Shenglan Gong

Point Mutation of the Proteasome β5 Subunit Gene Is an Important Mechanism of Bortezomib Resistance in Bortezomib-Selected Variants of Jurkat T Cell Lymphoblastic Lymphoma/Leukemia Line

To study the mechanism of acquired resistance to bortezomib, a new antitumor drug that is the first therapeutic proteasome inhibitor, we established a series of bortezomib-resistant T lymphoblastic lymphoma/leukemia cell lines, designated the JurkatBs, from the parental Jurkat line via repeated drug selection. There were no significant differences in the growth curves or colony formation between the JurkatB cells and parental Jurkat cells. The effects of bortezomib on cytotoxicity, cell cycle arrest, and induction of apoptosis were decreased in JurkatB cells compared with parental Jurkat cells. A mutation in the proteasome β5 subunit (PSMB5) gene (G322A), which encodes an amino acid change from Ala to Thr at polypeptide position 108, was detected by sequencing full-length cDNA clones and direct polymerase chain reaction products of the PSMB5 gene. Bortezomib caused less inhibition of chymotrypsin-like activity in resistant cells. When the G322A mutant PSMB5 was retrovirally introduced into parental Jurkat cells, it conferred bortezomib resistance to these cells, resulting in decreased cytotoxicity, apoptosis, and inhibition of chymotrypsin-like activity. The predicted structure of A108T-mutated PSMB5 shows a conformational change that suggests decreased affinity to bortezomib. In short, the G322A mutation of the PSMB5 gene is a novel mechanism for bortezomib resistance.

Overexpression of the PSMB5 gene contributes to bortezomib resistance in T-lymphoblastic lymphoma/leukemia cells derived from Jurkat line.

OBJECTIVE To study the mechanism of bortezomib resistance in JurkatB lines derived from T-lymphoblastic lymphoma/leukemia Jurkat line. MATERIALS AND METHODS Cytotoxicities of popular chemotherapeutic drugs to JurkatB cells were analyzed by trypan blue assay. Functional drug efflux in JurkatB cells was determined by flow cytometry utilizing daunorubicin and the expression of P-glycoprotein (P-gp) was detected by Western blot. mRNA expression levels of proteasome beta5 subunit (PSMB5) were measured by quantitation real-time reverse transcription polymerase chain reaction. In situ hybridization was performed to detect the amplification of PSMB5 gene. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin (AMC) from the substrate N-succinyl-Leu-Leu-Val-Tyr-AMC. Cytogenetic studies were performed using R-banded metaphases and fluorescence in situ hybridization (FISH) analysis. IkappaB-alpha levels were detected by Western blot. RESULTS No cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide was found in JurkatB cells. No evidence of drug efflux was found in JurkatB cells and the expression of P-gp was negative. The PSMB5 mRNA was overexpressed in highly resistant JurkatB5 and JurkatB1 lines compared with parental Jurkat, corresponding well with the increase of chymotrypsin-like activity and a karyotype of i(14q). Amplification of PSMB5 gene was demonstrated by in situ hybridization and FISH. The decreased IkappaB-alpha level in JurkatB5 cells after bortezomib treatment indicating an upregulation of nuclear factor-kappaB (NF-kappaB) activity. CONCLUSION The mechanism of bortezomib resistance is different from that of multidrug resistance. Overexpression of PSMB5 is an important mechanism for bortezomib resistance in JurkatB lines. NF-kappaB may play a critical role in evading the apoptotic effects.

Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line

OBJECTIVE To investigate the relationship between bortezomib resistance and mutations in the proteasome beta5 subunit (PSMB5) gene. MATERIALS AND METHODS Various bortezomib-resistant lymphoblastic lymphoma/leukemia lines were established by repeated cycles of bortezomib selection. Mutations were detected by sequencing the complementary DNA of the PSMB5 gene. Mutated clones were selected by limited dilution and cultured without bortezomib. Messenger RNA expression levels of PSMB5 in these mutated clones were measured by quantitative reverse transcription polymerase chain reaction. The degree of resistance was determined by cytotoxicity at various bortezomib concentrations. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin from the substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. RESULTS In addition to the previously reported PSMB5 G322A mutant (Ala49Thr), a C323T mutant (Ala49Val), and G322A, C326T conjoined mutant (Ala49Thr and Ala50Val) were selected and clones containing these mutations (JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T) were obtained. After being cultured without bortezomib for >2 months, no significant difference in PSMB5 messenger RNA levels was detected between these JurkatB cells and parental Jurkat cells. JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T clones displayed 22.0-fold, 39.4-fold, and 66.7-fold resistance, respectively, to bortezomib compared to Jurkat cells. There were no significant differences between the chymotrypsin-like activities of these mutants and Jurkat cells. The inhibitory effect of bortezomib on chymotrypsin-like activity was the weakest in JurkatB-G322A/C326T cells, and the strongest in JurkatB-G322A cells, with JurkatB-C323T cells falling in between. CONCLUSION Mutations of the PSMB5 gene resulting in substitutions of Ala49 and Ala50 of PSMB5 protein can confer varying bortezomib resistance.

FISH+CD34+CD38- cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome.

BackgroundIn acute myeloid leukemia (AML), the leukemia initiating cells (LICs) or leukemia stem cells (LSCs) is found within the CD34+CD38- cell compartment. The LICs subpopulation survives chemotherapy and is most probable the cause of minimal residual disease (MRD), which in turn is thought to cause relapse. The aim of this study was to determine the prognostic value of the percentage of LICs in blasts at diagnosis.Design and methodsThe percentage of LICs in the blast population was determined at diagnosis using a unique Flow-FISH analysis, which applies fluorescent in situ hybridization (FISH) analysis on flow cytometry sorted cells to distinguish LICs within the CD34+CD38- cell compartment. Fourty-five AML patients with FISH-detectable cytogenetic abnormalities treated with standardized treatment program were retrospectively included in the study. Correlations with overall survival (OS), events-free survival (EFS) and cumulative incidence of relapse (CIR) were evaluated with univariate and multivariate analysis.ResultsThe percentage of LICs is highly variable in patients with acute myeloid leukemia, ranged from 0.01% to 52.8% (median, 2.1%). High LIC load (≥1%) negatively affected overall survival (2-year OS: 72.57% vs. 16.75%; P = 0.0037) and events-free survival (2-year EFS: 67.23% vs. 16.33%; P = 0.0018), which was due to an increased cumulative incidence of relapse (2-year CIR: 56.7% vs. 18.0%; P = 0.021). By multivariate analysis, high LIC load retained prognostic significance for OS and EFS.ConclusionsIn the present study, we established the Flow-FISH protocol as a useful method to distinguish normal and leukemic cells within the CD34+CD38- cell subpopulation. The high percentage of LICs at diagnosis was significantly correlated with increased risk of poor clinical outcome.

Impact of clinical utility of MRD assessment with different techniques on survival in acute B lymphoblastic leukemia

Abstract We investigated the impact of minimal residual disease (MRD) obtained from different approaches on the outcomes of 141 B lymphoblastic leukemia (B-ALL) patients. Among 169 samples with more than 5% blasts by morphology, 3.6% (6/169) were Flow-MRD negative. Of the 212 positive molecular-MRD samples from Ph+ ALL patients, 55 (25.9%) were Flow-MRD negative. Before consolidation or allogeneic stem cell transplantation (allo-HSCT), negative Flow-MRD was associated with improved survival (p = .019 and .041, respectively) for Ph− ALL patients, but not for Ph+ ALL (p = .111 and .812, respectively). There was no difference in overall survival (OS) by achievement of complete molecular response at complete remission (CR, p = .333 and .863, respectively). Our results indicated that the results of MRDs detected with different methods varied. Flow-MRD can be used as a reliable prognostic marker for Ph− ALL patients. MRD either by flow cytometry or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) at CR did not affect OS or DFS for Ph+ ALL.

MYD88 L265P mutation promoted malignant B cell resistance against T cell-mediated cytotoxicity via upregulating the IL-10/STAT3 cascade

ABSTRACT The myeloid differentiation factor 88 (MYD88) signaling plays critical roles in the developments of B cells. Recent studies demonstrated that in the activated B cell subtype of diffuse large B cell lymphoma (DLBCL), approximately one‐third of the patients harbored somatically acquired MyD88 L265P mutation in their lymphomas. It remains unclear whether B cell lymphomas with MYD88 L265P mutation respond differently toward CD8+ T cell‐mediated cytotoxicity. Here, we demonstrated that, when incubated with autologous CD8+ T cells, the MYD88 L265P mutant lymphomas were more resistant to granzyme B‐ and perforin‐mediated killing than MYD88 wild‐type (WT) lymphomas. Interestingly, in the absence of autologous lymphomas, the granzyme B and perforin expression levels in CD8+ T cells from patients with MYD88 WT lymphomas and from patients with MYD88 L265P mutant lymphomas were comparable; however, in the presence of autologous lymphomas, the CD8+ T cells from patients with MYD88 L265P mutant lymphomas presented significantly lower granzyme B and perforin expression than CD8+ T cells from patients with MYD88 WT lymphomas. We further found that the IL‐10 expression level and the STAT3 activation level were significantly higher in MYD88 L265P mutant lymphomas than in MYD88 WT lymphomas. Suppressing IL‐10 significantly reduced STAT3 activation in both MYD88 WT and MYD88 L265P mutant lymphomas. Blocking either STAT3 or IL‐10 could significantly increase the susceptibility of MYD88 L265P mutant lymphomas toward CD8+ T cell‐mediated cytotoxicity. Together, these data revealed a mechanism of immune evasion in MYD88 L265P mutant lymphomas. HighlightsMYD88 L256P DLBCL showed higher survival capacity with autologous cytotoxic T cells.CD8+ T cells presented lower responses in DLBCL patients with MYD88 L256P mutation.MYD88 L256P mutant DLBCL showed higher STAT3 activation and IL‐10 expression.Blocking IL‐10/STAT3 increased cytotoxic T cell responses in MYD88 mutant DLBCL.

Flow cytometric analysis of CD64 expression pattern and density in the diagnosis of acute promyelocytic leukemia: a multi-center study in Shanghai, China

No unified immunophenotypic profiles and corresponding analytic strategies have been established for the rapid diagnosis of acute promyelocytic leukemia (APL) using flow cytometry (FCM). Here we describe a characteristic immunophenotypic panel that can rapidly and accurately distinguish APL from other types of adult acute myeloid leukemia (AML) using only FCM. By comparing APL cells and non-APL AML cells that share APL common immunophenotypes (CD34−CD117+HLA−DR−) we found that CD64 was a significant factor that differentiated APL from other AMLs. Further retrospective analyses of 205 APL and 629 non-APL AML patients from different hematology centers showed that either the CD64dim and homoCD13+homo CD33+homoMPO+ (myeloperoxidase) CD11c− panel or the CD64dim and homoCD13+homo CD33+homoMPO+ CD11c+CD10−CD117+ SSChigh (high side scatter signal) panel could distinguish APL from non-APL AML patients with nearly 100% sensitivity, specificity and accuracy. Moreover, relative quantification of CD64 expression enhanced the applicability of our APL diagnostic immunophenotypic panels (ADI-panels) in different hematology centers. Application of the ADI-panels will decrease diagnosis time and improve personalized treatment for APL, a life-threatening disease with very rapid progression.

[Acute promyelocytic leukaemia with translocations of t(15;17)(q22;q21) and rob(13;21): a case report and literatures review].

目的 报道1例初发伴有rob（13；21）t（15；17）（q22；q21）的急性早幼粒细胞白血病（APL）并探讨其临床和实验室特点。 方法 在常规核型分析和骨髓细胞形态学的基础上，应用双色双融合荧光原位杂交（DCDF-FISH）和RT-PCR技术进一步检测该患者的细胞遗传学异常和分子生物学异常。并结合文献分析此类伴少见变异易位APL患者的临床特点。 结果 患者的临床表现和骨髓细胞形态学检查均符合APL。初诊时免疫表型分析髓系标志呈阳性，R显带染色体分析显示患者核型为45，XX，rob（13;21）t（15;17）（q22；q21）[6]/45，XX，rob（13；21）[14]，FISH结果显示68.9%的细胞为典型的t（15；17）模式，RT-PCR结果显示PML-RARα融合基因水平为25.8%。经诱导化疗、巩固治疗后，达到完全缓解，核型为45，XX，rob（13；21）[20]，FISH检测PML-RARα融合基因阳性细胞率为2.5%，PML-RARα融合基因水平为0.003%。 结论 伴特征性的rob（13；21）t（15；17）（q22；q21）的APL十分罕见，患者临床表现和实验室检查基本符合APL的临床特点；该染色体异常对APL患者的预后判断价值仍需进一步的观察。OBJECTIVE To report an acute promyelocytic leukaemia (APL) case with translocation of rob (13;21) t(15;17) (q22;q21) and review its clinical and laboratory characteristics. METHODS Based on routine karyotype analysis and bone marrow morphology, we further used double color double fluorescent in situ hybridization (DCDF-FISH) and reverse transcriptase PCR (RT-PCR) to examine the patients abnormities on cytogenetic and molecular biology, and reveal the clinical characteristics of this rare translocation also from the related literatures. RESULTS The clinical manifestation and bone marrow morphology examination of this patient were in accordance with pathologic feature of APL. On first visit, immunophenotyping analysis showed positive myeloid markers. Through R-banding, the patients karyotype was confirmed as 45, XX, rob(13;21) t(15;17) (q22;q21) [6]/45, XX, rob(13;21) [14]. FISH results showed that 68.9% cells were typical t(15;17) pattern. The positive rates of fusion gene of PML-RARα detected by RT-PCR was 25.8%. Patient was treated by induction and consolidation therapy, the karyotype was 45, XX, rob(13;21 )[20] after complete remission. The positive rate of fusion gene of PML-RARα by FISH and its level were 2.5% and 0.003% respectively. CONCLUSION APL with rob (13;21) t(15;17) (q22;q21) was very rare, which was accorded with clinical and laboratory characteristics of APL. The value of chromosome abnormality as a prognostic marker in APL needs to be further observed..