PU.1 has previously been shown to be down-regulated in classical Hodgkin lymphoma (cHL) cells via promoter methylation. We performed bisulfite sequencing and proved that the promoter region and the ...−17 kb upstream regulatory element of the PU.1 gene were highly methylated. To evaluate whether down-regulation of PU.1 is essential for the growth of cHL cells, we conditionally expressed PU.1 in 2 cHL cell lines, L428 and KM-H2. Overexpression of PU.1 induced complete growth arrest and apoptosis in both cell lines. Furthermore, in a Hodgkin lymphoma tumor xenograft model using L428 and KM-H2 cell lines, overexpression of PU.1 led to tumor regression or stable disease. Lentiviral transduction of PU.1 into primary cHL cells also induced apoptosis. DNA microarray analysis revealed that among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428 cells after PU.1 induction. Stable knockdown of p21 rescued PU.1-induced growth arrest in L428 cells, suggesting that the growth arrest and apoptosis observed are at least partially dependent on p21 up-regulation. These data strongly suggest that PU.1 is a potent tumor suppressor in cHL and that induction of PU.1 with demethylation agents and/or histone deacetylase inhibitors is worth exploring as a possible therapeutic option for patients with cHL.
•PU.1 is a potent tumor suppressor in cHL cells and the induction of PU.1 is a possible therapeutic option for patients with cHL.
Zygomycosis is a lethal and invasive mold infection that is often associated with hematological malignancies. The keys for successful treatment include making a rapid diagnosis and appropriately ...administering antifungal agents. We herein report the early diagnosis of a case of zygomycosis in a patient with acute myeloid leukemia using a deoxyribonucleic acid sequence analysis. We successfully performed allogeneic hematopoietic stem cell transplantation with the use of high-dose liposomal amphotericin B and granulocyte transfusion.
A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors ...capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34(+)Sca-1(+)c-Kit(+) MPP fraction. The GATA-1(+) MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1(+) MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1(+) and PU.1(+) MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.
Summary
Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type‐I (HTLV‐I)‐infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine ...designed to augment an HTLV‐I Tax‐specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti‐ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate‐ to high‐risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax‐specific CTL responses were observed with peaks at 16–20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide‐pulsed DC vaccine is a safe and promising immunotherapy for ATL.
This prospective clinical study aimed to determine the efficacy and prognostic factors of adoptive activated αβT lymphocyte immunotherapy for various refractory cancers. The primary endpoint was ...overall survival (OS), and the secondary endpoint was radiological response.
The authors treated 96 patients. Activated αβT lymphocytes were infused every 2 weeks for a total of six times. Prognostic factors were identified by analyzing clinical and laboratory data obtained before therapy.
Median survival time (MST) was 150 days (95% confidence interval, 105–191), and approximately 20% of patients achieved disease control (complete response + partial response + stable disease). According to the multivariate Cox proportional hazards model with Akaike information criterion–best subset selection, sex, concurrent therapy, neutrophil/lymphocyte ratio, albumin, lactate dehydrogenase, CD4:CD8 ratio and T helper (Th)1:Th2 ratio were strong prognostic factors. Using parameter estimates of the Cox analysis, the authors developed a response scoring system. The authors then determined the threshold of the response score between responders and non-responders. This threshold was able to significantly differentiate OS of responders from that of non-responders. MST of responders was longer than that of non-responders (317.5 days versus 74 days). The validity of this response scoring system was then confirmed by internal validation.
Adoptive activated αβT lymphocyte immunotherapy has clinical efficacy in certain patients. The authors’ scoring system is the first prognostic model reported for this therapy, and it is useful for selecting patients who might obtain a better prognosis through this modality.
We describe 2 allogeneic stem cell transplantation patients who developed chronic graft-versus-host disease (GVHD) after dermatomal varicella-zoster virus (VZV) infection. Localized zoster did not ...respond to oral valaciclovir but did resolve with intravenous aciclovir. However, skin eruptions, eye/oral dryness, and liver dysfunction were observed at the healing stage of localized zoster, suggesting development of GVHD. Intensification of immunosuppressive therapy was required to control GVHD. Quantitative real-time PCR for VZV DNA was used to distinguish liver involvement by chronic GVHD from visceral dissemination of VZV in 1 patient. VZV infection may trigger chronic GVHD after allogeneic stem cell transplantation.
Background: Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Although most patients respond to initial ...therapy, relapse is inevitable and the overall prognosis is dismal. Therefore, we developed a novel immune therapy consisting of autologous dendritic cells (DC) pulsed with Tax peptides (ATL-DC-101) to prevent relapse. The used peptides are corresponding to the major epitopes of HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) that potentially act as anti-ATL effectors. Mogamulizumab is an anti-CCR4 antibody, which binds to ATL cells and regulatory T cells. A preceding pilot study suggested the safety of ATL-DC-101 monotherapy, and then we conducted a phase Ia/Ib study to investigate safety and efficacy of ATL-DC-101 combined with mogamulizumab.
Methods: ATL patients pretreated and stable for at least 4 weeks were enrolled. Peripheral blood mononuclear cells were obtained by apheresis and differentiated into DC, which was pulsed with Tax-peptides. The study consisted of 2 cohorts, i.e., Ia and Ib phase, in which patients were assigned ATL-DC-101 monotherapy and ATL-DC-101 combined with mogamulizumab, respectively. The protocol consisted of three subcutaneous injections of ATL-DC-101 at 2-week intervals. Mogamulizumab was administered once before ATL-DC-101 in Ib phase. The primary endpoint of the study was to evaluate safety and feasibility of ATL-DC-101. The secondary endpoints were anti-tumor effects, progression-free survival (PFS), overall survival (OS), and time to next treatment, estimated by Kaplan-Meier method. The safety and disease status were accessed at day 56 and followed until 2 years.
Results: Six aggressive ATL patients who were in stable condition by previous therapies were enrolled in this study from 2015 to 2016. Frequent DC- related toxicities were mild injection site reaction (n=5, 83.3%), decreased WBC count (n=3, 50%), and liver damage (n=2, 33.3%), all of which were grade 1 or 2. Notably, 2 patients (1 in each cohort) with partial response (PR) after chemotherapy were converted into complete response (CR) after ATL-DC-101. The 2-year PFS and OS were both 83.3%. All three patients who received ATL-DC-101 monotherapy (Ia) maintained CR for more than two years. Although one patient in phase Ib developed progressive disease after one year, the other two patients remained in CR. Interestingly ATL cells in a relapsed case harbored deletion of Tax gene, which resulted in the loss of the target peptide presentation. After a median follow-up of 29 months, 5/6 patients are alive without any additional chemotherapy after ATL-DC-101.
Conclusions: ATL-DC-101 was well tolerated and successfully maintained CR more than 2 years in 5/6 patients irrespective of additional mogamulizumab, indicating that this therapy could be a safe and effective long-lasting maintenance therapy even for elderly ATL patients. Currently, we are preparing Phase II trial to confirm anti-tumor effects of ATL-DC-101 monotherapy.
Shiratsuchi:Kyowa Hakko Kirin Co.Ltd: Research Funding; Chugai Pharmaceutical Co.Ltd: Research Funding; Daiichi Sankyo Co.Ltd: Research Funding. Fukuda:Chugai Pharmaceutical: Speakers Bureau. Ishida:Kyowa Hakko Kirin Co.Ltd: Honoraria, Research Funding; Celgene K.K: Honoraria, Research Funding; Bayer AG: Research Funding; Mundiparma K: Honoraria. Akashi:Ono Pharmaceutical: Research Funding; MSD: Research Funding; Taiho Pharmaceutical: Research Funding; sanofi: Research Funding; Celgene: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding; Novartis pharma: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Astellas Pharma: Research Funding; Asahi-kasei: Research Funding; Chugai Pharma: Research Funding. Matsuoka:Bristol Myers Squibb: Research Funding. Suehiro:Kyowa Hakko Kirin: Research Funding; Ono Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding.
Little is known about the appropriate management of transplant-eligible multiple myeloma (MM) patients primarily refractory to bortezomib (BOR), lenalidomide (LEN) and dexamethasone (DEX) that are ...frequently used as a standard therapy. We treated three patients with MM primarily refractory to BOR and LEN with carfilzomib (CFZ)/LEN/DEX as a re-induction therapy and high dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT). They achieved remission and CFZ/LEN/DEX followed by CFZ/DEX has been subsequently used as a maintenance therapy. They have been progression free for more than forty months without serious adverse events. Regimens including re-introduction and a long-term maintenance of CFZ with PBSCT may be appropriate for patients with BOR- and LEN- refractory transplant-eligible MM.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is caused by natural killer (NK) cells upon recognition of antigen-bound IgG
FcγRIIIa. This mechanism is crucial for cytolysis of ...pathogen-infected cells and monoclonal antibody (mAb)-mediated elimination of cancer cells. However, there is concern that mAb-based cancer therapy induces ADCC against non-target cells expressing antigens. To date, no strategy has been reported to enhance the selectivity of ADCC to protect non-target cells expressing antigens. Here, we introduce a model inhibitor which specifically blocks ADCC of anti-EGFR mAbs towards EGFR/folate receptor α (FRα) double positive cells. This inhibitor recruits mAbs on the FRα of the cell surface independent of Fab antigen recognition. The resulting ternary and/or quaternary complexes formed on the cell surface suppress signal transduction of FcγRIIIa in NK cells, consequently leading to more specific ADCC.
The epigenetic regulator dioxygenase Ten-eleven translocation 3 (TET3) catalyzes the oxidation of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and plays an important role in histone ...modification, thereby regulating the gene expression. Epigenetic aberrations play a key role in the pathophysiology of acute myeloid leukemia (AML), however the reasons for the highly distorted epigenome in AML are poorly known. Though in particular TET2 is widely studied in normal and malignant hematopoiesis, the role of TET3 in human myeloid leukemia is still unknown. First we quantified the expression of TET3 in normal murine and human hematopoiesis by quantitative real-time (qRT)-PCR. Among the Tet genes Tet3 showed highest expression in murine hematopoietic stem cells (HSCs) (2-fold and 3-fold compared to Tet1 and Tet2 respectively, p=0.01, p=0.006, n=3). Within the hematopoietic hierarchy highest expression of Tet3 was found in HSCs and lymphoid committed progenitors (CLPs) compared to committed myeloid progenitors (CMPs) (up to ~3.5-fold, p=0.003, n=3). In humans TET3 was higher expressed in CD34+ bone marrow (BM) cells compared to total BM cells (p=0.024, n=3) and within the lineage positive compartment the TET3 was higher expressed in CD33+ myeloid cells compared to lymphoid cells (CD19+ B cells, p=0.008 and CD3+ T cells, p=0.02, n=3). Our qRT-PCR expression data were in agreement with published microarray data demonstrating that TET3 is significantly higher expressed in myeloid cells compared to lymphoid cells (p<0.001, n=5) (Bagger FO et. al., Nucleic Acids Res. 2013). In AML patients TET3 showed a broad range of expression (ΔCT: -1.4 ± 1.4 SD, n=80), but was aberrantly expressed in majority of the AML patients compared to normal total BM (n=3) and CD33+ BM myeloid cells (n=3) (60% and 81% respectively, cut-off for higher expression: 1.5-fold SD of the median). TET3 was higher expressed in 30% of the cases in cytogenetically normal (CN)-AML (n=50) and in 95% of cases in PML-RARa+ AML (n=21) compared to total BM and CD33+ myeloid cells. The AML cell lines OCI-AML3 (NPM1 mutation+), NB4 (PML-RARa+)and KASUMI-1 (AML1-ETO+) showed higher expression of TET3 compared to other AML cell lines (EOL1, Molm 13, MONO-MAC-6 and THP-1) (p<0.001, n=3). shRNA mediated knockdown (KD) of TET3 in human AML cell lines (OCI-AML3, NB4 and KASUMI-1) adversely affected their cell growth (45%-80% reduction compared to scrambled (scr) control, p<0.001, n=3) and clonogenicity (57%-76% reduction, n=3) in vitro. Furthermore, TET3 KD significantly reduced the leukemic engraftment (median 32±17 and 5.6±6 for scr and shRNA respectively) of NB4 and OCI-AML3 in NSG mice (shRNA n=12; scr n=11, p<0.001). Histopathology showed absence or reduced infiltration of leukemic blast cells in different organs of mice transplanted with shRNA expressing AML cells. In contrast, shRNA mediated KD of TET3 in human normal CD34+ cord blood (CB) cells did not impact cell growth in vitro (n=3). TET3 depletion decreased total 5hmC level in the AML cell line NB4 and in parallel changed expression of genes involved in regulation of cell proliferation, MAP kinase activity and cell migration as determined by microarray gene expression analysis (n=3). Constitutive overexpression of wild-type TET3 in human AML cell lines significantly further enhanced their clonogenicity compared to empty vector control (p<0.05, n=3). Interestingly, overexpression of wild-type TET3 or the TET3-CDconstruct containing only the catalytic domain (CD) inhibited cell growth of human CD34+ CB cells and impaired myeloid differentiation in the CFC assays compared to empty vector (EV) (number of colonies EV=68± 5.7SD, TET3=23±1, TET3-CD=36±10, p<0.001, n=3) whereas erythroid colony formation was not affected. These results were also confirmed by flow cytometry with a decrease in the number of CD11b+CD14+ myeloid cells in the colonies (>70% reduction, n=3). In conclusion, our data indicates that ordered expression levels of TET3 are necessary for normal human HSC proliferation and differentiation. The growth and engraftment potential of AML cells are depending on high TET3 expression levels.
Akashi:Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Celgene: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy. Buske:Celltrion, Inc.: Consultancy, Honoraria.
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