Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics.

Resistance to chemotherapy-induced apoptosis and a multidrug-resistance (MDR) phenotype, mainly mediated by P-glycoprotein (P-gp), contribute to chemotherapy failure in hematologic malignancies. To study apoptosis-regulating factors in acute myeloid leukemia (AML), we investigated cell samples of adults with de novo AML by flow cytometry for constitutive expression levels of the apoptosis-related molecules CD95 (n = 135), Bcl-2 (n = 131), and Bax (n = 66), as well as spontaneous apoptosis in vitro (n = 104) and susceptibility to anti-CD95-induced apoptosis (CD95 sensitivity) (n = 93). We correlated these findings with P-gp function as detected by the rhodamine123-efflux test (n = 121), immunophenotype, FAB morphology, cytogenetics, and clinical data of the examined patients. Immature FAB M0/1 AML cells expressed significantly more Bcl-2 (P < 0.0002) and less CD95 (P < 0.0003) compared with AML cells of the more mature FAB M2-5 subtypes. No maturation-dependent difference in Bax expression was observed. FAB M2-5 AML cells were more susceptible to anti-CD95-induced apoptosis (P < 0.008) and showed a lower P-gp function (P < 0.002) than FAB M0/1 AML cells. Leukemic cells of AML patients who achieved a complete remission (CR) after induction chemotherapy expressed less Bcl-2 than non-responder (NR) (69 CR, 23 NR; P = 0.05). CR was associated with a higher extent of spontaneous apoptosis in vitro (58 CR, 17 NR; P=0.05) and a tendency towards a higher CD95 expression (73 CR, 23 NR; P = 0.08) compared to NR. CR also correlated with a low P-gp function (70 CR, 21 NR; P = 0.008) and a tendency towards CD34 negativity (73 CR, 23 NR; P = 0.08). No correlation between Bax expression and response to induction chemotherapy (49 CR, 12 NR) was observed. In stepwise logistic regression analyses, P-gp function and the extent of spontaneous apoptosis in vitro as well as CD95 sensitivity but not Bcl-2, CD95, Bax, and CD34 expression levels emerged as significant markers for response to induction chemotherapy. We conclude that the constitutive expression of CD95 and Bcl-2, as well as CD95 sensitivity and P-gp function but not constitutive Bax expression depend on the maturation stage of leukemic cells in adult de novo AML. P-gp function, the extent of spontaneous apoptosis in vitro and CD95 sensitivity are more predictive for response to induction chemotherapy in adult de novoAML than the constitutive expression levels of the apoptosis-related molecules CD95, Bcl-2 and Bax.     

Karyotype is an independent prognostic parameter in therapy-related acute myeloid leukemia (t-AML): an analysis of 93 patients with t-AML in comparison to 1091 patients with de novo AML.

The aim of this study was to compare the pattern of karyotype abnormalities of therapy-related acute myeloid leukemia (t-AML) (n=93) with de novo AML (n=1091), and to evaluate their impact on prognosis. Favorable, intermediate, and unfavorable cytogenetics were observed in 25.8, 28.0, and 46.2% of t-AML, and in 22.2, 57.3, and 20.4% of de novo AML. The median overall survival (OS) was shorter in t-AML than in de novo AML (10 vs 15 months, P=0.0007). Favorable and unfavorable cytogenetics had a prognostic impact with respect to OS in both t-AML (P=0.001 and 0.0001) and de novo AML (P<0.0001 and <0.0001). To define the overall prognostic impact of cytogenetics and t-AML, a multivariate Cox's regression analysis was performed for OS with favorable cytogenetics, unfavorable cytogenetics, t-AML, age, and white blood cell (WBC) count as covariates. All parameters proved to be independently related to OS (P=0.001 for t-AML, P<0.0001 for all other parameters). Within patients with t-AML, there were significant correlations between OS and both unfavorable (P<0.0001) and favorable cytogenetics (P=0.001), while age and WBC count had no impact on OS. In conclusion, these data indicate that cytogenetics are an important prognostic parameter in t-AML. Furthermore, t-AML is an unfavorable factor independent of cytogenetics with respect to survival.     

CD34+ cells from acute myeloid leukemia, myelodysplastic syndromes, and normal bone marrow display different apoptosis and drug resistance-associated phenotypes.

Myelodysplastic syndromes and acute myeloid leukemia (AML) are heterogeneous disorders in which conflicting results in apoptosis and multidrug resistance (MDR) have been reported. We have evaluated by multiparameter flow cytometry the expression of apoptosis- (APO2.7, bcl-2, and bax) and MDR-related proteins [P-glycoprotein (P-gp), multidrug resistance protein (MRP), and lung resistance protein (LRP)] specifically on bone marrow (BM) CD34+ cells, and their major CD32-/dim and CD32+ subsets, in de novo AML (n=90), high-risk myelodysplastic syndrome (n=9), and low-risk myelodysplastic syndrome (n=21) patients at diagnosis, and compared with normal BM CD34+ cells (n=6). CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients displayed higher expression of bcl-2 (P <0.0001) and lower reactivity for APO2.7 (P=0.002) compared with low-risk myelodysplastic syndrome and normal controls. Similar results applied to the two predefined CD34+ myeloid cell subsets. No significant differences were found in the expression of P-gp, MRP, and LRP between low-risk myelodysplastic syndrome patients and normal BM, but decreased expression of MRP (P <0.03) in AML and high-risk myelodysplastic syndromes and P-gp (P=0.008) in high-risk myelodysplastic syndromes were detected. Hierarchical clustering analysis showed that low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, whereas high-risk myelodysplastic syndromes were clustered together and mixed with the de novo AML patients. In summary, increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would be explained more appropriately in terms of an increased antiapoptotic phenotype rather than a MDR phenotype. In low-risk myelodysplastic syndromes abnormally high apoptotic rates would be restricted to the CD34- cell compartments.     

Morphologic dysplasia in de novo acute myeloid leukemia (AML) is related to unfavorable cytogenetics but has no independent prognostic relevance under the conditions of intensive induction therapy: results of a multiparameter analysis from the German AML Cooperative Group studies.

PURPOSE: On the basis of cytomorphology according to the French-American-British (FAB) classification, we evaluated the prognostic impact of dysplastic features and other parameters in de novo acute myeloid leukemia (AML). We also assessed the clinical significance of the recently introduced World Health Organization (WHO) classification for AML, which proposed dysplasia as a new parameter for classification. PATIENTS AND METHODS: We analyzed prospectively 614 patients with de novo AML, all of whom were diagnosed by central morphologic analysis and treated within the German AML Cooperative Group (AMLCG)-92 or the AMLCG-acute promyalocytic leukemia study. RESULTS: Patients with AML M3, M3v, or M4eo demonstrated a better outcome compared with all other FAB subtypes (P <.001); no prognostic difference was observed among other FAB subtypes. The presence or absence of dysplasia failed to demonstrate prognostic relevance. Other prognostic markers, such as age, cytogenetics, presence of Auer rods, and lactate dehydrogenase (LDH) level at diagnosis, all showed significant impact on overall and event-free survival in univariate analyses (P <.001 for all parameters tested). However, in a multivariate analysis, only cytogenetics (unfavorable or favorable), age, and high LDH maintained their prognostic impact. Dysplasia was not found to be an independent prognostic parameter, but the detection of trilineage dysplasia correlated with unfavorable cytogenetics. CONCLUSION: Our results indicate that cytomorphology and classification according to FAB criteria are still necessary for the diagnosis of AML but have no relevance for prognosis in addition to cytogenetics. Our results suggest that the WHO classification should be further developed by using cytogenetics as the main determinant of biology. Dysplastic features, in particular, have no additional impact on predicting prognosis when cytogenetics are taken into account.     

XIAP与Suvivin蛋白在弥漫性大B细胞淋巴瘤中的表达及其临床病理意义

 目的　研究XIAP在弥漫性大B细胞淋巴瘤（DLBCL）中的表达及其与Suvivin表达的相关性，并探讨其与临床预后的关系。方法　采用免疫组织化学法检测49例DLBCL中XIAP与Suvivin蛋白的表达水平。结果　XIAP、Suvivin蛋白在DLBCL中的阳性表达率分别为40.8 %（20／49）和44.9 %（22／49），高于良性淋巴结病变中阳性表达率，分别为15 %（3／20）和10 %（2／20），差异均有统计学意义（P＜0.05）；XIAP与Suvivin表达呈正相关（r＝0.382，P＝0.01）；XIAP表达的DLBCL患者的平均生存时间明显短于无表达的患者（P＜0.02）。结论　XIAP蛋白表达上调可能是DLBCL发生、发展的重要因素，并可能在抑制细胞凋亡过程中与Suvivin起协同作用，并可能是DLBCL一个新的预后不良因子。     

Reduced expression of FLIP SHORT in bone marrow of low risk myelodysplastic syndrome

Apoptosis is dysregulated in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML). FLIP (FLICE (FAS-associated death-domain-like IL-1 beta-converting enzyme)-inhibitory protein) has been described as an anti-apoptotic protein. Here, we characterize the expression level of FLIP(LONG) and FLIP(SHORT) mRNA in bone marrow aspirates from 61 patients diagnosed with MDS or AML. FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to high risk MDS, according to FAB classification (RA/RARS versus RAEB/RAEBt, P=0.0127) and IPSS (low risk/intermediate-1 versus intermediate-2/high risk, P=0.0345). Furthermore, FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to MDS-AML/AML de novo (P=0.0006), according to FAB classification. FLIP(LONG) expression did not differ between these groups. Increased levels of FLIP(SHORT) in RAEB and AML may be related to apoptosis resistance in these diseases and to MDS progression.     

Timed sequential therapy of acute myelogenous leukemia in adults: a phase II study of retinoids in combination with the sequential administration of cytosine arabinoside,idarubicin and etoposide

Clinical outcome in acute myeloid leukemia (AML) is unsatisfactory. One strategy to augment cytotoxicity is TST. All-trans retinoic acid (ATRA) down-regulates bcl-2 expression and heightens AML sensitivity to cytosine arabinoside (ara-C)-induced apoptosis in vitro. We designed a trial of ATRA plus ara-C-based TST in an attempt to enhance drug-induced apoptosis and clinical outcome. Between January 1998 and February 2000, 63 patients received induction TST (oral ATRA days 1-6, ara-C and idarubicin days 2-4, VP-16 days 9-11) followed by consolidation TST (ATRA, ara-C and idarubicin followed by a second ara-C infusion days 11-13). Complete remission (CR) was 60%, with higher rates for patients of <60 years (79%), de novo AML (70%), and non-adverse cytogenetics (81%). Median disease-free survival (DFS) for CR patients was 11.2 months (32% at 3+ years). For patients <60 years with de novo AML and non-adverse cytogenetics who underwent two-cycle TST, DFS was 67% at 3+ years. However, patients of age equal to 60 years and those with poor-risk disease features still have poor CR and DFS, despite the addition of ATRA.     

XIAP inhibitors induce differentiation and impair clonogenic capacity of acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) is a neoplasia characterized by the rapid expansion of immature myeloid blasts in the bone marrow, and marked by poor prognosis and frequent relapse. As such, new therapeutic approaches are required for remission induction and prevention of relapse. Due to the higher chemotherapy sensitivity and limited life span of more differentiated AML blasts, differentiation-based therapies are a promising therapeutic approach. Based on public available gene expression profiles, a myeloid-specific differentiation-associated gene expression pattern was defined as the therapeutic target. A XIAP inhibitor (Dequalinium chloride, DQA) was identified in an in silico screening searching for small molecules that induce similar gene expression regulation. Treatment with DQA, similarly to Embelin (another XIAP inhibitor), induced cytotoxicity and differentiation in AML. XIAP inhibition differentially impaired cell viability of the most primitive AML blasts and reduced clonogenic capacity of AML cells, sparing healthy mature blood and hematopoietic stem cells. Taken together, these results suggest that XIAP constitutes a potential target for AML treatment and support the evaluation of XIAP inhibitors in clinical trials.     

Expression of IAP family proteins in myelodysplastic syndromes transforming to overt leukemia

Bone marrow cells of patients with myelodysplastic syndromes (MDS) frequently undergo apoptosis, though the apoptotic cell ratio decreases when overt leukemia (OL) develops. Thus, we compared the expression of the inhibitor of apoptosis protein (IAP) gene family proteins in bone marrow samples from control, MDS, OL transformed from MDS (MDS --> OL), and de novo acute myelogenous leukemia (AML) subjects by the quantitative real-time RT-PCR method and an immunohistochemical approach. Overexpression of mRNA for survivin, cIAP1, NAIP and XIAP was significant in MDS bone marrow cells compared with control samples. However, the expression of mRNA for survivin, cIAP1 and cIAP2 exhibited a remarkable decrease after the development of OL (MDS --> OL). By immunohistochemistry, survivin was found to localize to the nucleus of myeloid cells in the majority of MDS cases. Next, the chronological changes in the expression of IAPs were determined in cases of MDS with evolution of OL. Although the expression of cIAP1 and cIAP2 revealed a sudden or gradual decrease as OL developed, survivin in many cases and XIAP in the majority of cases exhibited a peak of expression before a decline, indicating that these IAPs could be associated with the early events in the development of OL.     

AC133 expression on acute myeloid leukemia blasts: correlation to FAB and to CD34 expression and possible implications for peripheral blood progenitor cell purging in AML

AC133 is an antigen expressed on CD34+ hematopoietic progenitor cells. In acute myeloid leukemia (AML) it is expressed on leukemic blasts of most FAB subtypes. However, few data are available regarding coexpression of other surface antigens. We measured AC133 expression on AML blasts from 28 consecutive patients at initial diagnosis (n=26) or at diagnosis of first relapse (n=2) and on 26 leukapheresis products from 14 patients. In AML AC133 correlated with CD34 expression (Spearman r=0.4711, P=0.0114) and even stronger with combined CD34/CD33 expression (Spearman r=0.5083, P=0.0068). In leukapheresis products AC133 expression correlated with CD34 expression (Spearman r=0.7495, P=0.002) and the yield of the obtained amount of CD34+ cells (Spearman r=0.6484, P=0.0121). In conclusion AC133 expression is closely related to CD34 expression in AML. In leukapheresis products AC133 provides an additional marker for selection of PBPC autografts in AC133- AML.     

初治急性髓系白血病患者Bcl11a基因表达水平及其与预后的关系

目的 探讨急性髓系白血病（AML）患者Bcl11a基因表达水平及其临床意义.方法 通过实时荧光定量PCR方法检测80例初治AML患者及16例对照组Bcl11a基因的表达水平,分析其表达水平与疾病特征及临床疗效之间的关系.结果 初治AML患者Bcl11a表达水平高于对照组[0.039（0～ 0.504）比0.014（0.002 ～ 0.086）,P=0.004].以80例初治AML患者Bcl11a基因表达量的中位数作为分界点,将患者分为Bcl11a高表达组和低表达组各40例.Bcl11a低表达组AML患者中位年龄低于高表达组患者（29.5岁比41.0岁）,而初治AML患者在不同分层的性别、FAB分型、外周血象、骨髓原始细胞比例、遗传学危险度分层、免疫分型CD34表达间Bcl11a基因表达水平差异均无统计学意义（均P＞0.05）.Bcl11a基因低表达组患者完全缓解率高于高表达组[90％（36/40）比53％（21/40）,P=0.000],中位生存时间延长（268.0d比101.5 d,P=0.042）.结论 Bcl11a基因表达水平和AML患者年龄、疗效和预后存在一定的关系,Bcl11a基因高表达的AML患者预后较差.     

MRP3, BCRP, and P-glycoprotein activities are prognostic factors in adult acute myeloid leukemia.

PURPOSE: P-Glycoprotein (Pgp) is associated with poor outcome in acute myeloid leukemia (AML). We have investigated other ATP-binding cassette proteins such as BCRP, MRP1, MRP2, MRP3, and MRP5 for their potential implication in chemoresistance. EXPERIMENTAL DESIGN AND RESULTS: Eighty five AML patient samples were analyzed in this study. First, MRP3 function was higher in patients which had a high level of leukocytes (P = 0.01), a M5 FAB subtype (P = 0.04), and an intermediate or poor cytogenesis (P = 0.05). BCRP activity was not correlated with clinical or biological variables, but high Pgp activity was correlated with the following variables: CD34 expression (P = 0.002), FAB subtype (P = 0.002), intermediate or poor cytogenesis (P = 0.02), and elderly patients (P = 0.03). Second, Pgp, MRP3, and BCRP activities were correlated with complete remission (P = 0.02, P = 0.04, and P = 0.04, respectively), disease-free survival (P = 0.02, P = 0.03, and P = 0.25, respectively), and overall survival (P = 0.04, P = 0.04, and P = 0.05, respectively) in multivariate analysis. The patient samples expressing one or none of these Pgp, MRP3, or BCRP functional proteins have a better prognosis than the patients expressing two or three of these functional proteins (complete remission, P = 0.02; disease-free survival, P = 0.01; overall survival, P < 0.001). CONCLUSIONS: BCRP and MRP3 may also be involved in chemoresistance in AML, especially MRP3 in patients with M5 FAB. Additional modulation of BCRP or MRP3 to Pgp modulation may be necessary in some patients in order to improve the treatment outcome.     

复发/难治急性髓系白血病患者P-糖蛋白的表达及功能分析

 目的 研究复发／难治急性髓性白血病（AML）患者P-糖蛋白表达及其功能。方法 用免疫组化方法检测CD34表达,RT-PCR方法检测P-gp的表达,用流式细胞术检测P-gp的功能。结果 CD34表达与P-gp表达、功能呈正相关（r =0.621,P＜0.001;r =0.496,P＜0.001）,P-gp表达与功能呈正相关（r = 0.574,P＜0.01）;复发／难治组CD34阳性率（66.7 %）高于初治组（32.6 %）（P＜0.05）;复发／难治组P-gp功能阳性率（66.7 %）高于初治组（25.6 %）（P＜0.01）。结论 CD34表达、P-gp表达和P-gp功能三者间呈正相关;复发／难治AL组 CD34阳性率、P-gp功能阳性率高于初治组;P-gp功能的检测更具有临床指导意义。     

Sodium butyrate sensitizes human glioma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP

In TNF-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, co-treatment with nontoxic doses of sodium butyrate and TRAIL resulted in a marked increase of TRAIL-induced apoptosis. This combined treatment was also cytotoxic to glioma cells overexpressing Bcl-2 or Bcl-xL, but not to normal human astrocytes, thus offering an attractive strategy for safely treating resistant gliomas. Cotreatment with sodium butyrate facilitated completion of proteolytic processing of procaspase-3 that was partially blocked by treatment with TRAIL alone. We also found that treatment with sodium butyrate significantly decreased the protein levels of survivin and X-linked inhibitor of apoptosis protein (XIAP), two major caspase inhibitors. Overexpression of survivin and XIAP attenuated sodium butyrate-stimulated TRAIL-induced apoptosis, suggesting its involvement in conferring TRAIL resistance to glioma cells. Furthermore, the kinase activities of Cdc2 and Cdk2 were significantly decreased following sodium butyrate treatment, accompanying downregulation of cyclin A and cyclin B, as well as upregulation of p21. Forced expression of Cdc2 plus cyclin B, but not Cdk2 plus cyclin A, attenuated sodium butyrate/TRAIL-induced apoptosis, overriding sodium butyrate-mediated downregulation of survivin and XIAP. Therefore, Cdc2-mediated downregulation of survivin and XIAP by sodium butyrate may contribute to the recovery of TRAIL sensitivity in glioma cells.     

Expression of heat-shock proteins is associated with major adverse prognostic factors in acute myeloid leukemia

To identify prognostic factors alternative or additional to drug-resistance and apoptosis proteins, we studied the impact of the expression of heat-shock proteins (HSPs) in 98 newly diagnosed acute myeloid leukemia (AML). HSP27 was expressed by 39%, HSP60 by 26%, HSP70 by 58%, HSP90 by 41%, and HSP110 by 30% of cases. HSP expressions were correlated with that of differentiation antigens (CD34, CD14, CD15, CD33) and that of drug-resistance (MRP, MRK) and apoptosis (Bcl-2) proteins. HSP90 and HSP110 were correlated with FAB subtype and karyotypic grouping. Complete remission (CR) was obtained in 68 cases (69%). Median disease-free survival (DFS) of the 68 remitters was 18.1 months with a 3-year DFS rate of 41%. CR rates were higher in patients with lower expression of HSPs. Overall survival (OS) was significantly longer in patients with lower expression of HSPs. Cytogenetics, CD34 positive expression, MRK positive expression, and HSP110 positive expression remained as pejorative prognostic factors for OS in the multivariate analysis. When considering patients with intermediate risk cytogenetics, HSP110 and MRP positive expressions and CD33 negative expression were of poor outcome, while HSP27 and HSP60 positive expressions appeared of pejorative prognostic value in patients with unfavorable karyotypes.     

P-glycoprotein and terminal transferase expression identify prognostic subsets within cytogenetic risk classes in acute myeloid leukemia.

Clinical and biological features were assessed in 204 consecutive de novo adult acute myeloid leukemia (AML) patients who received intensive chemotherapy regimens. Multiparameter flow cytometric assays both of the multidrug resistance (MDR-1)-associated P-glycoprotein (PGP) using the UIC2 monoclonal antibody (MoAb), and of terminal transferase (TdT) were performed. Cytogenetic findings were obtained from 196 patients with high resolution banding. At onset, UIC2 and TdT positivities were detected in 58.5% and 24% of cases, respectively. There were strict correlations either between UIC2 negativity and FAB M3 or between TdT and FAB M0-M1 (P = 0.001 and < 0.0001, respectively). On the other hand, age was significantly associated with cytogenetic risk classes (P < 0.0001). CD34 positivity was highly correlated with TdT expression (P < 0.0001). Moreover, CD7 and CD11b were significantly represented in UIC2+ subset (P < 0.0001). Rhodamine 123 (Rh 123) efflux was significantly higher in 75 UIC2 positive patients compared to 65 UIC2 negative ones (P < 0.001). As regards to cytogenetics, TdT positivity was strongly related either to t(9;22) or single/associated anomalies of chromosome 7; on the other hand, most or all cases with t(8;21) or t(15;17) were UIC2 or TdT negative, respectively. The rate of first complete remission (CR) differed both between UIC2+ and UIC2- cases and between TdT+ and TdT- ones (40% versus 72%, P < 0.001; and 36% versus 61%, P = 0.001, respectively). The survival rates (Kaplan-Meier method) were significantly shorter either in UIC2+ or in TdT+ patients (P = 0.005 and = 0.011, respectively). UIC2 and TdT negative cases showed longer remission duration (P = 0.03 and = 0.22, respectively). The additional effect of UIC2 and TdT on prognosis allowed us to identify two subsets of patients, the first [UIC2- TdT-] at better and the second [UIC2+ TdT+] at worse clinical outcome compared to single UIC2 and TdT cases, concerning CR (P < 0.001), survival (P < 0.0001) and CR duration (P = 0.007). The combinations [UIC2+ TdT-] and [UIC2- TdT+] showed an intermediate clinical course. A strong difference was found between poor risk and intermediate/favorable risk cytogenetic classes with regard to CR rate (P < 0.0001), overall survival and CR duration (P < 0.001). Nevertheless, within the poor risk class, UIC2 positivity was able to identify patients at worst prognosis with regard to CR (P = 0.005), survival (P = 0.02) and CR duration (P = 0.015). On the other hand, UIC2 and TdT negativity allowed us to distinguish patients with longer survival (P = 0.012 and = 0.04, respectively) and CR duration (P = 0.04 and = 0.025, respectively) within the intermediate/favorable risk class. The independent prognostic value of UIC2, TdT and cytogenetic risk classes was confirmed in multivariate analysis. These results suggest that PGP and TdT expressions, together with cytogenetic findings, may represent a basic predictor of chemotherapeutic failure in AML.