The genetic spectrum of a Chinese series of patients with 46, XY disorders of the sex development.

PURPOSE: The etiology of 46, XY disorders of sex development (46, XY DSD) is complex, and studies have shown that different series of patients with 46, XY DSD has different genetic spectrum. In this study, we aimed to investigate the underlying genetic etiology in a Chinese series of patients with 46, XY DSD by whole exome sequencing (WES). METHODS: Seventy patients with 46, XY DSD were enrolled from the Peking Union Medical College Hospital (Beijing, China). The detailed clinical characteristics were evaluated, and peripheral blood was collected for WES to find the patients' rare variants (RVs) of genes related to 46, XY DSD. The clinical significance of the RVs was annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: A total of 57 RVs from nine genes were identified in 56 patients with 46, XY DSD, which include 21 novel RVs and 36 recurrent RVs. Based on the American ACMG guidelines, 43 variants were classified as pathogenic(P) or likely pathogenic (LP) variants and 14 variants were defined as variants of uncertain significance (VUS). P or LP variants were identified in 64.3% (45/70) patients of the series. Thirty-nine, 14, and 4 RVs were involved in the process of androgen synthesis and action, testicular determination and developmental process, and syndromic 46, XY DSD, respectively. The top three genes most frequently affected to cause 46, XY DSD were AR, SRD5A2, and NR5A1. Seven patients were found harboring RVs of the 46, XY DSD pathogenic genes identified in recent years, namely DHX37 in four patients, MYRF in two patients, and PPP2R3C in one patient. CONCLUSION: We identified 21 novel RVs of nine genes, which extended the genetic spectrum of 46, XY DSD pathogenic variants. Our study showed that 60% of the patients were caused by AR, SRD5A2 or NR5A1 P/LP variants. Therefore, polymerase chain reaction (PCR) amplification and Sanger sequencing of these three genes could be performed first to identify the pathogeny of the patients. For those patients whose pathogenic variants had not been found, whole-exome sequencing could be helpful in determining the etiology.

Identification of immune subtypes of Ph-neg B-ALL with ferroptosis related genes and the potential implementation of Sorafenib.

BACKGROUND: The clinical outcome of Philadelphia chromosome-negative B cell acute lymphoblastic leukemia (Ph-neg B-ALL) varies considerably from one person to another after clinical treatment due to lack of targeted therapies and leukemia's heterogeneity. Ferroptosis is a recently discovered programmed cell death strongly correlated with cancers. Nevertheless, few related studies have reported its significance in acute lymphoblastic leukemia. METHODS: Herein, we collected clinical data of 80 Ph-neg B-ALL patients diagnosed in our center and performed RNA-seq with their initial bone marrow fluid samples. Throughout unsupervised machine learning K-means clustering with 24 ferroptosis related genes (FRGs), the clustered patients were parted into three variant risk groups and were performed with bioinformatics analysis. RESULTS: As a result, we discovered significant heterogeneity of both immune microenvironment and genomic variance. Furthermore, the immune check point inhibitors response and potential implementation of Sorafenib in Ph-neg B-ALL was also analyzed in our cohort. Lastly, one prognostic model based on 8 FRGs was developed to evaluate the risk of Ph-neg B-ALL patients. CONCLUSION: Jointly, our study proved the crucial role of ferroptosis in Ph-neg B-ALL and Sorafenib is likely to improve the survival of high-risk Ph-neg B-ALL patients.

[Effect and Involved Mechanism of RSL3-induced Ferroptosis in Acute Leukemia Cells MOLM13 and Drug-resistant Cell Lines].

OBJECTIVE: To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells. METHODS: After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway. RESULTS: RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients. CONCLUSION: RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.

CD19 CAR-T cell treatment conferred sustained remission in B-ALL patients with minimal residual disease.

The persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor T (CD19 CAR-T) cells have shown promising responses in B-ALL. However, their role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T cells in MRD-positive B-ALL patients. From January 2018, a total of 14 MRD-positive B-ALL patients received one or more infusions of autogenous CD19 CAR-T cells. Among them, 12 patients achieved MRD-negative remission after one cycle of CAR-T infusion. At a median follow-up time of 647 days (range 172-945 days), the 2-year event-free survival rate in MRD-positive patients was 61.2% ± 14.0% and the 2-year overall survival was 78.6 ± 11.0%, which were significantly higher than patients with active disease (blasts ≥ 5% or with extramedullary disease). Moreover, patients with MRD had a lower grade of cytokine release syndrome (CRS) than patients with active disease. However, the peak expansion of CAR-T cells in MRD positive patients showed no statistical difference compared to patients with active disease. Five patients received two or more CAR-T cell infusions and these patients showed a decreased peak expansion of CAR-T cell in subsequent infusions. In conclusion, pre-emptive CD19 CAR-T cell treatment is an effective and safe approach and may confer sustained remission in B-ALL patients with chemotherapy-refractory MRD. The trials were registered at www.chictr.org.cn as ChiCTR-ONN-16009862 (November 14, 2016) and ChiCTR1800015164 (March 11, 2018).

Targeting Bcl-2 Proteins in Acute Myeloid Leukemia.

B cell lymphoma 2 (BCL-2) family proteins play an important role in intrinsic apoptosis. Overexpression of BCL-2 proteins in acute myeloid leukemia can circumvent resistance to apoptosis and chemotherapy. Considering this effect, the exploration of anti-apoptotic BCL-2 inhibitors is considered to have tremendous potential for the discovery of novel pharmacological modulators in cancer. This review outlines the impact of BCL-2 family proteins on intrinsic apoptosis and the development of acute myeloid leukemia (AML). Furthermore, we will also review the new combination therapy with venetoclax that overcomes resistance to venetoclax and discuss biomarkers of treatment response identified in early-phase clinical trials.

Umbilical cord blood-derived mesenchymal stromal cells promote myeloid-derived suppressor cell proliferation by secreting HLA-G to reduce acute graft-versus-host disease after hematopoietic stem cell transplantation.

BACKGROUND AIMS: Mesenchymal stem cells (MSCs) use multiple mechanisms to constrain both innate and adaptive immune responses to prevent graft-versus-host disease (GVHD). Myeloid-derived suppressor cells (MDSCs), as a heterogeneous population of early myeloid progenitor cells originating from bone marrow, are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses, indicating their potential for GVHD therapy. There is accumulating evidence that MSCs and MDSCs do not act independently, but rather establish crosstalk. However, the role of MSCs in MDSC expansion and activation in GVHD remains unexplored. METHODS: In vitro experiments included 2 groups: peripheral blood mononuclear cells (PBMCs) after mobilization and human umbilical cord blood-derived MSCs (UCB-MSCs) co-cultured with PBMCs. The number and functional difference of MDSCs in PBMCs were determined by flow cytometry. The culture supernatants of co-cultured cells were analyzed to identify cytokines involved in MDSC proliferation. The relationship between MSCs and MDSCs was clarified in GVHD and graft-versus-leukemia (GVL) animal models. RESULTS: In vitro experiments confirmed that UCB-MSCs secreted HLA-G protein to promote and maintain the proliferation of MDSCs in peripheral blood after granulocyte colony-stimulating factor mobilization, and UCB-MSCs mediated the function of MDSCs to inhibit the proliferation of T cells and promote the proliferation of regulatory T cells. UCB-MSCs overexpressing HLA-G induced MDSC production in recipient mice, improved the ability of MDSCs to suppress T cells and further reduced acute GVHD (aGVHD) symptoms and survival time without influencing GVL effects. CONCLUSIONS: UCB-MSCs expanded MDSCs via HLA-G/Ig-like transcript 4, reducing the severity of aGVHD without affecting GVL. The immunosuppressive potential of MSCs for the treatment of aGVHD significantly affects the development of MDSCs, thereby consolidating the position of MSCs in the prevention and treatment of aGVHD.

Rapid and simultaneous determination of ten anti-tuberculosis drugs in human plasma by UPLC-MS/MS with applications in therapeutic drug monitoring.

Tuberculosis remains a global challenge, particularly with a growing number of resistant cases, which may become an obstacle to eliminating this disease. Standardized short-course therapy composed of first-line anti-tuberculosis drugs isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA) is playing vital roles for curbing the rapid spread of tuberculosis. However, some patients have poor responses to standardized short-course therapy. As the number of drug-resistant tuberculosis increase, some other anti-tuberculous drugs are needed to achieve better treatment outcomes. In this study, we established a UPLC-MS/MS method for simultaneous detection of ten anti-tuberculosis drugs in human plasma including INH, EMB, PZA, RIF, rifampin, rifapentine as well as four second-line antituberculosis drugs, i.e. ethionamide, protionamide, thiosemicarbazone and clofazimine. This study contains almost all the commonly used anti-tuberculosis drugs. The plasma samples were treated with acetonitrile to precipitate proteins, and doped with the isotope internal standard. A Shiseido CAPCELL RAK-ADME (2.1 mm × 50 mm, 3 µm) column was used for chromatographic separation, and acetonitrile-water (containing 0.1% formic acid) was the mobile phase. The separation used gradient elution with a flow rate of 0.4 mL/min. The column temperature was 40 °C, and the sample volume was 1 µL. The electrospray ionization source (ESI) and the positive ion multiple reaction monitoring (MRM) mode were used for the detection. The analysis time was as short as 7 min. The results show a good linear relationship under optimized conditions in the range of 5.00-7.50 × 103, 1.00-1.50 × 103, 5.00-5.00 × 104, 5.00-7.50 × 103, 1.00-3.00 × 103, 1.00 × 101-1.00 × 104, 1.00-3.00 × 103, 1.00-3.00 × 103, 2.00-4.00 × 103, and 1.00 × 10-1-2.00 × 102 ng/mL for INH, EMB PZA, RIF, rifabutin, rifapentine, ethionamide, protionamide, thiosemicarbazone, and clofazimine, respectively, with a linear correlation coefficient of R > 0.99. Finally, 34 patients with pulmonary TB were tested for therapeutic drug monitoring. The results showed that the presented method have significant advances in sensitivity, separation efficiency and simplicity.

Corrigendum: Targeting Bcl-2 Proteins in Acute Myeloid Leukemia.

[This corrects the article .].

Anti-CD19 Chimeric Antigen Receptor T Cells in Combination With Nivolumab Are Safe and Effective Against Relapsed/Refractory B-Cell Non-hodgkin Lymphoma.

Chimeric antigen receptor (CAR) T cells are emerging as a novel treatment for patients with refractory/relapsed B-cell non-Hodgkin lymphoma (B-NHL), and combination with PD1 inhibitors may further improve the efficacy of anti-CD19 CAR (CD19 CAR)-T cells in the treatment of lymphomas. In a single-center study, we evaluated the safety and efficacy of a combination therapy with CD19 CAR-T cells and an anti-PD-1 antibody (nivolumab) in patients with relapsed/refractory B-NHL. A total of 11 patients with refractory/relapsed B-NHL were recruited and subsequently received CD19 CAR-T cells and nivolumab. The primary end points were safety and feasibility. The infusions were safe, and no dose-limiting toxicities occurred. Grade 1 or 2 cytokine release syndrome (CRS) was observed in 25% (3/11) and 50% (6/11) of the patients, respectively, and only one patient (1/11) experienced neurotoxicity. The objective response rate (ORR) and complete response (CR) rate were 81.81% (9/11) and 45.45% (5/11), respectively. The median follow-up time was 6 (1~15) months. The median progression-free survival (PFS) time was 6 months (1~14 months), and 3 patients continued to have a response at the time of this writing. Our study demonstrated that the combination of CD19 CAR-T cells and nivolumab was feasible and safe and mediated potent anti-lymphoma activity, which should be examined further in prospective clinical trials in refractory/relapsed B-NHL.

Progress and challenges of chimeric antigen receptor gene modified-T cell immunotherapy to cancer / 中国肿瘤生物治疗杂志

@# 过继细胞疗法（ACT）的飞速发展使其成为肿瘤治疗手段中的一项新热点，其中嵌合抗原受体修饰的T细胞（CAR-T 细胞）在治疗恶性血液肿瘤中取得的成果更是令人振奋，同时也为实体瘤的治疗提供了新策略。但是，目前CAR-T细胞免疫疗法 在肿瘤治疗过程中的局限性也日渐显露。本文旨在针对CAR-T细胞在肿瘤治疗中的研究进展及治疗中的挑战予以简要探讨。

Model Development for Threshold Voltage Stability Dependent on High Temperature Operations in Wide-Bandgap GaN-Based HEMT Power Devices.

Temperature-dependent threshold voltage (Vth) stability is a significant issue in the practical application of semi-conductor power devices, especially when they are undergoing a repeated high-temperature operation condition. The Vth analytical model and its stability are dependent on high-temperature operations in wide-bandgap gallium nitride (GaN)-based high electron mobility transistor (HEMT) devices that were investigated in this work. The temperature effects on the physical parameters-such as barrier height, conduction band, and polarization charge-were analysed to understand the mechanism of Vth stability. The Vth analytical model under high-temperature operation was then proposed and developed to study the measurement temperatures and repeated rounds dependent on Vth stability. The validity of the model was verified by comparing the theoretical calculation data with the experimental measurement and technology computer-aided design (TCAD) simulation results. This work provides an effective theoretical reference on the Vth stability of power devices in practical, high-temperature applications.