGF-b mediated upregulation of CD39 is counteracted by reactive oxygen species (ROS)-driven autophagy. In line, CD39+ peripheral blood Treg constitute a distinct lineage with low autophagic flux and absent ROS production. Patients with rare genetic defects in autophagy show supraphysiological levels of CD39+ Treg, validating our observations in vivo. These biological processes rely on a distinct transcriptional program with CD39+ Treg expressing low levels of two genes with putative involvement in autophagy, NEFL and PLAC8. Furthermore, the TGF-b downstream transcription factor SOX4 is selectively upregulated in CD39+ Treg. Overexpression of SOX4 in Treg strongly increases CD39 expression, while Crispr/Cas9-mediated knockout of SOX4 in Treg has the opposing effect. Thus, we identify a crucial role of SOX4 in immune regulation and provide new insights involving the interplay of tolerogenic cues and autophagy in Treg.

Biological properties of bone marrow plasma cells influence their recovery in aspirate specimens: impact on classification of plasma cell disorders and potential bias to evaluation of treatment response.

Methods to estimate bone marrow plasma cells (BMPC) basically include histopathology, cytomorphology, and flow cytometry. The present study compares the outcomes of these methods with special focus on the impact of BMPC-specific characteristics on their recovery by either method. Laboratory reports of diagnostic samples from 238 consecutive patients with suspected or known plasma cell disease were retrospectively analyzed. The median (IQR) proportion of BMPC was 30.0% (15.0-70.0%) by histological review (hBMPC), 7.0% (2.0-16.0%) by smear review (sBMPC), and 3.0% (0.8-10.0%) by flow cytometry (fBMPC). The disparity of results between core biopsy and aspirate smear was enhanced in case of poor quality of the smear, increased BM fiber content, higher grade cell atypia, expression of CD56 (all P < 0.0001), the number of cytogenetic aberrations (P = 0.0002), and abnormalities of the MYC gene (P = 0.0002). Conversely, expression of CD19 and a non-clonal plasma cell phenotype were associated with a lower difference between hBMPC and sBMPC (both P < 0.0001). The disparity between the percentages of sBMPC and fBMPC was associated with the quality of the smear (P = 0.0007) and expression of CD56 (P < 0.0001). Our results suggest that the recovery of BMPC in aspirate specimens not only is a matter of sampling quality but also depends on biological cell properties. Aspiration failure due to malignant type features of BMPC may lead to misclassification of plasma cell disorders and represent a bias for the detection of minimal residual disease after therapy.

Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia-A Retrospective Analysis in 337 Patients.

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

Umbilical Cord Blood Transplantation Is a Feasible Rescue Therapeutic Option for Patients Suffering from Graft Failure after Previous Hematopoietic Stem Cell Transplantation.

OBJECTIVE: Umbilical cord blood (UCB) is an important graft source for hematopoietic stem cell transplantation (SCT). Due to less stringent human leukocyte antigen (HLA) matching criteria compared to bone marrow or peripheral blood stem cells, UCB enables patients lacking an HLA-matched donor to receive potentially curative SCT. METHODS: We retrospectively analyzed the efficacy and safety of UCB transplantation (UCBT) at our center. RESULTS: Between June 2009 and June 2015, 27 UCBT were performed in 25 patients. Reasons for the use of UCB were lack of adequate related or unrelated stem cell donor (n = 20) and graft failure after previous SCT (n = 7). Median time to neutrophil engraftment was 22 days. Four patients experienced primary graft failure. Thirteen patients developed acute graft-versus-host disease (GVHD), whereupon 6 subsequently also developed chronic GVHD. After a median follow-up time of 19 months, 9 patients relapsed and 12 patients died. Cause of death was relapse in 8 and transplant-related events in 4 patients. Median overall survival and progression-free survival have not been reached yet. CONCLUSION: In our experience, UCBT is an alternative graft source for patients lacking a suitable related or unrelated donor and a feasible treatment option for patients experiencing graft failure after previous SCT.

In vitro and in vivo effects of JAK2 inhibition in chronic myelomonocytic leukemia.

In chronic myelomonocytic leukemia (CMML), colony-forming units granulocyte/macrophage (CFU-GM), which grow in vitro in the absence of exogenous growth factors, arise from the abnormal clone that is responsible for the overproduction of granulomonocytic cells. Previous in vitro findings including ours suggest that divergent molecular aberrations in CMML seem to converge within the GM-CSF signaling pathway. As JAK2 is a sentinel kinase in this pathway, JAK2 inhibition may be an attractive treatment approach in CMML. We investigated the in vitro effects of the specific JAK2 inhibitor TG101209 on the autonomous CFU-GM formation from peripheral blood mononuclear cells of patients with CMML. TG101209 was found to either block or strongly inhibit spontaneous CFU-GM growth in all 10 patients tested. This inhibitory effect was dose dependent and significantly more pronounced as compared to the inhibitory effect on stimulated CFU-GM growth from normal individuals. In a CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we can demonstrate a spleen response and the disappearance of constitutional symptoms which was associated with a decrease in autonomous CFU-GM formation ex vivo. Pharmacological JAK2 inhibition may be an interesting approach to be systematically studied in patients with CMML.

CD34(+)/CD38(-) stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin.

BACKGROUND: CD33 is a well-known stem cell target in acute myeloid leukemia. So far, however, little is known about expression of CD33 on leukemic stem cells in chronic leukemias. DESIGN AND METHODS: We analyzed expression of CD33 in leukemic progenitors in chronic myeloid leukemia by multi-color flow cytometry and quantitative polymerase chain reaction. In addition, the effects of a CD33-targeting drug, gemtuzumab/ozogamicin, were examined. RESULTS: As assessed by flow cytometry, stem cell-enriched CD34(+)/CD38(-)/CD123(+) leukemic cells expressed significantly higher levels of CD33 compared to normal CD34(+)/CD38(-) stem cells. Moreover, highly enriched leukemic CD34(+)/CD38(-) cells (>98% purity) displayed higher levels of CD33 mRNA. In chronic phase patients, CD33 was found to be expressed invariably on most or all stem cells, whereas in accelerated or blast phase of the disease, the levels of CD33 on stem cells varied from donor to donor. The MDR1 antigen, supposedly involved in resistance against ozogamicin, was not detectable on leukemic CD34(+)/CD38(-) cells. Correspondingly, gemtuzumab/ozogamicin produced growth inhibition in leukemic progenitor cells in all patients tested. The effects of gemtuzumab/ozogamicin were dose-dependent, occurred at low concentrations, and were accompanied by apoptosis in suspension culture. Moreover, the drug was found to inhibit growth of leukemic cells in a colony assay and long-term culture-initiating cell assay. Finally, gemtuzumab/ozogamicin was found to synergize with nilotinib and bosutinib in inducing growth inhibition in leukemic cells. CONCLUSIONS: CD33 is expressed abundantly on immature CD34(+)/CD38(-) stem cells and may serve as a stem cell target in chronic myeloid leukemia.

Improved outcome in patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation over the past 25 years: a single-center experience.

Although imatinib has become standard first-line therapy in chronic myelogenous leukemia (CML), allogeneic hematopoietic stem cell transplantation (HSCT) is still considered to be an important treatment alternative for patients with drug resistance or advanced disease. We retrospectively analyzed 175 adult CML patients who underwent HSCT at our institution between 1983 and 2007, with the aim to compare outcomes in patient subgroups and to identify prognostic variables. The median follow-up was 65 months. The probability of overall survival (OS) for all patients was 62%, with a significant improvement seen in the imatinib-era (2001-2007) compared to previous time periods (P <.05). Furthermore, a significantly better outcome for patients with chronic phase CML compared to patients with accelerated or blast phase could be observed (P < .05). Cumulative incidence (CI) of treatment-related mortality (TRM) was 9.7% at 100 days and 1 year after HSCT. CI of relapse was 5% at 1 year and 7.5% at 3 years after HSCT. Post-HSCT outcome was not influenced by pretreatment therapy with imatinib, donor type, or a conditioning regimen with total body irradiation (TBI). These data confirm earlier observations and suggest that allogeneic HSCT is still an important treatment option for high-risk patients with CML, and should thus remain an integral component in current and future treatment algorithms.

Immature and absolute platelet count changes and thrombocytopenia in malignant glioma.

BACKGROUND: Temozolomide (TMZ) is commonly used for the therapy of malignant glioma and induces thrombocytopenia in a fraction of patients. Currently, no biomarkers predicting TMZ-induced thrombocytopenia are available. In this study, we investigated whether changes in platelet count (PLT) or the immature platelet fraction (IPF) may serve as predictor of TMZ-induced thrombocytopenia in malignant glioma patients. METHODS: We prospectively included 52 malignant glioma patients receiving TMZ-containing therapy regimens in this study. Platelet counts and IPF were determined at each clinical follow-up visit (weekly during concomitant radiochemotherapy or at least monthly during TMZ monotherapy) using the Sysmex XE-2100 system. We explored the diagnostic utility of PLT change/day and IPF change/day from the last to the current follow-up visit for the prediction of clinically relevant thrombocytopenia (PLT < 100·000 µl(-1) ) at the next follow-up visit. RESULTS: Relevant thrombocytopenia was observed in 10 of 234 occasions. The areas under the receiver operating characteristic curves for PLT absolute change/day, PLT relative change/day and IPF relative change/day were 0·675, 0·703 and 0·663, respectively. The Youden indices (maximum sum of sensitivity and specificity minus one) were 0·31, 0·39, and 0·29, respectively. The corresponding positive predictive values were 16%, 57%, and 6·7%, and the negative predictive values were 97%, 97%, and 98%, respectively. CONCLUSIONS: The rather moderate diagnostic potential of our data indicate that the time course of PLT counts and IPF measured at routine clinical follow-up are not useful for the prediction of thrombocytopenia in glioma patients treated with TMZ.

Attenuation of canonical NF-κB signaling maintains function and stability of human Treg.

Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy.

Impact of HFE gene variants on iron overload, overall survival and leukemia-free survival in myelodysplastic syndromes.

Although iron overload is a clinical challenge, little is known about the clinical impact of HFE-variants in myelodysplastic syndromes (MDS) to date. We analyzed the HFE status in 167 MDS patients and 494 healthy controls. One or more of the 3 HFE-variants (H63D, C282Y, S65C) were found in 65/167 (38.9%) MDS patients and in 164/494 (33.2%) controls. At diagnosis, the median serum ferritin levels were higher in MDS patients with HFE-variants (409 µg/L; range: 23-7415) compared to those without HFE-variants (346.5 µg/L; range: 10-5450) (P=0.62). Moreover, 'HFE-mutated' patients had a slightly faster increase in serum ferritin in follow up examinations. The percentage of patients with HFE-variants was higher in refractory anemia (RA) (22/53=41.5%) or RA with ring sideroblasts (RARS) (17/39=43.6%) compared to RA with excess of blasts (RAEB) (16/46=34.8%) or RAEB in transformation (RAEB-T) (5/17=29.4%). Differences were also detectable when comparing low- and high-risk MDS variants defined by the World Health Organization classification. There was no significant correlation between HFE-variants and MDS-related somatic mutations. Progression-free survival was substantially longer in patients with HFE-variants compared to those without HFE-variants H63D and C282Y (P=0.089). Together, the HFE-variants H63D and C282Y are frequently detected in Austrian MDS patients. These patients have substantially higher ferritin levels at diagnosis, accumulate iron slightly faster and have a better progression-free survival than non-mutated patients.

Secondary basophilic leukemia in Ph-negative myeloid neoplasms: A distinct subset with poor prognosis.

During progression of myeloid neoplasms, the basophil compartment may expand substantially and in some of these patients, a basophilic leukemia is diagnosed. In patients with Ph-chromosome+ chronic myeloid leukemia, acceleration of disease is typically accompanied by marked basophilia. In other myeloid neoplasms, secondary leukemic expansion of basophils is rarely seen. We report on 5 patients who suffered from a myelodysplastic syndrome, myeloproliferative neoplasm, or acute leukemia and developed a massive expansion of basophils during disease progression. In 4 of 5 patients, peripheral blood basophil counts reached 40%, and the diagnosis "secondary basophilic leukemia" was established. As assessed by flow cytometry, neoplastic basophils expressed CD9, CD18, CD25, CD33, CD63, PD-L1, CD123, and CLL-1. In addition, basophils were found to display BB1 (basogranulin), 2D7, tryptase and KIT. In 4 of 5 patients the disease progressed quickly and treatment with azacitidine was started. However, azacitidine did not induce major clinical responses, and all patients died from progressive disease within 3 Y. In in vitro experiments, the patients´ cells and the basophilic leukemia cell line KU812 showed variable responses to targeted drugs, including azacitidine, venetoclax, hydroxyurea, and cytarabine. A combination of venetoclax and azacitidine induced cooperative antineoplastic effects in these cells. Together, secondary basophilic leukemia has a poor prognosis and monotherapy with azacitidine is not sufficient to keep the disease under control for longer time-periods. Whether drug combination, such as venetoclax+azacitidine, can induce better outcomes in these patients remains to be determined in future clinical studies.

Interference of Mycoplasma infection in a gene expression study: it was the environment and not the gene.

We show that short-term exposure to doxycycline, as used in tetracycline-inducible gene expression models, protects cells from stress-induced death in cultures infected with Mycoplasma arginini. Coinciding with the expected maximum level of gene activity, antimicrobial effects of tetracyclines might be mistaken for antiapoptotic properties of the expressed gene product.

Humoral immune response to tick-borne encephalitis vaccination in allogeneic blood and marrow graft recipients.

The aim of this prospective study was to characterize the humoral immune response to TBE vaccination after hematopoietic stem cell transplantation (HSCT). Nineteen adult patients 11-13 months after HSCT and 15 age-matched immunocompetent adults received up to three TBE vaccinations. Antibodies against TBE virus were measured by neutralization test (NT). As primary endpoint, the antibody response (NT titer of ≥10 and at least a twofold increase from baseline 4 weeks after second vaccination) was compared between patients and controls using Fisher exact test. Prior vaccination, 15 (79%) HSCT patients still had detectable neutralizing antibodies. At primary endpoint, the antibody response was significantly lower in patients than in controls (35% versus 93%; p < 0.001). The CD4+ cell count was a predictor for an antibody response in patients (p = 0.019). Interestingly, the majority of HSCT patients still had detectable antibodies prior vaccination. Following vaccination, antibody response in HSCT patients was associated with the CD4+ cell count.

Erratum: Author Correction: Humoral immune response to tick-borne encephalitis vaccination in allogeneic blood and marrow graft recipients.

[This corrects the article DOI: 10.1038/s41541-020-00215-1.].