WT1 Expression Levels Research Articles

Role of WT1 in Measurable Residual Disease Follow-Up in the Post Allogeneic Stem Cell Transplant Setting.

Objectives: The Wilms' tumor gene 1 (WT1) plays a critical role in cell development and the regulation of essential genes involved in cell growth and metabolism. In the context of hematopoietic tumors, including acute myeloid leukemia (AML), WT1 has been identified as a potential marker for measurable residual disease (MRD) assessment. Relapse after allogeneic hematopoietic stem cell transplantation (allo-SCT) remains a significant challenge in AML treatment, highlighting the importance of MRD monitoring for risk stratification and treatment decisions. This study aimed to investigate the clinical significance of WT1 as a molecular marker for MRD and its correlation with chimerism in AML patients post-allo-SCT setting. Methods: We have included 58 patients with WT1-expression-positive acute myeloid leukemia (AML) who received allo-SCT in our center between 2016-2022. The exclusion criteria are as follows: not having WT1 polymerase chain reaction (PCR) measurement at diagnosis, not receiving allo-SCT, and not having a serial measurement of WT1 post-transplant. Pre- and post-transplant assessments were made with flow cytometry, WT1 PCR, and bone marrow morphological evaluations. Statistical analyses were carried out to explore correlations between WT1 levels, MRD markers, and chimerism post-transplantation. Results: We found that WT1 had a significant correlation with flow cytometry and bone marrow morphological evaluation, but not with chimerism. Interestingly, high WT1 expressors exhibited a more robust correlation with chimerism compared to the general cohort. The negative predictive value for post-allo-SCT relapse was 91.8% for the whole WT1 cohort; for high WT1 expressors, it was similar, at 87.5%. The negative predictive value for post-allo-SCT relapse was high for the whole WT1 cohort; for high WT1 expressors, it was similar. The WT1 MRD assay showed a high negative predictive value for post-allo-SCT relapse, consistent across both the entire cohort (91.8%) and high WT1 expressors (87.5%). Conclusions: WT1 expression levels may serve as a valuable ancillary marker in MRD assessment and relapse prediction post-allo-SCT in AML patients, particularly for those lacking specific fusion genes or mutations. However, further large-scale, controlled studies are needed to standardize WT1 MRD assays and establish clear guidelines for their clinical application.

Clinical significance of WT1 in the evaluation of therapeutic effect and prognosis of non-M3 acute myeloid leukemia

ABSTRACT To explore the clinical significance and prognosis of acute myeloid leukemia (AML) patients with WT1 mutations.In total, the clinical data of 269 adult patients with non-M3 AML were considered retrospectively. From these patients, 153 carried WT1 mutation whereas 116 were negative. WT1 mutation positive patients were further divided into WT1 low expression and high expression groups base on the expression level of WT1 by qPCR at diagnosis (cut off: 170500). Survival and therapeutic effect analysis were performed for the above patients with different interfering factors such as co-mutations, the extent of WT1 log reduction and the chemotherapy regimens. Patients with high WT1 expression have higher rate of relapse. We can accurately identify patients with inferior outcomes when we take the following factors into consideration: the WT1 expression level at diagnosis; different prognostic factors including co-mutations (especially NPM1 and FLT3-ITD); the log reduction of WT1 after induction therapy and the risk of stratification. Idarubicin + Cytarabine (IA) regimen could reduce the expression level of WT1 after treatment, and Allo-HSCT played an important role in improving the prognosis of patients with WT1 high expression and patients with WT1 negativity. Among the relapsed patients, there existed a rising trend of WT1-MRD in advance than MFC-MRD and that of patients with continuous complete remission (CR). Different clinical background should be taken into consideration when we judge the prognosis and therapeutic effect of patients with WT1 mutations. In addition, WT1 may be an optional MRD marker, which needs regular monitoring.

BAALC-Expressing Leukemia Hematopoietic Stem Cells and Their Place in the Study of CBF-Positive Acute Myeloid Leukemias in Children and Adults

Background. Due to changing views on pathogenesis, risk factors and therapy strategies in prognostically favorable CBF-positive acute myeloid leukemias[1] (AML), the expression monitoring of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes, as an additional evaluation of treatment outcomes, appears to be insufficient. This indicates the need to improve the monitoring of the CBF+ AML course by means of parallel measurements of BAALC expression levels which roughly correlate with the mass of BAALC-expressing leukemia hematopoietic stem cells (BAALC-e LHSC).
 Aim. To improve the quality of assessing treatment outcomes with due account for expression levels of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes and the mass of BAALC-e LHSC and on this basis to pave the way for personalized CBF+ AML treatment.
 Materials & Methods. This study enrolled 39 adult patients aged 20–81 years (median 32 years) and 8 children aged 2–18 years (median 12 years). Among them there were 20 females and 27 males. AML with inv(16)(p13;q22)/t(16;16) was identified in 19 patients, t(8;21)(q22;q22) was detected in 28 patients. BAALC, WT1, RUNX1/RUNX1T1, CBFB/MYH11 expression levels were measured by quantitative real-time PCR and related to the expression of the ABL1 expert gene.
 Results. In 23 patients, inv(16) and t(8;21) appeared to be isolated. Additional multidirectional chromosomal changes were observed in 24 patients with inv(16) and in 18 patients with t(8;21). All enrolled patients showed increased BAALC expression. In the course of therapy, it was decreasing to the threshold value in 16/18 (89 %) patients. The evaluation of the mean BAALC expression levels in the pooled groups of children and adults with isolated findings of either inv(16) or t(8;21) showed the decrease of the BAALC-e LHSC mass only in children (p = 0.049). The comparison of the mean WT1 expression levels in the pooled groups of children and adults with isolated and additional chromosomal abnormalities revealed their significant decrease in patients with complicated variants (p = 0.023).
 Conclusion. The case reports provided in this paper show that the molecular monitoring with serial measurements of fusion genes and BAALC gene expression levels in CBF+ AML patients can lay the basis for further improvement of personalized treatment strategies for these patients. In all likelihood, parallel measurements of the above gene expression levels will allow to establish the framework for decision-making concerning treatment extent and timely HSC transplantation.

The Distribution and Significance of Activated T Cells and Lymphocyte Subsets in Myelodysplastic Syndrome

To investigate the distribution of bone marrow lymphocyte subsets in patients with myelodysplastic syndrome(MDS),the proportion of activated T cells with immunophenotype CD3+HLA-DR+ in the lymphocytes and its clinical significance, and to understand the effects of different types of MDS, different immunophenotypes, and different expression levels of WT1 on the proportion of lymphocyte subsets and activated T cells. The immunophenotypes of 96 MDS patients, the subsets of bone marrow lymphocytes and activated T cells were detected by flow cytometry. The relative expression of WT1 was detected by real-time fluorescent quantitative PCR, and the first induced remission rate (CR1) was calculated, the differences of lymphocyte subsets and activated T cells in MDS patients with different immunophenotype, different WT1 expression, and different course of disease were analyzed. The percentage of CD4+T lymphocyte in MDS-EB-2, IPSS high-risk, CD34+ cells >10%, and patients with CD34+CD7+ cell population and WT1 gene overexpression at intial diagnosis decreased significantly (P<0.05), and the percentage of NK cells and activated T cells increased significantly (P<0.05), but there was no significant difference in the ratio of B lymphocytes. Compared with the normal control group, the percentage of NK cells and activated T cells in IPSS-intermediate-2 group was significantly higher(P<0.05), but there was no significant difference in the percentage of CD3+T, CD4+T lymphocytes. The percentage of CD4+T cells in patients with complete remission after the first chemotherapy was significantly higher than in patients with incomplete remission(P<0.05), and the percentage of NK cells and activated T cells was significantly lower than that in patients with incomplete remission (P<0.05). In MDS patients, the proportion of CD3+T and CD4+T lymphocytes decreased, and the proportion of activated T cells increased, indicating that the differentiation type of MDS is more primitive and the prognosis is worse.

Combined expression of WT1/PRAME genes and BIRC5/PRAME genes and their clinical significance in adult patients with acute myeloid leukemia

Abstract Background Several tumor-associated antigens have been recently recognized that qualify to monitor tumor burden of acute myeloid leukemia (AML) or are used as antigen-specific approach for anticancer immunotherapy. Aim The current study was designed to assess the intracellular expression levels of the immunogenic tumor antigens ( WT1 , BIRC5, and PRAME genes) and clarify the prognostic effect of their combined overexpression, that is, WT1 /PRAME and BIRC5 / PRAME , on the clinical course, the outcome, and the response to treatment in patients with AML. Settings and design A total of 61 patients with AML were included in the study. All patients received induction chemotherapy. Detection of intracellular WT1, BIRC5, and PRAME genes in myeloid blast cells was done by real-time PCR on bone marrow samples. Their levels were correlated with the demographic data, the clinical and pathological data, the defined subgroups, the overall survival, and the response to treatment in patients with AML. Results WT1, BIRC5, and PRAME overexpression was detected in 73.8, 28, and 38.6% of patients, respectively. Nearly half of the patients (51.7%) were WT1+/PRAME− and significantly associated with absence of both favorable cytogenetic prognosis and complete remission. In addition, it expression was correlated with female sex, higher count of blasts in peripheral blood, positive CD34 expression, and higher FAB classification (M4) but with no statistically significant difference. Most patients were double negative BIRC5−/PRAME− (45.6%). A significant relation was found between BIRC5+/PRAME− and older age patients (P=0.003) and a lower overall survival (P=0.005) and also associated with higher count of blast cells but not statistically significant. Most BIRC5−/PRAME+ (72.7%) achieved complete remission at 28 days, followed by double negative BIRC5−/PRAME− (56.6%) at 28 days, most of BIRC5+/PRAME− patients died (77.8%). Conclusion In patients with AML, the expression levels of genes WT1, BIRC5, and PRAME were high, and these genes had a substantial correlation with poor prognostic factors, including a poor cytogenetic prognosis, incomplete remission, and a reduced survival rate. Therefore, they could be considered as targets for anticancer immunotherapy as well as possible markers for monitoring AML.

TNF-α and IL-1β Promote Renal Podocyte Injury in T2DM Rats by Decreasing Glomerular VEGF/eNOS Expression Levels and Altering Hemodynamic Parameters.

Diabetic nephropathy (DN) is a serious microvascular complication in those with type 2 diabetes mellitus (T2DM). Evidence confirms that serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the T2DM stage are proposed as prognostic markers for DN development, but it is unclear how they affect renal podocyte-associated nephrin and WT-1 expression. In the presence of podocyte injury, glomerular vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and hemodynamic parameters are dysregulated. The current research aimed to clarify the relationship of TNF-α and IL-1β with podocyte injury by altering VEGF/eNOS expression and hemodynamic parameters. A high-fat diet/streptozotocin-induced DN rat model was established. Serum TNF-α and IL-1β levels were tracked in the pre-T2DM, T2DM and DN stages. In the DN stage, the mRNA and protein expression levels of renal TNF-α, IL-1β, VEGF, eNOS, nephrin and WT-1 were studied. Renal hemodynamic parameters, including peak systolic velocity, end-diastolic flow velocity and mean velocity were measured with a color Doppler ultrasound technique. Compared to those in the normal control (CTL) group, serum TNF-α and IL-1β levels increased significantly in the pre-T2DM stage (obesity, insulin resistance and hyperlipidemia), T2DM stage (hyperglycemia) and DN stage (abnormal renal functions) (all: P < 0.05) in the DN group. Serum TNF-α and IL-1β levels in the T2DM stage were significantly higher than those in the pre-T2DM stage (two: P < 0.05). Compared to the CTL group, renal nephrin, WT-1, TNF-α, IL-1β, eNOS and VEGF expression and hemodynamic parameters in the DN stage all showed significant differences separately (all: P < 0.05). Increased serum and renal TNF-α and IL-1β levels played important roles in reducing renal nephrin and WT-1 expression levels, which may be related to the fact that the former affected renal VEGF/eNOS expression and blood flow parameters in the DN rats.

Prognostic Relevance of Minimal Residual Disease in Therapy Related and Secondary Acute Myeloid Leukemia Receiving CPX-351 or Fludarabine-Based Induction

Background: The high prognostic value of minimal residual disease (MRD) assessment in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy has been confirmed by several studies. The most widely adopted MRD techniques include multicolor flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan-leukemic marker WT1. However, mostly younger patients receiving a conventional 3+7 induction are included in clinical studies evaluating MRD in AML. As a consequence, secondary AML, evolving from a previous myelodisplastic syndrome (s-AML) or therapy-related AML (t-AML), which usually affect elderly AML patients are usually under-represented in those trials. Recently, CPX-351 was approved for the treatment of s-AML and t-AML, following the positive results of the Phase III trial by Lancet et al. However, as MRD was not assessed in this trial, only a limited ammount of data are available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: In order to assess the prognostic value and to define the best time points for MRD assessment, MFC-based and WT1 based MRD was evaluated in a cohort of elderly s-AML or t-AML patients receiving CPX-351 induction. Timepoints for assessment were after induction (TP1), after first consolidation (TP2) and at the end of treatment (TP3). As a comparison, we also evaluated MRD in a cohort of elderly s-AML and t-AML patients who received an age-adjusted fludarabine-containing regimen (FLAI3). MRD analysis in this cohort was performed with the same methodics and at the same timepoints. Methods: A total of 151 elderly (>60 year, median age 68, range 60-77) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=50) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels. All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria. Results: After first induction cycle, 95 patients (59%) achieved CR. Patients treated with CPX-351 had a significantly higher CR rate, if compared to patients receiving FLAI (40/50, 80% and 55/101, 54.5%, respectively, p<0.05). Adding GO to FLAI did not increase CR rate. Overall, in the two cohorts, an MRD negative CR was achieved in 51 (53.7%) and 53 responding patients (55.8%) assessed with MFC or WT1-based methods, respectively. The probability of achieving MFC MRD negativity was significantly higher among patients treated with CPX-351, when compared to patients receiving FLAI with or without GO (MFC MRD negativity rate of 26/40, 65% and 25/55, 45%, respectively, p<0.05). In both cohort, the most informative timepoint in terms of survival prediciton was after first cycle (TP1). WT1-based MRD assessment led to superimposable results. In multivariate analysis, MRD was significantly correlated with an higher probability of longer Overall Survival (OS) in all treatment group (2-year OS of 34% and 77% in patients with or without residual MFC MRD after induction, respectively, p<0.05). Allogeneic stem cell transplantation consolidation (HSCT) was also significantly correlated with longer OS in both treatment groups (p<0.05). Notably, 12/40 (30%) CR patients treated with CPX 351 underwent HSCT, whereas only 8 CR patients proceeded to HSCT in the FLAI group (15%) Conclusions: MRD assessment has a strong prognostic value also in the context of elderly s-AML and t-AML patients. WT1-based and MFC-based MRD assessment lead to similar conclusions thus allowing to obtain MRD data from virtually all AML patients treated in the selected time period. Overall, CPX-351 resulted in higher CR rate with deeper MRD responses, if compared to FLAI3. This increased efficacy translated in a significantly higher number of elderly AML patients proceeding to HSCT in CR, which is the only curative approach in this particular AML setting.

The nephroprotective action of Passiflora edulis in streptozotocin-induced diabetes

In the present study, we aimed to evaluate the therapeutic effect of Passiflora edulis fruit peel aqueous (AFA) extract as an adjuvant to insulin to confer nephroprotection against streptozotocin-induced diabetes. Male Wistar rats were divided into four groups based on treatment received for 60 days: diabetic (DB), control (CTL), insulin (INS), and insulin + AFA extract (INS + AFA). mRNA and protein expression levels of podocyte (nephrin, podocin, and WT1) and tubular (megalin) proteins were measured in kidney tissue specimens and urine. Biochemical parameters and kidney histopathology were also examined. Herein, the INS + AFA group showed superior glycemic control, which resulted in the reduction of urinary albumin/creatinine ratio, maintenance of baseline levels of Nphs1, Nphs2, Wt1, and Lrp2 mRNA expression, prevention of protein loss from the kidney tissue into the urinary space, along with the maintenance of glomerular basement membrane thickness, hyalinization, glomerular and tubulointerstitial fibrosis at values approximating those of the CTL group and significantly lower than those in the DB group. Therefore, these results suggest that, as an anti-diabetic agent, the AFA extract adjuvant to insulin could reduce and potentially prevent diabetic kidney disease.

Immunophenotypic Features and Clinical Prognosis of Patients with Mixed Phenotype Acute Leukemia

To retrospectively analyze the laborotary test results and clinical data of 31 patients with mixed phenotype acute leukemia (MPAL) in order to summarize and discuss the biological characteristics, curative effect, and prognosis of each subtype of MPAL based on immunophenotype results. MPAL patients diagnosed and treated in our hospital from July 2013 to January 2019 were selected to analyze the data of cell morphology, immunophenotyping, cytogenetics, molecular biology (MICM), and routine blood at initial diagnosis. Follow-up was carried out until the last discharge time. Among 31 patients, there were 19 males and 12 females, with a median age of 41(12-76) years old. According to the results of immunophenotyping and EGIL score, there were 16 cases of myeloid-T lymphoid mixed phenotype (myeloid-T group), 9 cases of myeloid-B lymphoid mixed phenotype (myeloid-B group), 5 cases of T-B lymphoid mixed phenotype (T-B group), and 1 case of myeloid-T-B lymphoid mixed phenotype. Compared between different subtypes, the antigen expression characteristics were the highest positive rate and expression rate of HLA-DR in myeloid-B group, and the positive rate of CD2 in T-B group was significantly higher than that in the myeloid-T group. Meanwhile, the expression rates of CD7 and cCD3 (cytoplasmic CD3) in T-B group were higher than those in myeloid-T group, and cCD79a was positive in all cases of myeloid-B group and T-B group. The median WBC of T-B group was 81.92×109/L, which was significantly higher than that of the other two groups (P<0.05). The quantitative results of WT1 were higher than 10-4 in 92.6% of the patients, and the WT1 expression level in myeloid-B group was significantly lower than the other two groups (P<0.01). Among the 9 patients with myeloid-B mixed phenotype, 5 cases showed BCR-ABL positive. Among 28 patients followed up, 21 cases achieved complete remission (CR), the median time to first obtain CR was 32.5(9-75) days, and the median follow-up time was 16 months (range from 21 days to 6 years). The CR rate and median overall survival (OS) time in myeloid-B group were 88.9% and 40 months, which were higher than the other two groups. The CR rate and 3-year OS rate in T-B group were relatively lower (50.0%, 0). WT1 gene is highly expressed in patients with MPAL, and each subgroup of MPAL based on immuophenotype has its unique antigen expression characteristics. Compared with myeloid-T group and T-B group, myeloid-B group can acquire higher remission rate and have better prognosis.

Gene expression analysis and the risk of relapse in favorable histology Wilms’ tumor

ABSTRACT Introduction and Objectives Wilms’ tumor (WT) relapse occurs in 15% of patients. We aim to investigate the association between the expression of several genetic markers and WT relapse risk. Materials and methods The study included 51 children treated for WT at a tertiary center between 2001 and 2019: 23 patients had disease relapse (group A) and 28 remained relapse-free after at least 2 years of follow-up (group B). Patients with syndromic, bilateral synchronous or anaplastic WT were excluded. Autologous renal tissue from 20 patients served as control. Total RNA was isolated from tumor tissue and control. Gene expression levels of WT1, HIF1α, b-FGF, c-MYC and SLC22A18 were assessed using quantitative RT-PCR and normalized to GAPDH. Immunohistochemical staining for WT1 and gene expression levels were compared between the study groups. Results Median patient age was 3 (IQR = 2–5) years and 36 (70.6%) had stage I disease. Baseline characteristics were similar between study groups. Relapse occurred at a median of 6.8 (2.8–24.7) months, predominantly in the lungs (11/23, 47.8%). Tumors that relapsed expressed significantly higher levels of WT1, HIF1α, b-FGF and c-MYC and lower levels of SLC22A18 (p < 0.001). Strong immunohistochemical staining for WT1 was seen in 73.9% of group A and 14.29% of group B (p < 0.001). These associations retained statistical significance irrespective of patient and tumor characteristics. Conclusions Higher expression levels of WT1, HIF1 α, b-FGF and c-MYC and lower level of SLC22A18 are associated with increased risk of WT relapse. These genetic markers can serve as future prognostic predictors and help stratify patients for treatment.

P-793 The effect of Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1) in human ovarian somatic cell development

Abstract Study question Can an inducible NR5A1 (encoding Steroidogenic Factor 1) cell line be used to study the functional role of NR5A1 in human female gonadal differentiation? Summary answer Conditional induction of NR5A1 in a human pluripotent stem cell (hPSC)-based model of gonadal differentiation allows us to study its effect on the differentiating cells. What is known already According to animal studies, NR5A1 is a critical factor in gonadal development. Previous research in our lab showed that CRISPR/Cas9 induced upregulation of NR5A1 during gonadal differentiation in male (XY) hPSCs increases expression of early Sertoli cell markers such as Inhibin A and Anti-Müllerian hormone (AMH) and thus pushes the cells into a more mature gonadal cell type. However, the role of NR5A1 in female gonadal differentiation has yet to be determined. Study design, size, duration The female (XX) human embryonic stem cell (hESC) line, H9, was edited using CRISPR/Cas9 to conditionally induce NR5A1. These cells were subjected to a 10/20-day gonadal differentiation protocol where NR5A1 was induced through addition of Doxycycline (DOX) and Trimethoprim (TMP) from day 4 until day 10/20. Starting from day 4 technical duplicates/triplicates from every second day were subjected to qRT-PCR. Technical triplicates from day 4, 6, 8 and 10 were subjected to bulk RNA-sequencing. Participants/materials, setting, methods The cell line that was used in this project is a female hESC line, H9 (WiCell). A previously established protocol for gonadal differentiation (Sepponen et al., 2017) was followed wherein different cell culture media and matrices were supplemented with small molecules, inhibitors and growth factors. The CRISPR/Cas9 genome-editing system was used to generate the NR5A1 inducible cell line. Cell characterisation was performed using qRT-PCR and bulk RNA sequencing (NovaSeq S4) methods. Main results and the role of chance qRT-PCR results showed that without NR5A1 induction there was almost no expression of this gene, but its expression increased drastically upon addition of DOX and TMP to the culture media. NR5A1 induction seemed to upregulate FOXL2 and WNT4, which are crucial genes for female gonadal differentiation, although the increase was not statistically significant. AMH showed a significant increase on day 8 of differentiation after NR5A1 induction. We also saw an increase in the expression of steroidogenic genes such as STAR, CYP11A1, CYP17A1 and CYP19A1 after NR5A1 induction. Subjecting the cells to NR5A1 induction for an additional 10 days resulted in the upregulation of bipotential gonadal markers GATA4, WT1 and EMX2 after NR5A1 induction. Similarly, the female gonadal markers RSPO1 and WNT4 were upregulated over time and WNT4 expression further increased upon NR5A1 induction. NovaSeq S4 data showed that on day 8, after induction of NR5A1, genes related to reproductive system development and female pregnancy were upregulated. These included DHH, NR5A1, CYP19A1, AR, INHA and NR0B1. On day 10 genes related to reproductive system development such as FOXL2, NR5A1, INHA, LHCGR, and NR0B1 were also upregulated after NR5A1 induction. Limitations, reasons for caution The effect of NR5A1 induction on expression levels of bipotential gonadal markers GATA4, LHX9, WT1, EMX2 and female gonadal markers FOXL2, RSPO1, WNT4 was small in comparison to differentiation without additional NR5A1 induction. Moreover, we lack a protocol to generate bipotential gonadal cells expressing NR5A1 without inducing NR5A1 through CRISPR/Cas9. Wider implications of the findings Taken together the results will provide valuable insight and novel information on the function of NR5A1 in early female gonadal development and more in general on human ovarian development and sex determination. This knowledge will increase our understanding of pathological gonadal development, which often underlies disorders of sex development. Trial registration number Not applicable

The effect of ω-6 fatty acid on WT1 and WIF-1 genes expression and inducing apoptosis in A375 melanoma cell line

BackgroundAn exciting question as to whether nutritional linoleic acid (LA, Omega-6, or ω-6) fatty acid interferes with tumorigenesis is remained to be entirely determined in well experimentally qualified investigations. To elucidate the particular roles of LA in tumorigenesis of melanoma skin cancer, we evaluated the effect of this fatty acid on a melanoma cell line, A375, in terms of tumor growth characteristics by MTT and Flow Cytometry. In particular, we have analyzed the expression levels of two genes (WIF-1 and WT1) involved during Wnt signaling pathway as a molecular base for the formation and progression of many cancer types. MethodsIn order to analyze the cell viability, MTT assay was performed following the treatment of cultured human melanoma cell line, A375, by various concentrations of ω-6 in 24 h, 48 h, and 72 h time intervals, and apoptosis was determined using Flow Cytometry. The expression of WIF-1 and WT1 genes were measured using quantitative real-time PCR, which were further analyzed by Rest 2.0.13 Software. ResultsOur findings revealed that during treatment of cells with all the concentrations of ω-6 in various time intervals, the expression of the WIF-1 gene was significantly (p < 0.05) increased in contrast to decreasing WT1 expression levels in a time- and concentration-dependent manner. The cell viability was also shown to be decreased with an IC50 value of 1000 μM. The differences were more substantial following 72 h of treatment. DiscussionOur results supported the contributory role of ω-6 on the tumor growth of melanoma cells, highlighting the potential of this fatty acid as an influencing element which at least in part exerts its inhibitory roles by interfering with Wnt signaling molecules WIF-1 and WT1.

The relationship between CA125 status and P53, WT1 and clinical features in epithelial ovarian cancer

To investigate the relationship between ER status, P53, WT1, CA125 and clinical characteristics of epithelial ovarian cancer, and the differential diagnosis value of low grade and high grade serous ovarian cancer. 34 patients with epithelial ovarian cancer admitted to our hospital were selected as the research object, and divided into low grade group and high grade group according to the histological grade of ovarian cancer. The immunohistochemical expressions of ER, P53 and WT1 in the two groups were compared and analyzed, and their correlation and clinicopathological parameters were analyzed. The total positive rates of P53 and WT1 in ovarian cancer were 55.88% and 41.18%. The proportion of P53 positive expression in high-grade group was significantly higher than that in low-grade group, while the WT1 positive rate was lower than that in low-grade group, the difference was statistically significant (P &lt; 0.05). The WT1 (+) &amp;P53 (+) group had a higher level of CA125 than the non-expression group, and the difference was statistically significant (P &lt; 0.05). WT1 protein was significantly correlated with pathological stage, degree of differentiation, pathological type and lymph node metastasis of epithelial ovarian cancer (P &lt; 0.05),which could be used as an important prognostic factor for patients. The expression level of P53 is higher in high-grade serous ovarian cancer, while the expression level of WT1 is lower. The use of P53 and WT1 can diagnose high-grade serous ovarian cancer, and WT1 has higher diagnostic value for low-grade serous ovarian cancer. The CA125 status was significantly correlated with the expression of P53 and WT1.

TTP protects against acute liver failure by regulating CCL2 and CCL5 through m6A RNA methylation.

Tristetraprolin (TTP), an important immunosuppressive protein regulating mRNA decay through recognition of the AU-rich elements (AREs) within the 3′-UTRs of mRNAs, participates in the pathogenesis of liver diseases. However, whether TTP regulates mRNA stability through other mechanisms remains poorly understood. Here, we report that TTP was upregulated in acute liver failure (ALF), resulting in decreased mRNA stabilities of CCL2 and CCL5 through promotion of N6-methyladenosine (m6A) mRNA methylation. Overexpression of TTP could markedly ameliorate hepatic injury in vivo. TTP regulated the mRNA stabilization of CCL2 and CCL5. Interestingly, increased m6A methylation in CCL2 and CCL5 mRNAs promoted TTP-mediated RNA destabilization. Moreover, induction of TTP upregulated expression levels of WT1 associated protein, methyltransferase like 14, and YT521-B homology N6-methyladenosine RNA binding protein 2, which encode enzymes regulating m6A methylation, resulting in a global increase of m6A methylation and amelioration of liver injury due to enhanced degradation of CCL2 and CCL5. These findings suggest a potentially novel mechanism by which TTP modulates mRNA stabilities of CCL2 and CCL5 through m6A RNA methylation, which is involved in the pathogenesis of ALF.

Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia

Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients.A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d'Aragona”, Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines.In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment.Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. DisclosuresNo relevant conflicts of interest to declare.

Low-Density Granulocytes Are Decreased in Acute Myeloid Leukemia and in Myelodysplastic Syndromes with Negative Prognostic Factors

Introduction. Myelodysplastic syndromes (MDS), a group of clonal hematological diseases, are characterized by ineffective hematopoiesis, progressive peripheral blood (PB) cytopenia(s), and increased risk of developing acute myeloid leukemia (AML). Classification and risk stratification are constantly under revision for a better estimation of prognosis in those patients. Investigation of immune biomarkers is needed, because immune dysregulation also plays an important role in dysplastic hemopoiesis and immunological escape of neoplastic clones. Here, we studied frequency of low-density granulocytes (LDGs), a neutrophil subset with immunoregulatory functions, in MDS and AML at diagnosis and during treatments.Methods. A total of 17 patients (M/F, 14/12; median age, 69 years old; range, 21-84 years) and seven healthy subjects were enrolled at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d'Aragona”, Salerno, Italy, between October 2020 and July 2021. Patients were diagnosed with AML (N = 7), or MDS (N = 10) according to the 2016 World Health Organization criteria. For immunophenotyping, fresh EDTA whole PB was stained with the ollowing antibodies: CD45; HLA-DR; CD15; CD3; CD56; CD19; CD11b; CD33; CD34; CD14; and CD16 (all from Beckman Coulter, Brea, CA). Acquisition was carried out using a Navios EX flow cytometer, and Navios software v1.3 (Beckman Coulter). Post-acquisition compensation and analysis were performed using FlowJo software (v.10.7.1, Becton Dickinson). LDGs were identified as CD3-CD56-CD19-CD11b+CD33+CD14-CD15+ cells, following previously published gating strategies (Rahman S, et al. Ann Rheum Dis. 2019). Data were analyzed using Prism (GraphPad software, La Jolla, CA). A P < 0.05 was considered statistically significant.Results. Frequencies of circulating LDGs were significantly reduced in AML patients at diagnosis compared to controls (P = 0.0018) and MDS (P = 0.0077) and were slightly decreased compared to AML in complete remission (P = 0.1605). MDS patients were then divided based on Revised International Prognostic Scoring System (IPSS-R), and very-low and low-risk MDS patients displayed significantly higher circulating LDG frequencies compared to AML at diagnosis (P = 0.0083), while no differences were described between AML at baseline and intermediate-risk MDS (P = 0.1103). Subsequently, LDGs were correlated with clinical and phenotypic features by correlation analysis showing significant negative correlations between LDGs and blasts identified by flow cytometry (r = -0.5463; P = 0.0057) but not by cytology (P = 0.1346), between LDGs and lymphocytes (r = -0.4407; P = 0.0311) or flow cytometric normalized blast count (NBC; r = -0.5283; P = 0.0096) as previously defined (Giudice V, et al. Biomedicines. 2021). A slight negative correlation was described between LDGs and WT1 expression levels (r = -0.5369; P = 0.0719), particularly evident in MDS patients (r = -0.9980; P = 0.0402), supporting our previous findings of negative prognostic impact of WT1 expression in MDS and AML. Finally, we investigated CD16 expression on LDGs, because CD16 is essential for neutrophil degranulation. Despite no differences were described between percentage of LDG subsets among patients' groups, various correlations were identified by Pearson analysis. In particular, CD16+ LDGs negatively correlated with blasts (P = 0.0229), while positively correlated with lymphocytes (P = 0.0404) detected by flow cytometry. Conversely, CD16int and CD16- LDGs negatively correlated with lymphocytes (P = 0.0109 and P = 0.0021, respectively) and positively correlated with granulocytes identified by flow cytometry (P = 0.0024 and P = 0.0008, respectively). In addition, CD16int LDGs negatively correlated with blasts detected by flow cytometry (r = -0.65; P = 0.0414).Conclusions. Our preliminary results suggested a possible role of LDGs in prognostic definition of AML and MDS patients especially when combined with other biomarkers, such as WT1 expression levels or NBC. Moreover, our data supported the hypothesis of biological heterogeneity of granulocytes, as LDG subsets variously correlated with lymphocytes and leukemic cells suggesting different roles in suppression or activation of immune responses. However, our findings need further validation in larger cohorts and in in vitro studies. DisclosuresNo relevant conflicts of interest to declare.

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Applicability of the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) to Predict Outcomes of Acute Myeloid Leukemia Patients Older Than 60 Years Undergoing Non-Myeloablative Allogeneic Stem Cell Transplantation

Background: For most acute myeloid leukemia (AML) patients an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of cure and long-term survival. Higher age and/or comorbidities may confine this option in a considerable number of AML patients due to toxic myeloablative preparation regimens. Non-myeloablative conditioning (NMA) regimes were developed to allow HSCT in these patients. The HSCT comorbidity index (HCT-CI) has been shown to predict outcome after allogeneic HSCT in cohorts of unselected patients. However, there is no separate evaluation of its prognostic significance in older (> 60 years) AML patients undergoing less-toxic NMA-HSCT. We evaluated the prognostic impact of the HCT-CI in a large, homogenously treated AML patient cohort that received NMA-HSCT.Methods: We retrospectively analyzed 289 AML patients older than 60 years (median age 66, range 60-77 years) at the time of NMA-HSCT. The conditioning regimen consisted of fludarabine 30 mg/m 2 for 3 consecutive days in addition to 2 (n=278) or 3 Gy (n=6) total body irradiation (TBI), five patients received 2 Gy TBI alone. Graft versus host disease prophylaxis contained cyclosporine and mycophenolate mofetil, none of the patients received in vivo or in vitro T-cell depletion. Disease risk at diagnosis was assessed according to the European LeukemiaNet (ELN) 2017 classification. In patients transplanted in morphologic remission, measurable residual disease (MRD) at HSCT was assessed based on NPM1 mutations and BAALC, MN1, and WT1 expression levels. The pre-transplant HCT-CI was calculated prior to the start of the conditioning regimen. Median follow up after HSCT was 3.8 years.Results: 97% of the patients (n=281) engrafted. 36% of the patients (n=104) had a low risk (0 points), 31% (n=90) an intermediate risk (1-2 points), and 33% (n=95) a high risk (≥3 points) HCT-CI score. The performance score at HSCT (ECOG, 0/1 vs. 2/3) was not different between HCT-CI risk groups (P=.86). Incidences of chronic graft-versus-host disease (GvHD) were 16% limited and 52% extensive disease. The non-relapse mortality (NRM) did not differ significantly between HCT-CI risk groups after NMA-HSCT (P=.56, Figure 1A, at 5 years HCT-CI low 24% vs. HCT-CI intermediate 20% vs. HCT-CI high 27%, respectively). Likewise, neither the cumulative incidence of relapse (P=.88, Figure 1B, at 5 years 46% vs. 45% vs. 43%) nor the overall survival (P=.70, Figure 1C, at 5 years 40% vs. 44% vs. 41%) differed according to the HCT-CI risk groups. The HCT-CI also did not impact outcomes in separate analyses according to ELN2017 risk at diagnosis (OS, P=.20, P=.30, and P=.70 for favorable, intermediate, and adverse, respectively) or MRD status prior to HSCT (OS, P=1 and P=.30 for MRD-negative and MRD-positive patients, respectively). In the favorable risk subgroup of patients being MRD-negative at the time of NMA-HSCT, the median overall survival reached 49% at 5 years after NMA-HSCT, irrespective of the HCT-CI.Conclusion: In the here presented cohort of older AML patients a higher HCT-CI did not have a negative impact on NRM or survival. In general, the NRM following NMA-HSCT was relatively low. Our data indicates that comorbidities per se - reflected by a higher HCT-CI score - should not impede NMA-HSCT. Independently from the HCT-CI score MRD-negative patients had notably good survival of 49 % at 5 years following NMA-HSCT. As the incidences of chronic graft-versus host disease were relatively high, alternative immunosuppressive strategies may help to further improve outcomes. Our data aid in informed clinical decisions regarding HSCT consolidation in older AML patients since we show that HSCT with this reduced toxicity regimen represents a feasible treatment option in older and comorbid AML patients also with higher HCT-CI scores. [Display omitted] DisclosuresBackhaus: Bayer: Other: Current Employment of Family Member. Jentzsch: Astellas: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria. Vucinic: Janssen: Honoraria, Other: Travel Sponsoring; Novartis: Honoraria; Gilead: Honoraria, Other: Travel Sponsoring; Abbvie: Honoraria, Other: Travel Sponsoring; MSD: Honoraria. Franke: BMS: Honoraria; Gilead: Honoraria, Other: Travel Sponsoring; Jazz Pharmaceuticals: Honoraria, Other: Travel Sponsoring; MSD: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Platzbecker: Celgene/BMS: Honoraria; AbbVie: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Geron: Honoraria; Takeda: Honoraria. Schwind: Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

Total Flavones of Abelmoschus manihot Ameliorates Podocyte Pyroptosis and Injury in High Glucose Conditions by Targeting METTL3-Dependent m6A Modification-Mediated NLRP3-Inflammasome Activation and PTEN/PI3K/Akt Signaling.

Background: The total flavones of Abelmoschus manihot (TFA), a compound that is extracted from Abelmoschus manihot, has been widely used in China to reduce podocyte injury in diabetic kidney disease (DKD). However, the mechanisms underlying the therapeutic action of this compound have yet to be elucidated. Podocyte pyroptosis is characterized by activation of the NLRP3 inflammasome and plays an important role in inflammation-mediated diabetic kidneys. Regulation of the PTEN/PI3K/Akt pathway is an effective strategy for improving podocyte damage in DKD. Previous research has also shown that N6-methyladenosine (m6A) modification is involved in DKD and that m6A-modified PTEN regulates the PI3K/Akt pathway. In this study, we investigated whether TFA alleviates podocyte pyroptosis and injury by targeting m6A modification-mediated NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. Methods: We used MPC-5 cells under high glucose (HG) conditions to investigate the key molecules that are involved in podocyte pyroptosis and injury, including activation of the NLRP3 inflammasome and the PTEN/PI3K/Akt pathway. We detected alterations in the levels of three methyltransferases that are involved in m6A modification. We also investigated changes in the levels of these key molecules in podocytes with the overexpression or knockdown of methyltransferase-like (METTL)3. Results: Analysis showed that TFA and MCC950 protected podocytes against HG-induced pyroptosis and injury by reducing the protein expression levels of gasdermin D, interleukin-1β, and interleukin-18, and by increasing the protein expression levels of nephrin, ZO-1, WT1 and podocalyxin. TFA and 740Y-P inhibited activation of the NLRP3 inflammasome via the PI3K/Akt pathway by inhibiting the protein levels of NIMA-related kinase7, NLRP3, ASC, and caspase-1, and by increasing the protein expression levels of p-PI3K and p-Akt. TFA improved pyroptosis and injury in HG-stimulated podocytes by regulating METTL3-dependent m6A modification. Conclusion: Collectively, our data indicated that TFA could ameliorate pyroptosis and injury in podocytes under HG conditions by adjusting METTL3-dependent m6A modification and regulating NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. This study provides a better understanding of how TFA can protect podocytes in DKD.

Pre-Transplant Minimal Residual Disease Evaluation Performed By Molecular WT1-Based or Multicolor Flow Cytometry Assessment Overcomes the Prognostic Value of Cytogenetic and Molecular Risk Factors at Diagnosis in Advanced Acute Myeloid Leukemia Patients

BACKGROUND AND AIMSAllogeneic stem cell transplantation (HSCT) offers the only chance of cure for patients with advanced acute myeloid leukemia (AML). High levels of pre-HSCT minimal residual disease (MRD) have been reported to predict relapse risk after HSCT even in patient transplanted in first complete remission (CR). WT1 expression levels and multicolor flow cytometry (MFC) are the most common tools to evaluate MRD. The aim of this study was to analyze the role of pre-HSCT combined MRD assessment as predictor for the post-transplant relapse risk in patient transplanted beyond first CR.MATERIALS AND METHODSWe retrospectively analyzed the outcome of 92 consecutive AML patients receiving HSCT in 2nd or 3rd CR. Pre-HSCT marrow samples were analyzed for WT1 expression and MFC as MRD evaluation. Median age at transplant was 45 years. Disease phase was CR2 in 63 (68%) and CR3 in 29 patients (32%). ELN risk category at diagnosis was low in 28 (30%), intermediate in 44 (48%) and high in 20 (22%) patients. Sixty-six (71%) patients received myeloablative conditioning, whereas 26 patients (29%) received reduced intensity conditioning. Stem cell source was bone marrow in most cases and donor was an HLA-identical sibling in 18 (20%), haploidentical sibling (HAPLO) in 24 (26%) and unrelated donor in 50 (54%). Median follow-up was 64 months (95% CI 39.8 - 88.2 months).A positive MFC MRD was defined by the presence of at least 1x10-3 residual leukemic cells by 4 or 8 (since 2011) color flow-cytometry. WT1 copy number/Abl copy number 250x104 was used as cut-off value for abnormal WT1 expression.RESULTSRelapse occurred in 30 patients (33%) and two years non-relapse mortality was 29%. Three-year estimate of OS was 47.9% (median 19 months). The survival probability was significantly affected by donor source (better for HAPLO, p<0.05), ELN risk at diagnosis (better for ELN low risk, p<0.01), MRD status before HSCT measured with any method (p <0.01 for WT1-based MRD, p<0.03 for MFC based MRD) and CR status at HSCT (better for CR2, p<0.05).Specifically, among MRD negative patient a relatively low rate of relapse was observed regardless of risk at diagnosis (2-years OS of 62.2% and 52.7% among MFC MRD negative patient with ELN risk low or intermediate/high, respectively, Fig.1).The predictive value of MRD resulted independent from all other analyzed variables. However, patients with positive MRD did slightly better when undergoing HAPLO HSCT. Multivariate OS analysis revealed that the MRD evaluation by any method was the only independent predictor of OS (p <0.05 for both methods of analysis).MRD evaluation was also a strong predictor of cumulative incidence (CI) of relapse in competitive risk analysis (p<0.01 and p<0.03, respectively, for WT1 and MFC MRD). Multivariate CI of relapse analysis showed that donor source and MRD evaluation significantly influenced relapse risk (p <0.05 and <0.01, respectively).CONCLUSIONSPre transplant MRD evaluation by both WT1 and MFCon bone marrow samples is a reliable predictor of relapse risk which may overcome the prediction based on ELN risk at diagnosis. Both methods were able to efficiently predict post-transplant disease reoccurrence risk: patients with negative pre-HSCT MRD markers have a significantly better OS, while patients with positive MRD markers show an higher risk of relapse, irrespective of lower or higher ELN risk at diagnosis.Furthermore, our results seem to suggest that HAPLO donor may have a positive impact in reducing relapse rate in advanced AML patients, in particular in patients with positive MRD.Refining the baseline risk assessment based on ELN risk in patient undergoing HSCT beyond CR1 who have an higher risk of relapse is very important when evaluating optimal stem cell source and conditioning intensity and could allow to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches. [Display omitted] DisclosuresAngelucci:Roche: Other: Advisory board: biosimilars; Novartis Oncology: Other: Protocol Telesto: sterring committee Chair; Bluebird Bio: Other: Advisory board: Gene therapy in Thalassemia; Jazz: Other: Advisory board: AML; Celgene: Other: protocoll ACE 536 B-Thal 001: DMC Chair; Novartis Oncology: Other: Advisory board: iron toxicity; Novartis Oncology Swiss: Other: Invited speakers sponsored satellite meeting during ; Celgene: Honoraria, Other: Advisory: research project .