BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

Allogeneic stem cell transplantation for mantle cell lymphoma-update of the prospective trials of the East German Study Group Hematology/Oncology (OSHO#60 and #74).

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Implementation of Palliative Care in Clinical Practice in German Units for Allogeneic Stem Cell Transplantation: A Nationwide Survey.

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for otherwise fatal diseases, however it is associated with a considerable morbidity and mortality. In consequence, it can be assumed that a considerable percentage of patients would benefit from high-quality palliative care (PC) during their course of disease. To assess the standard of PC in German transplant centers, a questionnaire was sent out to all German centers recognized from the EBMT membership list and the annually ZKRD report (n = 52). The response rate was not as high as expected with n = 27 (51,9%), even after reminding by phone calls or by e-mails. In brief, palliative care after allogeneic stem cell transplantation shows a wide variation in Germany. This is true for structures, processes and measures. A national standard for SCT-patients has not been established so far and there are no pre-conditions concerning palliative care after alloSCT for a certification by the EBMT according the JACIE standards. There is a considerable need for a crosslinking of alloSCT with PC. Clear standards should be established by the scientific societies concerning personnel, structure and processes.

Targeting IL-11 in the treatment of BK virus-associated haemorrhagic cystitis-A promising new approach.

The BK polyomavirus (BKPyV) has pathogenic relevance especially in immunocompromised patients. No causal therapy has been established yet. Therefore, new therapeutic targets need to be identified in experimental studies. A 3D organotypic cell culture model with primary urothelial cells and fibroblasts was used as infection model. The detection of virus replication was performed with quantitative polymerase chain reaction (qPCR), and immunohistochemistry (IHC) was also used for analysis. Interleukin levels were measured by enzyme-linked immunosorbent assay (ELISA). Interestingly, the signal transducer and activator of transcription 3 (STAT3) pathway seems to be activated during infection with BKPyV, for example phosphorylated STAT3 is significantly (P < 0.0001) elevated on day 6 following infection. Therefore, we performed ELISAs for involved interleukins in STAT3 pathway. Interleukin 11 (IL-11) was significantly (P = 0.026) elevated at day 9. Subsequently, 3D cultures were treated with IL-11 neutralizing antibody. At day 9 following infection, the median virus replication rate is 4.4 × 106 copies/ml. The difference to replication rate without treatment was significantly lower at day 6 (P < 0.0001) and at day 9 (P < 0.0001), respectively. STAT3 pathways seem to be involved during BKPyV infection and need further investigation in experimental studies. A very promising target for treatment might be IL-11.

Allogeneic transplantation in multiple myeloma: long-term follow-up and cytogenetic subgroup analysis.

This phase 3 trial compared tandem autologous stem cell transplantation (autoSCT) versus autoSCT followed by reduced-intensity conditioning allogeneic stem cell transplantation (auto/alloSCT) in patients with newly diagnosed multiple myeloma (MM) with deletion of (del) chromosome 13q (del13q). The availability/absence of a human leukocyte antigen-matched-related or matched-unrelated donor (MUD) determined the nature of the second SCT. The primary endpoint was progression-free survival (PFS) in the intention-to-treat population (n = 199). Auto/alloSCT was performed in 126 patients; 74 received MUD allografts. After 91 months median follow-up, median PFS with auto/allo versus tandem autoSCT was 34.5 versus 21.8 months (P = 0.003; adjusted hazard ratio 0.55, 95% confidence interval 0.36-0.84). Median overall survival (OS) was 70.2 versus 71.8 months (P = 0.856). Two-year non-relapse mortality with auto/allo versus tandem autoSCT was 14.3% versus 4.1% (P = 0.008). In patients harboring both del13q and del17p, median PFS and OS were 37.5 and 61.5 months with auto/allo (n = 19) versus 6.1 and 23.4 months with tandem autoSCT (n = 6) (P = 0.0002 and 0.032). Our findings suggest that auto/alloSCT significantly extends PFS versus tandem autoSCT in del13q MM, and indicate some survival benefit for first-line alloSCT in high-risk MM.

Ruxolitinib for Therapy of Graft-versus-Host Disease.

OBJECTIVE: Steroid-resistant graft-versus-host disease (GvHD) is a major challenge after allogeneic stem cell transplantation and associated with significant morbidity and mortality. There is no therapeutic standard defined beyond calcineurin inhibitors (CNI) and steroids. Furthermore, some patients may have contraindications against CNI or high-dose steroids. Efficacy of ruxolitinib against GvHD has been described recently. METHODS: Ruxolitinib was used for treatment of acute or chronic GvHD in eight patients. The patients either needed intensification of therapy or had contraindications against use of CNI or high-dose steroids. RESULTS: Supplementation of therapy in acute GvHD with severe diarrhea with ruxolitinib was unsuccessful. All these patients died from acute GvHD. Introduction of ruxolitinib into therapy and relapse prophylaxis in other patients was successful in 4/4 cases (CR=3, PR=1). Indications for ruxolitinib were contraindications against CNI due to aHUS in two cases and the need for steroid sparing in two other cases. None of these patients suffered from diarrhea at the initiation of ruxolitinib. CONCLUSION: Ruxolitinib was effective for therapy of acute and chronic GvHD in higher lines in patients without severe diarrhea. Ruxolitinib could replace successfully CNI and high-dose steroids. Further investigations are necessary to define the position of ruxolitinib in GvHD-therapy.

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line.

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.

Recovery from hypogonadism and male health in adult allogeneic stem cell transplantation.

OBJECTIVE: There is a substantial lack of data about men`s health in adult allogeneic stem cell transplantation. METHODS: We conducted prospective unicentric non-interventional clinical study on men's health with a follow-up time of 1 year. RESULTS: Between 11/2013 and 12/2015, we were able to include 27 patients. AML was the most frequent underlying disease (25.9%), and we mainly used intermediate intense conditioning protocols (77.8%). Erectile dysfunction, loss of libido, and loss of efficiency were the most frequent symptoms of hypogonadism. At inclusion of the study, hypogonadism was already frequent. Primary hypogonadism was found in eight cases (29.6%) and secondary hypogonadism in one case (3.7%). We did not observe hypogonadism 6 months after inpatient treatment anymore, but there might still be the impairment of fertility because of still rising FSH levels at the end of the observation period. There were no significant associations of hypogonadism with myeloablative conditioning or kind of donor. Interestingly, there is a significant association with nicotine abuse (P = .049). CONCLUSIONS: On the whole, male hypogonadism was found in one-third of the patients who underwent allogeneic stem cell transplantation.

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

Depletion of the Third Complement Component Ameliorates Age-Dependent Oxidative Stress and Positively Modulates Autophagic Activity in Aged Retinas in a Mouse Model.

The aim of the study was to investigate the influence of complement component C3 global depletion on the biological structure and function of the aged retina. In vivo morphology (OCT), electrophysiological function (ERG), and the expression of selected oxidative stress-, apoptosis-, and autophagy-related proteins were assessed in retinas of 12-month-old C3-deficient and WT mice. Moreover, global gene expression in retinas was analyzed by RNA arrays. We found that the absence of active C3 was associated with (1) alleviation of the age-dependent decrease in retinal thickness and gradual deterioration of retinal bioelectrical function, (2) significantly higher levels of antioxidant enzymes (catalase and glutathione reductase) and the antiapoptotic survivin and Mcl-1/Bak dimer, (3) lower expression of the cellular oxidative stress marker-4HNE-and decreased activity of proapoptotic caspase-3, (4) ameliorated retinal autophagic activity with localization of ubiquitinated protein conjugates commonly along the retinal pigment epithelium (RPE) layer, and (5) significantly increased expression of several gene sets associated with maintenance of the physiological functions of the neural retina. Our findings shed light on mechanisms of age-related retinal alterations by identifying C3 as a potential therapeutic target for retinal aging.

High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization.

Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 µm thickness, and interconnected pore size of 150 µm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017.

Genetic rearrangements result in altered gene expression and novel fusion transcripts in Sézary syndrome.

Sézary syndrome (SS) is an aggressive, leukemic cutaneous T-cell lymphoma variant. Molecular pathogenesis of SS is still unclear despite many studies on genetic alterations, gene expression and epigenetic regulations. Through whole genome and transcriptome next generation sequencing nine Sézary syndrome patients were analyzed in terms of copy number variations and rearrangements affecting gene expression. Recurrent copy number variations were detected within 8q (MYC, TOX), 17p (TP53, NCOR1), 10q (PTEN, FAS), 2p (DNMT3A), 11q (USP28), 9p (CAAP1), but no recurrent rearrangements were identified. However, expression of five genes involved in rearrangements (TMEM244, EHD1, MTMR2, RNF123 and TOX) was altered in all patients. Fifteen rearrangements detected in Sézary syndrome patients and SeAx resulted in an expression of new fusion transcripts, nine of them were in frame (EHD1-CAPN12, TMEM66-BAIAP2, MBD4-PTPRC, PTPRC-CPN2, MYB-MBNL1, TFG-GPR128, MAP4K3-FIGLA, DCP1A-CCL27, MBNL1-KIAA2018) and five resulted in ectopic expression of fragments of genes not expressed in normal T-cells (BAIAP2, CPN2, GPR128, CAPN12, FIGLA). Our results not only underscored the genomic complexity of the Sézary cancer cell genome but also showed an unpreceded large variety of novel gene rearrangements resulting in fusions transcripts and ectopically expressed genes.

Hypermethylation of FOXP3 Promoter and Premature Aging of the Immune System in Female Patients with Panic Disorder?

Immunological abnormalities associated with pathological conditions, such as higher infection rates, inflammatory diseases, cancer or cardiovascular events are common in patients with panic disorder. In the present study, T cell receptor excision circles (TRECs), Forkhead-Box-Protein P3 gene (FOXP3) methylation of regulatory T cells (Tregs) and relative telomere lengths (RTLs) were investigated in a total and subsamples of 131 patients with panic disorder as compared to 131 age- and sex-matched healthy controls in order to test for a potential dysfunction and premature aging of the immune system in anxiety disorders. Significantly lower TRECs (p = 0.004) as well as significant hypermethylation of the FOXP3 promoter region (p = 0.005) were observed in female (but not in male) patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and older persons within the patient group. The presently observed reduced TRECs in panic disorder patients and FOXP3 hypermethylation in female patients with panic disorder potentially reflect impaired thymus and immunosuppressive Treg function, which might partly account for the known increased morbidity and mortality of anxiety disorders conferred by e.g. cancer and cardiovascular disorders.

In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.

Hypothermic, initially oxygen-free, controlled limb reperfusion for acute limb ischemia.

BACKGROUND: Controlled limb reperfusion has been shown to prevent the deleterious effects of ischemia-reperfusion (IR) syndrome following revascularization of acute limb ischemia (ALI). To reduce the production of cell-toxic oxygen-free radicals, we have established a new initially oxygen-free, hypothermic, heparin-coated perfusion and hemofiltration system and report on our first results. METHODS: In a retrospective single-center study, controlled limb reperfusion was applied in 36 patients (64.7 ± 15 years) with ALI of category IIA to III (33.7 ± 20.7 hr ischemic time). 52.8% had central (aortic and bifurcation) and 47.2% had peripheral (common iliac artery and distal) vascular occlusions. The common femoral artery and vein were cannulated, and a hypothermic (22°C), initially oxygen-free, potassium-free ringer's solution was perfused using a heparin-coated extracorporeal membrane oxygenation (ECMO) and hemofiltration system with low-dose heparinization. Thirty-day mortality, clinical recovery of neurological dysfunction, limb amputation, and fasciotomy rate were analyzed. Laboratory parameters associated with ischemia and IR injury were determined. RESULTS: Average perfusion time was 94 ± 35 min. Thirty-day mortality was 27.8%. 55.5% of patients showed complete recovery of motor and sensory dysfunction. A total of 27.8% of patients developed a compartment syndrome and required fasciotomy. Lower leg amputation was necessary in 11.1% of patients. Lactate levels were reduced in ischemic limbs by 25.3% within 60 min (P < 0.05). Preoperative negative base excess of -1.96 ± 0.96 mmol/L was equalized after 12 hr (P < 0.05), while pH stayed balanced at 7.4. Serum potassium stayed within normal limits throughout 24 hr, and therefore systemic hyperkalemia was prevented and imminent metabolic acidosis was corrected. CONCLUSIONS: An initially oxygen-free, hypothermic, heparin-coated ECMO counteracts local and systemic effects of IR injury. Reduced mortality and morbidity might result from this new treatment, although this could not be conclusively proven in our study. A prospective, randomized controlled trial is needed to prove superiority of this new concept.

Characteristics of A20 gene polymorphisms in T-cell acute lymphocytic leukemia.

A20 is a repressor of NF-κB and was recently shown to be frequently inactivated by deletions or mutations in several types of lymphomas including T-cell lymphoma. Little is known about the characteristics of A20 mutations in T-cell acute lymphoblastic leukemia (T-ALL). In this study, we analyzed A20 polymorphisms and characterized their features in 11 cases with T-ALL, 30 samples from healthy Chinese individuals, and 3 cells lines including CCRF-CEM, Molt-4, and Toledo cells. Two frequent A20 polymorphisms were found: a CCT deletion at position 12384 and a nucleotide exchange (A to C) at position 13751 (rs2307859 and rs661561). The homozygous form (CC) of rs661561 was detected in all 10 cases with detectable T-ALL, while only 80% (24/30) of the healthy controls had this genotype. We found one T-ALL case without the above frequent single-nucleotide polymorphisms (SNPs) in which a T to G mutation at position 12486 was found, which results in an amino acid exchange (Phe127Cys; rs2230926). Similar results were found in Molt-4 cells, which lack the frequent SNPs but have a heterozygous polymorphism at position 13749 (C > T) (rs5029948). Interestingly, the T-ALL case with the Phe127Cys mutation and Molt-4 cells demonstrated a high A20 copy number as measured by real-time polymerase chain reaction amplification with three primer sets that cover different regions of the A20 gene, corresponding to a high A20 and low NF-κB expression level. In conclusion, we characterized the features of A20 polymorphisms in T-ALL, and found that a low frequency A20 mutation, which was thought to be involved in malignant T-ALL development, might function differently in T cell lymphomas.