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.

Platelet Doubling After First Decitabine Cycle Predicts Response and Survival of Myelodysplastic Syndrome Patients.

OBJECTIVE: Although the effect of decitabine on myelodysplastic syndrome (MDS) has been demonstrated, merely a proportion of patients respond to therapy, and no well-recognized predictors have been identified. This study was conducted to investigate the effectiveness of decitabine in real-world clinical practice, and determine the predictive factors of response and overall survival (OS) in MDS patients. METHODS: Clinical and pathological data were collected from 94 patients and analyzed. These patients were reclassified according to the 2016 World Health Organization classification criteria, and restratified by International Prognostic Scoring System prognostic scores. The response evaluation was performed according to the 2006 modified International Working Group response criteria. RESULTS: In this study, 62% of patients responded to decitabine. Among these patients, 15 patients (16%) obtained complete remission (CR), 15 patients (16%) obtained marrow CR with hematologic improvement (HI), 20 patients (21%) obtained marrow CR without HI, and 8 patients (9%) only obtained HI, and no patient botained partial remission. The OS of the responders was significantly longer than that of non-responders (67 months vs. 7 months, P<0.001). The OS in patients with and without platelet doubling was significantly different in both the low/intermediate and high/very high risk groups (P=0.0398 and P=0.0330). The multivariate analysis revealed that platelet doubling after the first decitabine cycle is an independent predictor of response and OS in MDS patients (P=0.002 and P=0.008). CONCLUSION: Decitabine is effective for treating MDS patients in real-world clinical practice. Furthermore, platelet doubling after the first decitabine cycle can be used as a predictor of response and survival in MDS patients.

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial).

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO-based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA-anthracycline for low-/intermediate-risk patients, or ATRA-ATO-anthracycline versus ATRA-anthracycline-cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of -5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI -0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan-Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA-chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

Clinical Features and MicroRNA Expression Patterns Between AML Patients With DNMT3A R882 and Frameshift Mutations.

Background: DNA methyltransferase 3A (DNMT3A) plays a unique role in hematopoiesis and acute myeloid leukemia (AML) pathogenesis. While the influences of DNMT3A mutation subtypes are still under debate. Purpose: Exploration of the clinical and molecular differences between AML patients carrying DNMT3A R882 mutations and DNMT3A frameshift mutations. Methods: Next generation of sequencing (NGS) and clinical data of 118 AML patients in our center were analyzed and compared. NGS, mRNA and miRNA profiling and clinical data from 12 patients in TCGA database were integrative analyzed. Results: Among all patients enrolled, 113 patients were positive for the variants of interest. Overall, a total of 295 variants were discovered, among which 24 DNMT3A mutations were detected, including 1 non-sense, 20 missense, 3 frameshift mutations. And 7 DNMT3A R882 mutations (3 R882H, 2 R882C, and 2 R882P) were found. Clinical analysis from our cohort and TCGA database indicated that patients carrying DNMT3A R882 mutation exhibited significantly higher levels of peripheral blood hemoglobin and non-significantly inferior prognosis compared with patients with DNMT3A frameshift mutations. Integrative analysis indicated that miR-10b, miR-143, and miR-30a were significantly decreased in the DNMT3A R882 group. High miR-143 expression is significantly associated with better prognosis in AML patients with DNMT3A mutations. Conclusion: Different molecular and clinical characteristics existed between patients with DNMT3A variant subtypes. The distinct microRNA expression pattern for DNMT3A R882 AML patients might not only act as markers to predict disease prognosis, but also could be further investigated to develop novel therapeutic targets for patients with DNMT3A mutations.

Prognostic Value of D-Dimer in Patients with Diffuse Large B-cell Lymphoma: A Retrospective Study.

This study evaluated the significance of serum D-Dimer for predicting survival of patients with diffuse large B-cell lymphoma (DLBCL). We analyzed the clinical data from 113 patients who were newly diagnosed with DLBCL at Tongji Hospital from January 2012 to January 2016. The results indicated that there were higher levels of D-Dimer in DLBCL patients with the following characteristics: stage III/IV, lymphocyte monocyte ratio (LMR) <2.27, lactate dehydrogenase (LDH) > upper limit of normal (ULN), albumin (ALB) < 35 g/L, and anemia. After the first chemotherapeutic regimen, D-Dimer was significantly decreased concomitantly with LDH. Cox univariate regression analysis showed that the overall survival (OS) was negatively affected by the following factors: age > 60 years, stage III/W, LDH > ULN, LMR < 2.27, anemia and D-Dimer > 0.92. Multivariate analysis showed that only LDH > ULN (P=0.038) and age > 60 years (P=0.047) were independent adverse prognostic factors. However, it was suggested that D-Dimer could be regarded as a marker of high tumor burden and a potential prognostic screening tool for patients with DLBCL, not otherwise specified (NOS).

Efficacy and Safety of Generic Dasatinib as a Second-line Treatment for Patients with Chronic Myeloid Leukemia: a Multicenter Retrospective Study in Hubei Province, China.

Dasatinib is a second-generation tyrosine kinase inhibitor (TKI) and it could be used as a second-line treatment for patients with chronic myeloid leukemia (CML). Yinishu, a generic dasatinib made in China, was approved by the China Food and Drug Administration in 2013 and it costs much less than the patented dasatinib SPRYCEL. The present study aimed to examine the efficacy and safety of Yinishu as a second-line treatment for CML by comparing the baseline clinical characteristics, rates of adverse events and efficacy between Yinishu and SPRYCEL groups. The results showed that there were no significant differences in the rates of optimal response between Yinishu and SPRYCEL for patients who started second-line treatment because of treatment failure. For patients who started second-line treatment because of intolerance of first-line treatment, their levels of BCR-ABL1/ABL1 on the international scale (BCR-ABLIS) was maintained very low throughout the course of Yinishu treatment. Drug-related adverse events occurred with the same frequency in these two groups. It was confirmed that Yinishu was effective and safe as a secondline treatment for CML patients. Yinishu may be more suitable for patients who are economically unable to pay for the patented dasatinib SPRYCEL.

[Effect of 5-Aza-CdR on biological activity and inhibitor of DNA binding 4 gene expression in human erythroleukemia cell line K562].

This study was aimed to investigate the effect of 5-Aza-CdR on the biological activity of human erythroleukemia cell line K562 and the expression of inhibitor of DNA binding 4 (ID4). ID4 methylation in K562 cell line was detected by methylation-specific PCR. RQ-PCR was used to analyze the expression levels of ID4 mRNA in K562 cell line treated by different concentrations of 5-Aza-CdR. Cell apoptosis rate and cell cycle were analyzed by flow cytometry. The result showed that ID4 gene methylation existed in K562 cells, ID4 mRNA expression in K562 cells treated with 5-Aza-CdR increased in a concentration-dependent manner, the difference between experimental groups was statistical significant (p < 0.01). The 5-Aza-CdR could enhance the apoptotic rate of K562 cells in time and dose-dependent manner, the apoptotic rate of K562 cells highly correlated to relative expression level of ID4 mRNA (r = 0.95). After the K562 cells were treated by 5-Aza-CdR for 48 hours, cells in G(0)/G(1) phase increased, cells in G(2)/M phase decreased along with enhancement of drug concentration. It is concluded that methyltransferase inhibitor 5-Aza-CdR can re-express the silent ID4 gene in K562 cells. The upregulation of ID4 may be a key factor to give rise to cell apoptosis, and the cell cycle of K562 cells can be arrested by 5-Aza-CdR.

[Diagnostic role of BIOMED-2 multiplex polymerase chain reaction for antigen receptor genes rearrangement in lymphoproliferative disorders].

OBJECTIVE: To establish a sensitive and effective method for detection of immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangement,and to explore its role in diagnosis and differential diagnosis of lymphoproliferative disorders. METHODS: Fifty-eight lymphoid tissue samples from 54 patients with lymphoproliferations were evaluated by the novel BIOMED-2 multiplex polymerase chain reaction (PCR) for antigen receptor genes rearrangement. RESULTS: Multiplex PCR demonstrated monoclonal Ig/TCR gene rearrangements in 22 of 25 (88.0%) B-cell malignancies and 8 of 15 (53.3%) T-cell malignancies. Among 17 benign lymphoproliferations confirmed histopathologically, polyclonal rearrangements were detected in 14 cases (82.4%). In total, the clonality analysis and the final clinico-histopathological diagnosis were concordant in 77.2%. Combination detection of Iglambda and TCR delta gene rearrangements did not increase the detection rate of monoclonal rearrangement of Ig/TCR, but might help to the detection of Iglambda+ or TCR delta+ lymphomas. CONCLUSION: The novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.

[Mild type of hemophilic pseudotumor with injury of femoral nerve (one case report)].

To explore the diagnoses and treatment of hemophilic pseudotumor, one case with hemophilic pseudotumor misdiagnosed and treated by operation, was observed and analyzed. The result showed that the final diagnosis of this case was following: hemophilia A (mild type) and hemophilic pseudotumor with injury of femoral nerve. The final diagnosis was given from inquiring case history and family history additionally, and drawing assistance from laboratory examination and computed tomography. After operation, the patient's wound healed very well through supplying coagulation factors positively. In conclusion, it was important for inquiring case history and family history particularly and thinking highly of laboratory examination to reduce the misdiagnosis and error of therapy for this case. If paying attention to preoperative preparation, the danger of hemorrhage during operation can be reduced and wound after operation can heal more rapidly.

Activity of telomerase and extracellular regulated protein kinases in parental and drug resistant cells of leukemia and ovarian cancer.

In order to study the role of telomerase and extracellular regulated protein kinase (ERK) in drug-resistance of leukemia and ovarian cancer cells, telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis were used for qualitative analysis or quantitative detection of telomerase activity respectively, and Western blot was used to detect the expression level of phosphorylatedly activated ERK(1) and ERK(2) protein in the parental and drug resistant cells of leukemia and ovarian cancer. In addition, chemotherapy sensitivity to HRT or DDP was evaluated by MTT assay. The difference of cell cycle distribution between parental cell and drug-resistant cell was analyzed by flow cytometry. The results showed that the drug resistant cells were of higher percentage in G(0)/G(1) phase compared with the parental cell lines. Telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level were higher in drug-resistant cells than in parental cell. It is suggested that the increasing number of the drug resistant cells in G(0)/G(1) phase may be considered as a sign of drug resistance. The up-regulation of telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level may play an important role in drug resistance of leukemia and ovarian cancer cell lines.

Inhibition of NF-kappaB by mutant IkappaBalpha enhances TNF-alpha-induced apoptosis in HL-60 cells by controlling bcl-xL expression.

BACKGROUND: The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism. METHODS: The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). RESULTS: Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner. CONCLUSIONS: NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.

[Analysis of the distribution characteristics and drug sensitivity of infection pathogens in hematology department ward].

The objective of this study was to investigate clinical distribution characteristics and drug sensitivity of infection pathogens in hematology department wards of our hospital during recent two years. The isolation and cultivation of pathogens from samples were performed by routine methods. Drug sensitivity tests of bacteria were performed by Kirby-Bauer method. Drug sensitivity tests of fungi were performed by ATBFUNGUS Drug sensitivity strips. The results showed that 102 strains of pathogens were isolated from all detected samples. The composition ratio of Gram-positive bacteria, Gram-negative bacteria and fungi was 42.2%, 34.3%, 3.5%, respectively. 58.8% of pathogens were isolated from samples of malignant hematopathy patients. 27.5% were isolated from samples of the patients with fever of unknown origin (FUO). 51.0% of pathogens were isolated from samples of the patients who suffered from agranulocytosis or leucocytopenia. Isolated fungi were mostly sensitive to anti-fungal drugs. G+ bacteria were most sensitive to vancomycin. G- bacteria were most sensitive to imipenem. Most bacteria were resistant to multiple antibiotics. It is concluded that the infection in hematology department wards was related with many conditions, such as weakened resistance of patients, leucocytopenia or agranulocytosis, tumor loading, etc. The prompt microbiological examination and drug sensitivity tests are important to rationally select antibiotics, reduce infection incidence and mortality rate, and decrease the occurrence of drug resistant strains.

[To inhibit ERK for enhancing chemotherapy sensitivity of drug-resistance cell lines of leukemia and ovarian carcinoma].

The aim was to study the roles of extracellular regulated protein kinases (ERK) and telomerase activity in drug resistance of human leukemia and ovarian carcinoma cells. Flow cytometry was used to analyze apoptosis rate. Telomere repeat amplification protocol (TRAP) and bioluminescence analysis method were used for detection of telomerase activity. The phosphorylated ERK(1/2) protein expression was observed by Western blot method. The results showed that the specific inhibitor PD98059 of ERK kinase 1 (MEK(1)) enhanced the sensitivity of HL-60/E6 leukemia cell lines to harringtonine (HRT) or COC1/DDP ovarian carcinoma cell lines to cis-dichlorodiamine platinum (DDP). Both PD98059 and chemotherapy drugs HRT and DDP reduced the phosphorylated ERK(1) and ERK(2) protein expression level, and down-regulated the telomerase activity. The sole action of each was inferior to the combination action of PD98059 and HRT or DDP. In conclusion, ERK and telomerase serve a function to some extent in drug resistance of leukemia and ovarian carcinoma cells. The inhibition of ERK signal transduction pathways led to reduction of phosphorylated ERK(1) and ERK(2) protein expression level, and successionally down-regulated the telomerase activity. The final result was to enhance the sensitivity of HL-60/E6 to HRT or COC1/DDP to DDP.

[Regulative function of extracelluar regulated protein kinases and telomerase in apoptosis of hepatocarcinomatous cell SMMC-7721].

OBJECTIVE: To study the changes of telomerase activity and protein expression of phosphorylated (activated) extracellular regulated protein kinases (ERK1 and ERK2) in the course of inhibiting hepatocarcinomatous cell proliferation and inducing cell apoptosis by three kinds of chemotherapy drugs: Harringtonine (HRT), Vincristine (VCR), and Etoposide (Vp16). To discuss the regulative function to hepatocarcinomatous cell apoptosis and interrelation of telomerase and ERK. METHODS: Cytotoxicity assay, flow cytometry analysis, telomerase repeat amplification protocol assay (TRAP), bioluminescence analysis, and western blot were used in this experiment. RESULTS: HRT, VCR, and Vp16 could inhibit cell proliferation (0.28% 0.08%, 0.25% 0.16%, 0.24% 0.11%), induce apoptosis (21.12%, 28.83%, 12.30%), inhibit telomerase activity, and down-regulate the protein expression of phosphorylated ERK. CONCLUSIONS: It might be through ERK signal transduction pathways that chemotherapy drugs down-regulate telomerase activity and induce apoptosis.

Regulative function of extracellular regulated protein kinases and telomerase in apoptosis of hepatocarcinomatous and leukemic cell lines.

In order to investigate the change of telomerase activity and phosphorylated (activated) extracellular regulated protein kinases (ERK) 1 and 2 in hepatocarcinomatous cell line SMMC7721 and leukemic cell line K562 proliferation inhibition and apoptosis, three kinds of chemotherapeutic drugs harringtonine (HRT), vincristine (VCR) and etoposide (VP-16) were selected as inducers; and MTT assay, flow cytometry analysis, telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis were used. The results showed that after treatment of HRT, VCR and VP-16 for 24 hours, the cell proliferation was inhibited, apoptosis was induced, and telomerase activity and the protein expression of phosphorylated ERK1/2 were down-regulated. In HRT treated groups, the descendent grade was the most obvious. It was concluded that the common molecular mechanism of these chemotherapeutic drugs killing SMMC7721 and K562 cell lines might be through inhibiting ERK signal transduction pathways, cutting down ERK activity, reducing the transcription of target genes of ERKs, then indirectly down-regulate telomerase activity, and cell apoptosis is the final result of durative loss of telomere.

[Antisense Oligodeoxynucleotide Directed to NF-kappaB-RelA Down-Regulates bcl-x(L) mRNA in Drug-Resistant Leukemia Cell Line HL-60/E6]

In order to explore the effect of nuclear factor-kappa B (NF-kappaB) on bcl-x gene transcrtiption in drug-resistant leukemia cell line HL-60/E6, first of all, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin, and then bcl-x(L) mRNA levels were detected by RT-PCR after exposing HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA at different times. Morever, indirect immuno-fluorescence and FCM were used to demonstrate the location of NF-kappaB-RelA in HL-60/E6 cells and the efficiency of liposome-mediated ODN transfection. The results showed that RelA kept active and located at the nuclei of HL-60/E6 cells, and the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P < 0.01 in 4, 6 and 12 h). Exposure of HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA led to a maximal 40% decline of bcl-x(L) mRNA levels. No significant change of bcl-x(L) mRNA level occurred in control group. It was concluded that NF-kappaB was involved in regulating bcl-x transcription. Suppressing NF-kappaB-RelA by antisense technology may down-regulate level of bcl-x(L) mRNA.