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.

High expression levels of x-linked inhibitor of apoptosis protein and survivin correlate with poor overall survival in childhood de novo acute myeloid leukemia.

PURPOSE: Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. EXPERIMENTAL DESIGN: Using flow cytometry and Western blot analysis, we, therefore, investigated 45 leukemic cell samples from children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins [CD95, Bcl-2, Bax, Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP-1), survivin]. RESULTS: XIAP (P < 0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by French-American-British (FAB) morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (P < 0.01) and Bcl-xL (P < 0.01) expression was lower in patients with favorable rather than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP [30 (n = 10) versus 41 months (n = 34); P < 0.05] and survivin [27 (n = 10) versus 41 months (n = 34); P < 0.05]. CONCLUSIONS: We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups, and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics, and poor overall survival should be confirmed within prospective pediatric AML trials.

Differences in the expression pattern of apoptosis-related molecules between childhood and adult de novo acute myeloid leukemia.

Distinct expression patterns of pro- and anti-apoptotic proteins may contribute to different prognoses and therapy outcomes in adult versus childhood acute myeloid leukemia (AML). Therefore, we investigated whether expression levels of apoptosis-related proteins CD95, Bcl-2, Bax, Bcl-xL, procaspase-3, XIAP, cIAP-1, and survivin differ between children and adults with de novo AML.

XIAP expression correlates with monocytic differentiation in adult de novo AML: impact on prognosis.

Antiapoptotic proteins like the inhibitor of apoptosis proteins (IAPs) are molecular markers potentially useful for the characterization of acute myeloid leukemia (AML). We screened 92 adults with de novo AML for the protein expression of various IAPs, Bcl-2 family members and the proform of Caspase-3 using quantitative immunoblot and flow cytometry. XIAP expression correlated with myelomonocytic French-American-British (FAB) subtypes M4/M5 (P < 0.05) and expression of monocytic markers (CD 14, CD 36; P < 0.05; CD 4, HLA-DR; P < 0.01) in AML blasts. In addition, XIAP was overexpressed in normal monocytes but undetectable in granulocytes. In AML, XIAP expression was significantly lower in patients with favorable than intermediate or poor cytogenetics (n = 74; P < 0.05). In total, 62 of the examined patients were treated according to the German AML Cooperative Group (AMLCG) 92 protocol. These patients were analyzed for prognostic significance of apoptosis-related proteins. Patients expressing low levels of XIAP enjoyed better overall survival than patients expressing high amounts of XIAP (mean, 9 (n = 41) versus 19 months (n = 21); P < 0.05). Other IAPs, most importantly Survivin, were of no prognostic value. We conclude that XIAP but not other IAP family members is associated with monocytic differentiation in normal and malignant myelopoiesis, and may be of prognostic significance for overall survival in adult de novo AML.

Correlation of protein expression and gene expression in acute leukemia.

BACKGROUND: Flow cytometry (FC) is a standard method for diagnosing and subclassifying acute myeloid (AML) and acute lymphoblastic (ALL) leukemias and allows the analysis of cell surface and intracellular proteins. In the future, diagnostic procedures may include oligonucleotide microarray analysis (MA) to detect expression patterns of large numbers of specific genes. METHODS: For comparison between methods, we performed FC and MA by using the Affymetrix GeneChip HG-U133A microarray in parallel and correlated protein expression levels and mRNA abundance of 39 relevant genes in 113 patients with newly diagnosed AML and ALL and four normal bone marrow samples. RESULTS: In 1,512 of 2,187 (69.1%) comparisons between methods, congruent results were obtained with regard to positivity or negativity of expression, respectively. Specifically, there was a significant correlation between protein expression and mRNA abundance for genes essential for diagnosing and subclassifying AML and ALL with regard to positivity and expression. CONCLUSIONS: These data suggest that protein expression is highly correlated to mRNA abundance in AML and ALL. Further, expression patterns of specific genes provide important information at diagnosis for patients with AML and ALL that may be used for the discrimination from other leukemias.

In vivo expression of survivin and its splice variant survivin-2B: impact on clinical outcome in acute myeloid leukemia.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in most human cancers, but undetectable in normal differentiated adult tissue in vivo. Because of this cancer-related expression, survivin is a promising target for cancer therapy. To determine the expression and prognostic role of survivin in acute myeloid leukemia (AML), we investigated the mRNA expression pattern of survivin and of the splice variants survivin-2B and survivin-DeltaEx3 in adult (n = 74) and children (n = 31) with de novo AML using RT-PCR. Survivin was the predominant transcript variant in AML cells, whereas significantly lower levels of survivin-2B and survivin-DeltaEx3 were observed (p < or = 0.0001). Neither expression of survivin nor of any splice variant correlated with maturation stage (FAB subtypes, immunophenotype) or cytogenetic risk groups. For AML cases treated according to AMLCG92 (adult) and AML-BFM93 (children) protocols, respectively, expression patterns were correlated with clinical data: in adult AML (n = 51), low expression of survivin-2B correlated with a better overall survival (p = 0.05; mean survival time 19 months vs. 9 months) and a better eventfree survival (p < or = 0.01; 27 months vs. 10 months). In childhood AML (n = 31), high survivin-DeltaEx3 expression was associated with a shorter overall survival (p < or = 0.05; 24 months vs. 43 months). We conclude that certain survivin splice variants have potential prognostic impact for long-term therapy outcome in adult as well as childhood de novo AML.

[Expression and significance of apoptosis protein inhibitor survivin and XIAP, in patients with myelodysplastic syndromes and in the cell line MUTZ-1].

OBJECTIVE: To investigate the expression of apoptotic protein inhibitors, survivin and XIAP, in patients with myelodysplastic syndromes (MDS) and in the cell line MUTZ-1, as well as to explore the possible mechanisms of homoharringtonine (HHT) in the treatment of MDS. METHODS: Bone marrow samples from 47 patients with de novo MDS at diagnosis were examined and bone marrow samples from 15 normal donors were used as control. A MDS-RAEB cell line MUTZ-1 was used as in vitro model. Detection of apoptotic cells and cell cycle analysis were performed with flow cytometry (FACS). The expression of apoptotic protein inhibitor survivin and XIAP in the MDS cells were detected by RT-PCR technique. MUTZ-1 were treated with antisense oligodeoxynucleotide (AS-ODNs) of survivin and or HHT, the effects were evaluated by cell viability and cell apoptosis. RESULTS: Survivin mRNA positive rate in MDS were significantly higher than that in normal controls (38.3% and 0, respectively, P < 0.01), and the positive rate in high risk group (RAEB, RAEBT and CMML) was significantly higher than that in RA/RAS group (53.6% and 16.7%, respectively, P < 0.05). XIAP was expressed in all untreated MDS and healthy controls. XIAP mRNA expression in high risk group was significantly higher than that in RA/RAS subtypes and healthy controls (1.55 +/- 0.34, 0.74 +/- 0.24, and 1.01 +/- 0.28, respectively, P < 0.01). However, XIAP mRNA expression was significantly lower in RA/RAS subtypes than in healthy control (0.74 +/- 0.24 and 1.01 +/- 0.28, P < 0.054). Apoptosis peak detected by FACS analysis and positive Annexin V FITC staining on cell membrane indicated that HHT could induce MUTZ-1 cell undergoing apoptosis in dose- and time-dependent manners. Treatment of MUTZ-1 cells with HHT revealed that HHT could significantly down-regulate survivinexpression but had no significant effect on XIAP expression in the cells. AS-ODNs of survivin could inhibit MUTZ-1 cells growth, induce them to apoptosis and sensitize them to HHT. CONCLUSION: The expression levels of survivin; Institute of Hematology, Oncology and Tumor Immunology, Robert Roessle Clinic, Humboldt University, Berlin, Germany (Wolf Dieter Ludwig, Christian Wuchter) and XIAP vary in different subtypes of MDS patients, suggesting that the proteins may play an important role in the pathogenesis of the disease. Down-regulation of survivin in MUTZ-1 cells may be one of the mechanisms that HHT induces apoptosis of MDS cells.

In vitro susceptibility to dexamethasone- and doxorubicin-induced apoptotic cell death in context of maturation stage, responsiveness to interleukin 7, and early cytoreduction in vivo in childhood T-cell acute lymphoblastic leukemia.

Within childhood T-cell acute lymphoblastic leukemia (T-ALL), patients with a cortical (CD1a(+)) immunophenotype have been identified as a subgroup with favorable outcome in the acute lymphoblastic leukemia-Berlin-Frankfurt-Münster (ALL-BFM), Cooperative study group for childhood acute lymphoblastic leukemia (COALL) and Pediatric Oncology Group studies. We investigated in leukemic samples of children with T-ALL (n = 81) whether the different in vivo therapy response could be linked to differential in vitro susceptibility to apoptotic cell death. The extent of dexamethasone- as well as doxorubicin-induced apoptosis, detected by annexin V staining, positively correlated with the expression levels of CD1a (Spearman correlation coefficient, r(s) = 0.3 and 0.4, respectively; P <.01). When compared to cortical T-ALL, mature (CD1a(-), surface CD3(+)) T-ALL were significantly more resistant to doxorubicin, and immature, pro-/pre-T-ALL were more resistant to both drugs (P <.05). Apoptosis-related parameters (Bax, Bcl-2, CD95, and CD95-induced apoptosis) did not account for differential susceptibility to drug-induced apoptosis. By contrast, an interleukin 7-induced rescue of leukemic cells from spontaneous apoptosis, recently proposed to reflect distinct developmental stages and apoptotic programs in T-ALL, was highly associated with susceptibility to dexamethasone- but not doxorubicin-induced apoptosis (P <.001 versus P =.08). Analysis of clinical data showed that in vitro susceptibility to dexamethasone (but not to doxorubicin) closely correlated with early in vivo therapy response characterized by percentages of blast cells in bone marrow on day 15 (r(s) = -0.46, P =.001). Taken together, the in vitro assessment of drug-induced apoptosis revealed maturation-dependent differences within childhood T-ALL. The enhanced sensitivity to both drugs in cortical T-ALL might account for the better in vivo treatment response of this prognostically favorable T-ALL subgroup.

Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease.

FLT3 length mutation (FLT3-LM) is a molecular marker potentially useful for the characterization of acute myeloid leukemia (AML). To evaluate the distribution of FLT3-LM within biologic subgroups, we screened 1003 patients with AML at diagnosis for this mutation. FLT3-LM was found in 234 (23.5%) of all patients and thus is the most frequent mutation in AML described so far. Of all positive patients, 165 (70.5%) revealed a normal karyotype. Of the 69 patients with chromosome aberrations, 24 (34.8%) had a t(15;17). The mutation was rare in AML with t(8;21), inv(16) 11q23 rearrangements, and complex karyotypes. FLT3-LM was not distributed equally within different French-American-British (FAB) subtypes and was correlated with a high peripheral blood count in FAB M1, M2, and M4 (P <.0001). In addition, the median age of patients with the mutation was lower (54.9 vs 57.6 years; P =.043), and, at a ratio of 1.36:1 (P =.023), the mutation was more frequent in females than in males. Within the AMLCG study, FLT3-LM was of intermediate prognostic significance. The complete remission rate of 70.3% in patients with FLT3-LM was similar to that (70.4%) in patients without FLT3-LM. Overall survival was not different between patients with or without FLT3-LM. In contrast, patients with FLT3-LM had a significantly shorter event-free survival (7.4 vs 12.6 months; P =.0072) because of a higher relapse rate. Besides the importance of FLT3-LM for biologic and clinical characterization of AML, we show its value as a marker for disease monitoring based on 120 follow-up samples of 34 patients.