Orelabrutinib for the treatment of relapsed or refractory MCL: a phase 1/2, open-label, multicenter, single-arm study.

Relapsed or refractory (r/r) mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a poor prognosis. Bruton tyrosine kinase (BTK) is a mediator of B-cell receptor signaling and is associated with the development of B-cell lymphomas. Patients with r/r MCL were enrolled in this phase 1/2 study and treated with orelabrutinib, a novel, highly selective BTK inhibitor. The median number of prior regimens was 2 (range, 1-4). The median age was 62 years (range, 37-73 years). Eligible patients received oral orelabrutinib 150 mg once daily (n = 86) or 100 mg twice daily (n = 20) until disease progression or unacceptable toxicity. A dose of 150 mg once daily was chosen as the preferred recommended phase 2 dose. After a median follow-up duration of 23.8 months, the overall response rate was 81.1%, with 27.4% achieving a complete response and 53.8% achieving a partial response. The median duration of response and progression-free survival were 22.9 and 22.0 months, respectively. The median overall survival (OS) was not reached, and the rate of OS at 24 months was 74.3%. Adverse events (AEs) occurring in >20% of patients were thrombocytopenia (34.0%), upper respiratory tract infection (27.4%), and neutropenia (24.5%). Grade ≥3 AEs were infrequent and most commonly included thrombocytopenia (13.2%), neutropenia (8.5%), and anemia (7.5%). Three patients discontinued treatment because of treatment-related adverse events (TRAEs), but no fatal TRAEs were reported. Orelabrutinib showed substantial efficacy and was well tolerated in patients with r/r MCL. This trial was registered at www.clinicaltrials.gov as #NCT03494179.

Evaluation of orelabrutinib monotherapy in patients with relapsed or refractory Waldenström's macroglobulinemia in a single-arm, multicenter, open-label, phase 2 study.

Background: Orelabrutinib is a novel, small molecule, selective irreversible Bruton tyrosine kinase inhibitor. The purpose of this study was to evaluate the efficacy and safety of orelabrutinib in patients with relapsed or refractory Waldenström's macroglobulinemia (R/R WM). Methods: This is a prospective, multicenter study of orelabrutinib in patients with WM who had at least one prior line of treatment. Orelabrutinib was administered orally at a daily dose of 150 mg until disease progression or unacceptable toxicity. The primary endpoint was major response rate (MRR) assessed by the Independent Review Committee (IRC) according to IWWM-6. This study is registered with ClinicalTrials.gov, NCT04440059. This trial was also registered on Center for Drug Evaluation (www.chinadrugtrials.org.cn) in March 2019, with a number of CTR2019036. Findings: Between August 2019 and December 2020, 66 R/R WM patients were assessed for eligibility. Forty-seven eligible patients were evaluated for efficacy at a median follow-up of 16.4 months (interquartile range: 12.5, 19.5). As assessed by IRC, the MRR was 80.9%, and the overall response rate was 89.4%. The median time to at least a minor response was 1.9 months. The PFS rates was 89.4% at 12 months. For patients with MYD88L265P /CXCR4NEG, MYD88L265P /CXCR4 S338X, and MYD88NEG /CXCR4NEG mutations, the MRRs were 84.6%, 100%, and 25.0%. Most adverse events were Grades 1 or 2 (91.0%). The common grade 3 or higher adverse events occurring were neutropenia (10.6%), thrombocytopenia (6.4%), and pneumonia (4.3%). Serious adverse events (SAE) occurred in 10 patients (21.3%). One treatment-related death was reported (hepatitis B reactivation). Interpretation: Orelabrutinib has shown good efficacy and manageable safety profiles in patients with R/R WM. Funding: InnoCare Pharma.

[Level of Microparticles in Patients with Essential Thrombocythemia and Its Relation with JAK2V617F Mutation].

OBJECTIVE: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients. METHODS: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF+MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 µm. Standard fluorescent microbeads (0-0.8 µm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations. RESULTS: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/µl vs 21.3±18.4/µl), PMP (1378.9±2454/µl vs 113.8±97.1/µl), TF+MP (123±354.6/µl vs 21±15.7/µl) and EMP (1434.7±2601.9/µl vs 291.7±297.7/µl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/µl vs 55.6±63.8/µl), PMP (1853±1874.7/µl vs 444.7±712/µl), TF+MP (120.4±112.5/µl vs 60.3±128.3/µl) and EMP (1637.1±1563.5/µl vs 629.4±1000.2/µl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/µl vs 59.3±51/µl), PMP (2944.8±3767.6/µl vs 334.8±420.2/µl), TF+MP (162.9±166.8/µl vs 31.0±28.7/µl) and EMP (3031.8±4048.8/µl vs 701.5±729.2/µl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF+MP (154.7±516.3/µl vs 100.5±126.6/µl) (P<0.05). CONCLUSION: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF+MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

[Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients].

OBJECTIVE: To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. METHODS: Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. RESULTS: ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). CONCLUSION: The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

[Clinical Efficacy of Autologous Transplantation of Hematopoietic Stem Cells Cryopreserved by Ladder-style Freezing from Low Temperature Refrigerator to Liquid Nitrogen].

OBJECTIVE: To investigate the efficacy of hematopoietic stem cells cryopreserved by ladder-style freezing from low temperature refrigerator to liquid nitrogen in treatment of hematological malignancies, and to analyze the survival condition of patients after hematopoietic stem cell transplantation. METHODS: The coyoprotectant formed by 3% hydroxyethyl starch, 4% albumin and 5% dimethyl sulfoxide (DMSO) was need for cryopreservation of hematopoietic stem cells,which were first placed in -800C low temperature refrigerator and then were stored in -1960C liquid nitrogen tank. 98 cases of hemafologic malignancies (io cases of ALL, 24 cases of AML, L-cases of MM and 53 case of malignant lymphoma) were selected from January 2002 to December 2016, and recived transplantatin auto-hematopoiehc stem cells cryopresorved by above-mentined method. The overall survival rate (OS), progression-free survival (PFS) were analyzed statistically. RESULTS: One case failed in implantation due to intracranial hemorrhage and the other 97 cases all succeeded in hematopoietic reconstitution. The average time needed for neutrophil count ≥0.5×109/L was 9.24±1.89 d, and the average time needed for blood platelet ≥20×109/L without platelet transfusion for 3 days was 11.04±1.84 d. The median survival time was 47.6 months (1-80 months). The 3 and 5 year OS rates were (97.2±1.9) %, (84.2±4.6) % and (77.8±5.6) %, respectively. 3- and 5-year PFS of patients were (74.4±5.1)% and (61.2±6.2)%. CONCLUSION: ladder-style freezing from low temperature refrigerator to liquid nitrogen can reach the same clinical transplantation effect with traditional programmed cooling freezing method in autologous hematopoietic stem cells transplantation. moreover the incidence of complications after transpeantatim does not show increase.

Complete mitochondrial genome of the Algerian honeybee, Apis mellifera intermissa (Hymenoptera: Apidae).

In this study, the complete mitochondrial genome sequence of Algerian honeybee, Apis mellifera intermissa, is analyzed for the first time. The results show that this genome is 16,336 bp in length, and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (43.2%), C (9.8%), G (5.6%), and T (41.4%), so the percentage of A and T (84.6%) is considerably higher than that of G and C. All the genes are encoded on H-strand, except for four subunit genes (ND1, ND4, ND4L, and ND5), two rRNA genes (12S and 16S rRNA), and eight tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in A. m. intermissa.

The complete mitochondrial genome of black-footed ferret, Mustela nigripes (Mustela, Mustelinae).

In this study, the complete mitochondrial genome sequence of black-footed ferret, Mustela nigripes, is determined for the first time. This mitogenome is 16,556 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (32.9%), C (26.1%), G (13.8%), and T (27.2%), so the percentage of A and T (60.1%) is higher than that of G and C. Most of the genes are encoded on H-strand, except for the ND6 subunit gene and six tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in M. nigripes.

[Effects of human bone marrow mesenchymal stem cells on cytokines secretion from allogeneic dendritic cell activated cytokine-induced killer cells].

AIM: to study the effect of human bone marrow derived mesenchymal stem cells (hMSCs) on cytokines secretion (IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2) of allogeneic DC-CIK cells (in co-culture of CIK cells with DC), which investigate the mechanism of immunoregulation induced by hMSCs. METHODS: the hMSCs from bone marrow were isolated, expanded and identified by cell morphology, differentiation into neuron-like cells with NSE, fat-like cells with red-oil stain, and expression of CD29, CD44. The DC and CIK cells from peripheral blood were isolated, expanded and identified by CD1α, HLA-DR or CD3(+);CD56(+);. The hMSCs were co-cultured with DC-CIK cells according to ratio 1:10. The expression of the six cytokines in supernatant was evaluated by flow cytometry after 4 days of DC-activated CIK cells in co-culture with hMSCs. RESULTS: the hMSCs displayed a fibroblast-like morphology and the positive cells of CD29 and CD44 were 96.6%, 94.6%, which have the capacity of differentiation into neuron-like cells with expressed NSE as well as fat-like cells with red-oil stain positive. The expression of CD1α, HLA-DR in DC was (91.9 ± 10.04)% and (88.8 ± 8.92)%. The CD3(+);CD56(+); double positive cells in DC-CIK cells was (29.23 ± 12.23)% compared to CIK cells with (15.98 ± 2.49)%. The cytokines secretion of DC-CIK cells in co-culture with hMSCs was IFN-γ (135.05 ± 48.19) ng/L; TNF-α (11.33 ± 1.42) ng/L; IL-10 (10.15 ± 2.25) ng/L; IL-6 (494.63 ± 235.222) ng/L; IL-4 (7.07 ± 2.30) ng/L and IL-2 (1074.6 3 ± 303.74) ng/L. In control group (DC-CIK cells) the secretion of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 was (717.6 ± 248.15) ng/L; (17.78 ± 7.52) ng/L; (29.95 ± 12.76) ng/L; (8.03 ± 0.21) ng/L, (9.08 ± 3.07) ng/L as well as IL-2 1 250 ng/L. CONCLUSION: the secretion of IFN-γ and IL-10 were down-regulated. It probably implied that hMSCs had the effect of immunoregulation on DC-CIK cells.

[Protection of cryopreserved platelets by dimethyl sulfoxide combined with trehalose].

This study was aimed to investigate the protective effects of dimethylsulfoxide (DMSO) combined with trehalose on the cryopreserved platelets. The platelets were preserved at -80 degrees C. The experiments were divided into 5 groups: blank control group composed of apheresis platelet suspension; trehalose group composed of apheresis platelet suspension and 0.25 mol/L trehalose; DMSO group composed of apheresis platelet suspension and 5% DMSO; 5% combined group composed of apheresis platelet suspension, 5% DMSO and 0.25 mol/L trehalose; 2.5% combined group composed of apheresis platelet suspension, 2.5% DMSO and 0.25 mol/L trehalose. All the groups were thawed at 37 degrees C in a waterbath. The recovery rate of platelets and mean platelet volume (MPV) were assayed by using hemocytometer; the ultrastructural changes were examined by electron microscopy; the expressions of CD41, CD42b, CD61 and CD62p on platelets were detected by flow cytometry. The results indicated that single use of trehalose had no strong effect in increasing the recovery rate of platelets, but the morphology of platelets was close to normal. The DMSO showed significant effect in increasing the recovery rate of platelets and maintaining the intact property of platelets, however, the shape of platelets tended to sealing, and partial platelets still displayed heteromorphic changes. The combination of DMSO and trehalose revealed the protective effect on the external morphology and internal structure of platelets to be close to the normal homeostasis, and ensured an ideal recovery rate of the cryopreserved platelets and higher expression levels of CD41, CD42b, CD61 and CD62p in the same time. It is concluded that the combined use of DMSO and trehalose possesses the synergistic protective effect on the cryopreserved platelets, therefore, the combined use of both as the protective agent is hopeful to further raise the effectiveness of clinical infusion of the cryopreserved platelets.