Brain And Acute Leukemia, Cytoplasmic Research Articles

Heritable polymorphism predisposes to high BAALC expression in acute myeloid leukemia

Overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is implicated in myeloid leukemogenesis and associated with poor outcome in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia patients. Additionally, high BAALC expression occurs in glioblastoma, melanoma, and childhood gastrointestinal stroma tumors, suggesting an oncogenic role for BAALC. However, the mechanisms underlying the deregulated expression are unknown. We hypothesized that a common heritable genetic feature located in cis might account for overexpression of BAALC in an allele-specific manner. By sequencing the genomic region of BAALC we identified nine informative single nucleotide polymorphisms (SNPs) and tested them for a possible association with BAALC expression levels. We show that BAALC overexpression occurs in the presence of the T allele of SNP rs62527607[GT], which creates a binding site for the activating RUNX1 transcription factor in the BAALC promoter region. The mechanism is demonstrated experimentally in vitro using luciferase reporter assays and electrophoretic mobility shift assay (EMSA) analysis. The association of high BAALC expression with the T allele and its correlations with RUNX1 expresser status are shown in vivo in a test set (n = 253) and validation set (n = 105) of samples from cytogenetically normal AML patients from different populations. Thus, we identify a heritable genomic feature predisposing to overexpression of an oncogene, thereby possibly leading to enhanced AML leukemogenesis. Our findings further suggest that genomic variants might become useful tools in the practice of personalized medicine.

ShRNA-Mediated BAALC knockdown affects proliferation and apoptosis in human acute myeloid leukemia cells

Brain and acute leukemia, cytoplasmic (BAALC) is a novel molecular marker indicating an inferior outcome in acute myeloid leukemia (AML) with normal cytogenetics. The biological function of BAALC is largely unknown. In this study, BAALC gene expression in an acute myeloid leukemia cell line KG1a was knocked down by a small hairpin RNA (shRNA). The expression of BAALC mRNA and protein in the knockdown cells was significantly inhibited. The proliferation and apoptosis status in the knockdown cells were investigated. The growth curves and FACS analysis demonstrated that BAALC gene knockdown resulted in decreased proliferation and enhanced apoptosis in KG1a cells. These results indicated that BAALC may act as an adverse prognostic factor through prompting proliferation and inhibiting apoptosis in leukemia cells.

BAALC Regulates Hematopoietic Stem Cells Through P53 and Promotes Leukemia with Impaired P53 Function

Abstract 48Elevated expression of BAALC (brain and acute leukemia, cytoplasmic) is an adverse prognostic factor in patients with cytogenetically normal acute myeloid leukemia (CNAML). However, its precise role in normal hematopoiesis and leukemogenesis remains to be elucidated. To address this issue, we evaluated the effect of BAALC overexpression upon hematopoiesis and leukemogenesis and generated BAALC-deficient mice.First, we examined the effect of BAALC overexpression upon hematopoiesis. BAALC-transduced bone marrow (BM) cells had decreased colony-forming capacity. Bone marrow transplantation (BMT) assay demonstrated that the donor chimerism was lower in transplanted mice with BAALC-overexpressed cells than with control cells. To examine the mechanism, we performed gene expression analysis. This revealed that p53 pathway is up-regulated in BAALC-overexpressed cells. Colony-forming assay and BMT assay using p53-deficient BM cells demonstrated that the blockage of proliferation by BAALC overexpression is canceled in p53-deficient cells in vitro and in vivo, suggesting that the inhibition of proliferation by BAALC overexpression is dependent on p53 pathway.Next, we generated BAALC-conventionally deficient mice. Most BAALC-deficient female mice died between E11.5 and E12.5 because of defects of placental development. BAALC-deficient E11.5 yolk sac cells had increased colony-forming capacity. Transplantation assay showed that the donor chimerism at 4 weeks after transplantation was higher in transplanted mice with BAALC-deficient E12.5 fetal liver cells than with wild fetal liver cells. Moreover, the frequency of G0/G1 phase cells was decreased in BAALC-deficient yolk sac. Thus, the proliferation is activated and the frequency of quiescent cells is reduced in BAALC-deficient embryonic hematopoietic cells. In addition, we examined the role of BAALC in adult hematopoiesis using BAALC-conditionally deficient mice with Mx-Cre system. In BAALC-deficient (Mx-Cre+BAALC flox/flox) BM, long-term hematopoietic stem cells (HSCs), identified as CD34−Flt3−cKit+Sca1+Lin− cells, were reduced as compared with control (Mx-Cre−BAALC flox/flox) BM. BAALC-deficient adult BM had impaired long-term repopulating ability in serial competitive BMT. Moreover, BAALC-deficient mice showed higher mortality rates after weekly 5-FU injections. On the other hand, the ability of colony forming unit spleen (CFU-S) was enhanced and the recovery after myelosuppression induced by single 5-FU injection was faster in BAALC-deficient mice, indicating that BAALC-deficient HSCs have enhanced short-term repopulating ability. Next, to address the relationship between BAALC and p53 protein levels, we performed intracellular flow cytometric analysis and immunocytochemical staining. These experiments revealed that p53 protein is reduced in BAALC-deficient HSCs. Immunoprecipitation revealed that BAALC interferes with the binding of MDM2 to p53, which leads to stabilization of p53 protein.Finally, we tested the effect of BAALC overexpression in leukemia mouse models. BAALC overexpression promoted leukemia in mice in cooperation with TEL/PDGFBR-AML1/ETO (TPAE). BM cells transduced with TPAE and BAALC had increased colony-forming capacity as compared with TPAE and mock-transduced cells. By contrast, BAALC cannot accelerate myc/BCL2-induced leukemia. Western blot showed that p53 is inactivated in TPAE leukemic cells but not in myc/BCL2 leukemic cells. BM cells transduced with myc/BCL2 and BAALC had decreased colony-forming capacity as compared with myc/BCL2 and mock-transduced cells. However, BAALC overexpression promoted colony-forming capacity of p53-deficient cells transduced with myc/BCL2. Similarly, BAALC overexpression promoted the colony-forming capacity of BM cells transduced with BCR/ABL which is known to inactivate p53. Promoted proliferation of BCR/ABL-transduced cells by BAALC overexpression was canceled by the MDM2 antagonist, Nutlin-3. Taken together, BAALC overexpression accelerates leukemia with impaired p53 pathway.In conclusion, these data suggest that BAALC activates p53 pathway and maintains the stemness in normal HSCs. Once p53 pathway is impaired, however, BAALC promotes proliferation of leukemic cells. Our data for the first time uncovered a novel function of BAALC, which may lead to novel therapeutic strategy for CNAML with poor prognosis. Disclosures:No relevant conflicts of interest to declare.

In Acute Promyelocytic Leukemia (APL) Low BAALC Gene Expression Identifies a Group of Patients with Favorable Outcome in Overall Survival and Relapse Free Survival,

Abstract 3543 Introduction:Acute promyelocytic leukemia (APL) is characterized by the translocation t(15;17) which results in the PML-RARα fusion protein. ATRA based treatment regimens lead to long term survival in approximately 75% of cases. However, relapse occurs in about 15% of APL patients. Moreover, it is not yet known which underlying pathomechanisms drive relapse of the disease. In addition, to avoid over- or undertreatment of patients, respectively, risk stratification of patients according to biological or molecular criteria would most probably improve current treatment modalities. As opposed to other leukemias, in APL no molecular markers have been established for risk stratification as yet. The gene brain and acute leukemia, cytoplasmic (BAALC) has been shown to be of prognostic relevance in other leukemias such as T acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Therefore, the aim of our study was to evaluate whether BAALC expression could be of prognostic value in APL.Materials and Methods: Bone marrow mononuclear cells were retrospectively evaluated in 86 APL patients after informed consent. 50 patients were female and 36 patients were male. Median age was 47 years (range: 19–82 years). All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Glucose-6-phosphate-isomerase (GPI) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res; 2001) after determination of PCR efficiencies. BAALC expression groups were defined as follows: BAALC expression lower than the 25th percentile (BAALClow) and higher than the 25th percentile (BAALC high). Time to complete remission, relapse free survival (RFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). For statistical analysis the Statistical Analysis Software version 9.2 was used. Results:In univariate analysis low BAALC expression was significantly associated with a superior OS. Patients in the BAALClow group showed an OS at 10 years of 82% as compared to 60% in the BAALChigh group (p=0.025). Moreover, RFS in the BAALClow group was significantly higher at 10 years as compared to the BAALChigh group (86% vs. 59%; p=0.0087). Time to complete remission was not correlated to BAALC expression levels. Conclusion:Low BAALC expression identifies a group of APL patients with a highly superior outcome indicated by a better OS and RFS in these patients. However, these findings need to be validated in an independent prospective study and it should be investigated whether this marker is independent of the applied treatment regimen and other known relevant markers. However, since the translocation t(15;17) by itself is not able to induce leukemia BAALC might be an interesting target for further investigation. Disclosures:No relevant conflicts of interest to declare.

WT1 overexpression at diagnosis may predict favorable outcome in patients with de novo non-M3 acute myeloid leukemia

We reviewed the frequency and prognostic significance of FLT3 (fms-like tyrosine kinase receptor-3) and NPM (nucleophosmin) gene mutations and WT1 (Wilms’ tumor) and BAALC (brain and acute leukemia, cytoplasmic) gene expression in 100 consecutive patients with intermediate and poor cytogenetic risk de novo acute myeloid leukemia (AML) receiving conventional anthracycline–AraC based therapy. We observed a strict relationship between unfavorable karyotype and BAALC >1000 (p = 0.0001). Multivariate analysis of 81 patients with intermediate karyotype revealed that younger age (p = 0.00009), NPM gene mutation (p = 0.002), and WT1 >75th percentile (>2365) (p = 0.003) were independent, positive factors for complete remission (CR). WT1 expression above 2365 was correlated also to longer event-free survival (EFS) and overall survival (OS) in the same subset of patients (p = 0.003 and p = 0.02, respectively); the same finding occurred in younger patients with AML with intermediate karyotype (p = 0.008 and p = 0.01, respectively). In patients with intermediate karyotype, FLT3 internal tandem duplication (ITD) negatively affected EFS (EFS at 30 months: 30% vs. 6% in FLT3-ITD negative and FLT3 positive patients, respectively; p = 0.01) and OS (OS at 30 months: 38% vs. 20%, p = 0.03). The positive prognostic value of high WT1 expression does not have a clear explanation; it may be implicated either with WT1 anti-oncogenic function, or with the stimulating effect of WT1 oncogene on the leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.

BAALC (brain and acute leukemia, cytoplasmic)

Review on BAALC (brain and acute leukemia, cytoplasmic), with data on DNA, on the protein encoded, and where the gene is implicated.

BAALC-associated gene expression profiles define IGFBP7 as a novel molecular marker in acute leukemia

Over expression of BAALC (brain and acute leukemia, cytoplasmic) predicts an inferior outcome in acute myeloid leukemia (AML) and acute lymphoblastic leukemia patients. To identify BAALC-associated genes that give insights into its functional role in chemotherapy resistance, gene expression signatures differentiating high from low BAALC expressers were generated from normal CD34(+) progenitors, T-acute lymphoblastic leukemia (T-ALL) and AML samples. The insulin-like growth factor binding protein 7 (IGFBP7) was one of the four genes (CD34, CD133, natriuretic peptide receptor C (NPR3), IGFBP7) coexpressed with BAALC and common to the three entities. In T-ALL, high IGFBP7-expression was associated with an immature phenotype of early T-ALL (P<0.001), expression of CD34 (P<0.001) and CD33 (P<0.001). Moreover, high IGFBP7-expression predicted primary therapy resistance (P=0.03) and inferior survival in T-ALL (P=0.03). In vitro studies revealed that IGFBP7 protein significantly inhibited the proliferation of leukemia cell lines (Jurkat cells: 42% reduction, P=0.002; KG1a cells: 65% reduction, P<0.001). In conclusion, IGFBP7 was identified as a BAALC coexpressed gene. Furthermore, high IGFBP7 was associated with stem cell features and treatment failure in T-ALL. In contrast to BAALC, which likely represents only a surrogate marker of treatment failure in acute leukemia, IGFBP7 regulates the proliferation of leukemic cells and might be involved in chemotherapy resistance.

Prognostic significance of the BAALC isoform pattern and CEBPA mutations in pediatric acute myeloid leukemia with normal karyotype: a study by the Japanese Childhood AML Cooperative Study Group

High BAALC (brain and acute leukemia, cytoplasmic) gene expression may indicate an adverse prognosis for adults who have acute myeloid leukemia (AML) and a normal karyotype, but its prognostic significance for pediatric AML cases is unclear. Whether different BAALC isoform patterns are of prognostic significance is also unclear. Newly diagnosed AML patients with normal karyotype who were treated by the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed in terms of their BAALC expression levels (n = 29), BAALC isoforms (n = 29), and CEBPA mutations (n = 49). Eleven and 18 patients exhibited high and low BAALC expression, respectively, but these groups did not differ significantly in terms of overall survival (54.6 vs. 61.1%, P = 0.55) or event-free survival (61.4 vs. 50.0%, P = 0.82). Three of these 29 patients (10.3%) expressed the exon 1-5-6-8 BAALC isoform along with the expected 1-6-8 isoform and had adverse clinical outcomes. Novel CEBPA mutations were also identified in four of 49 patients (8.2%). All four patients have maintained complete remission for at least 5 years. Thus, 1-5-6-8 isoform expression may be associated with an adverse prognosis in pediatric AML with normal karyotype. CEBPA mutations may indicate a favorable prognosis.

BAALC and ERG expression in acute myeloid leukemia with normal karyotype: impact on prognosis

Cytogenetic aberrations are important prognostic factors in acute myeloid leukemia (AML). About 45% of de novo AML lack cytogenetic abnormalities, so identification of predictive molecular markers might improve therapy. We studied the prognostic impact of brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) expression in AML with normal karyotype. Pretreatment bone marrow samples from 30 cytogenetically normal AML patients were analysed for BAALC and ERG expression using real time RT-PCR. The patients were dichotomized at BAALC and ERG mean expression into low and high expression. BAALC showed high expression in 70% of patients and its expression did not correlate with the clinical parameters of patients. ERG was high in 33.3% of patients and its expression was associated with lower ages and higher white cell counts. With follow-up for 2 years, patients with high BAALC and high ERG had low rates of clinical remission (P < 0.005) and inferior overall survival (OS) (P < 0.001 and <0.002 for BAALC and ERG respectively). No significant association was observed between the increase in BAALC and ERG expression (P = 0.398). Multivariable analysis confirmed high BAALC expression as an independent risk factor for OS. Overexpression of BAALC and ERG either separate or concomitant predict adverse clinical outcome and may define important risk factor in cytogenetically normal AML.

BAALC is an important predictor of refractoriness to chemotherapy and poor survival in intermediate-risk acute myeloid leukemia (AML)

We have analyzed brain and acute leukemia, cytoplasmic (BAALC) gene expression and other genetic markers (ERG, EVI1, MN1, PRAME, WT1, FLT3, and NPM1 mutations) in 127 intermediate-risk acute myeloid leukemia (AML) patients: 98 cytogenetically normal and 29 with intermediate-risk cytogenetic alterations. High versus low BAALC expressers showed a higher refractoriness to induction treatment (31% vs 10%; p = .005), lower complete remission rate after salvage therapy (82% vs 97%; p = .010), and lower 3-year overall (23% vs 58%, p < .001) and relapse-free survival (26% vs 52%, p = .006). Similar results were found when cytogenetic subgroups were analyzed separately. Multivariate models confirmed the unfavorable prognosis of this marker. In conclusion, BAALC is a relevant prognostic marker in intermediate-risk AML.

Overexpression of BAALC Indicates Drug Resistance and Inferior Survival in Adult B-Precursor Acute Lymphoblastic Leukemia.

Abstract 1581▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-607 IntroductionHigh expression of the Brain And Acute Leukemia, Cytoplasmic (BAALC) gene is associated with primary chemotherapy resistance and inferior outcome in adult patients with cytogenetically normal (CN) acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (ALL). Due to the prognostic significance of BAALC in different leukemic lineages, we investigated its expression pattern and prognostic impact in adult B-precursor ALL, with a focus on patients lacking the molecular high-risk factors BCR-ABL and MLL-AF4. MethodsBAALC expression was determined by quantitative real-time RT-PCR in pretreatment bone marrow samples of 368 adult patients with newly diagnosed B-precursor ALL treated on the 06/99 and 07/03 GMALL trials. Patients were grouped into tertiles (T1-T3) according to BAALC expression levels. The median follow-up was 44 months. Patients who received stem cell transplantation (SCT) in first remission (CR1) were censored at the time of SCT for survival analyses. In an additional set of 51 adult B-precursor ALL specimens, gene-expression profiling (GEP) was performed using HG-U133 Plus 2.0 (Affymetrix) to obtain insights into the biological function of BAALC and its potential role in drug resistance. Samples were divided into tertiles (t1-t3) by the median expression of the two probe sets representing BAALC (218899_s_at, 222780_s_at). ResultsHigher BAALC expression (T3 vs T2 vs T1) was associated with a higher age (P<0.001), a higher white blood cell (WBC) count (P=0.008), CD34 positivity (P=0.001), presence of BCR-ABL (P<0.001), and of MLL-AF4 (P<0.001) in the overall cohort. In multivariate analysis including clinical and molecular variables, higher BAALC expression was the only predictive factor for primary therapy resistance in the overall cohort [OR 2.4 (95% CI 1.4-4.3); P=0.002]. In the BCR-ABL- and MLL-AF4-negative subgroup, higher BAALC expression independently predicted primary resistant disease [OR 4.2 (95% CI 1.4-12.3); P=0.01], as did the immunophenotype. BCR-ABL- and MLL-AF4-negative patients with higher BAALC expression had a significantly shorter overall survival [OS; 5-year OS: BAALC T3: 38% (95% CI 22-54), BAALC T2: 52% (95% CI 36-67), BAALC T1: 70% (95% CI 59-81); P=0.004]. Upon multivariate analysis, BAALC was of independent prognostic significance for OS in BCR-ABL- and MLL-AF4-negative patients [HR 1.4 (95% CI 1.0-2.0); P=0.03], as well as the factors WBC and age. Furthermore, we identified high BAALC expression as an independent predictive factor for inferior OS in the GMALL standard risk (SR) group [BCR-ABL- and MLL-AF4-negative patients with early therapy response and WBC '30/nl at initial diagnosis; HR 1.5 (95% CI 1.0-2.3); P=0.04]. The other variable in the final model predicting OS for SR patients was age. Interestingly, BCR-ABL- and MLL-AF4-negative patients allocated to allogeneic SCT in CR1 with high BAALC expression showed a particular survival benefit with a 5-year OS of 62%. In GEP analysis, high BAALC expression (t3 vs t1) was associated with the up-regulation of hematopoietic stem cell markers (CD34, CD99, FZD6) and genes implicated in chemoresistance (TSPAN7, LYN). Moreover, GEP revealed down-regulation of genes in the high BAALC group that had been related to a favourable outcome in adult ALL and AML (CDKN1C/p57Kip2, RGS2). ConclusionsIn addition to T-ALL and CN-AML, we have shown that BAALC predicts an unfavourable response to induction chemotherapy and inferior OS in adult B-precursor ALL patients. These clinical observations are further supported by the GEP signature associated with high BAALC expression. The lineage-independent association of BAALC with an immature, highly proliferative, and resistant leukemic phenotype underscores a possible involvement of BAALC in leukemogenesis and its putative role in chemoresistance. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Gene Expression Profiling Identifies IGFBP7 as a BAALC Co-Expressed Gene with a Functional Role in Acute Leukemia.

Abstract 2629▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-605The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) is a molecular marker of immature hematopoietic cells. In normal hematopoiesis, BAALC is highly expressed in CD34 positive progenitors and down-regulated with cell differentiation. Moreover, high mRNA expression levels of BAALC have shown to be an adverse risk factor and were associated with chemotherapy resistance in adult patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute T-lymphoblastic leukemia (T-ALL). While the prognostic value of BAALC expression has been validated, the function of BAALC in leukemia remains unknown. The aim of this study was to identify genes correlated with BAALC to gain further insights into the underlying pathways of the BAALC-associated chemotherapy resistant leukemic phenotype. Therefore, we generated gene expression profiles (GEP) using pre-treatment T-ALL samples (n=86) that differentiated high versus low BAALC expressers (HG-U133 Plus 2.0, Affymetrix). Samples were divided into quartiles (Q1-Q4) according to BAALC expression and QB1-3 was defined as low BAALC and QB4 as high BAALC. Differentially expressed genes between QB1–3 and QB4 were defined by a minimum expression change of three-fold (P'0.05). In order to identify BAALC-associated genes common to T-ALL, CN-AML and normal hematopoiesis we compared these BAALC-derived GEP in T-ALL with BAALC-associated profiles of CN-AML (Langer C et al, Blood 2008) and of normal CD34 positive progenitors (Komor et al, Stem Cells 2005). Only four genes (CD34, CD133, NPR3, IGFBP7) were common to the BAALC-associated GEP of all three entities (T-ALL, CN-AML, and CD34 positive progenitors). Of these genes, the human gene Insulin-like Growth Factor Binding Protein 7 (IGFBP7) was further investigated as the Insulin-like growth factor signaling plays an important role in various human cancers. Next we determined IGFBP7 expression levels by real-time RT-PCR in an independent cohort of 219 adults with newly diagnosed T-ALL, registered within the German Multicenter Acute Lymphoblastic Leukemia Study 06/99 and 07/03 protocols. In T-ALL samples, IGFBP7 showed a heterogeneous expression pattern compared to normal bone marrow (expression range: 0–79.1 vs 0.6–2.3). For correlation of IGFBP7 expression with clinical and molecular features, samples were divided into quartile groups according to IGFBP7 expression and defined as low IGFBP7 with expression levels in QI1 to QI3 (n=158) and as high IGFBP7 with expression levels in QI4 (n=53). High expression levels of IGFBP7 were associated with an immature phenotype of early T-ALL (62% of patients in QI4 showed an early T-ALL immunophenotype as compared to only 15% in QI1–3; P<0.001), high cell surface expression of CD34 (mean: 36% vs 15%; P<0.001) and CD33 (mean: 24% vs 3%; P<0.001). Moreover, high IGFBP7 expression significantly predicted a higher frequency of refractory disease (11% vs 3%; P=0.03) and an inferior overall survival (OS; 4-year OS: IGFBP7 QI4: 42% vs IGFBP7 QI1–3: 55%; P=0.03). In vitro, studies revealed that treatment with rIGFBP7 significantly inhibited proliferation of KG1a leukemic cells (65% reduction of proliferation compared to untreated cells; P<0.001) determined by Cell Proliferation Reagent WST-1. Moreover, a 26% reduction (P<0.001) of the DNA synthesis phase was detected by BrdU incorporation after rIGFBP7 treatment of KG1a cells. Taken together, we identified IGFBP7 as a lineage-independent, BAALC co-expressed gene involved in leukemia. High IGFBP7 expression was associated with stem cell features and treatment failure in T-ALL. In contrast to BAALC which likely represents only a surrogate marker of treatment failure in acute leukemias, IGFBP7 might play functional role and contribute to the molecular basis of BAALC-associated drug resistance. As rIGFBP7 inhibits proliferation of leukemic cells, secreted IGFBP7 protein may therefore promote resistance to cell cycle dependent cytostatic drugs targeting highly proliferate blast cells. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Molecular Monitoring of ERG and BAALC as Minimal Residual Disease Markers in Patients with Acute Leukemia.

Although expression of the genes ERG (ETS-related gene) and BAALC (brain and acute leukemia, cytoplasmic) predicts outcome in acute leukemia, there is little information about their utility as markers of minimal residual disease (MRD). A real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) was used to examine relative levels of ERG and BAALC gene expression in 11 patients with acute leukemia. Pretreatment and post-chemotherapy bone marrow (BM) samples were analyzed to quantify these transcripts. Peripheral blood (PB) samples were also obtained to evaluate the correlation of ERG and BAALC expression levels in BM and PB samples. Significant levels of ERG gene were expressed in all patients. In contrast with ERG, the levels of BAALC gene expression were significantly lower in some patients. Continuous monitoring of the ERG mRNA was performed and assessed a correlation between MRD levels and subsequent clinical courses. In patients with relative high expression of BAALC transcripts (>7x10exp-3/GAPDH), high levels of ERG gene expressions were consistently observed whether they were in remission status or not. In 2 patients with acute promyelocytic leukemia (APL), MRD was simultaneously monitored by RQ-PCR using primers for PML/RAR alpha. Changes in expression levels of PML/RAR alpha and ERG presented poor correlation. While, in other patients with relative low expression of BAALC transcripts (<7x10exp-3/GAPDH), ERG gene expression levels gradually decreased after the induction therapy and following consolidation treatments. In patients unable to attain remission, ERG expression levels remained high. Thus, the quantitation of the ERG gene expression made it possible to assess MRD in patients with acute leukemia (except for APL) carrying low BAALC expression. To our knowledge, this is the first report concerning the use of ERG mRNA expression for MRD monitoring.

Prognostic Impact of BAALC Expression in the Context of Other Molecular Markers in Cytogenetically Normal Acute Myeloid Leukemia.

Background: In previous studies, high blood expression values of BAALC (brain and acute leukemia cytoplasmic) have been shown to be an adverse risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). So far, the prognostic value of BAALC expression has not been evaluated in the context of other relevant molecular markers such as mutations (mut) in the NPM1 or CEBPA genes.Aims: To evaluate the prognostic impact of BAALC expression in the context of NPM1, FLT3-ITD (internal tandem duplication)/TKD (tyrosine kinase domain mutations at codons 835/836), CEBPA, and MLL-PTD (partial tandem duplication) mutation status on response to induction therapy, relapse-free (RFS) and overall survival (OS).Methods: Patients (pts) 16 to 60 years of age were entered on three treatment trials conducted by the German-Austrian AML Study Gorup (AML HD93, AML HD98A, AML04–07). In all three trials, a genetic randomization was performed assigning all pts with an HLA-matched family donor to allogeneic stem cell transplantation (SCT) in first complete remission (CR). BAALC expression was determined in peripheral blood from 339 CN-AML pts by real-time RT-PCR as recently described; gene mutation screening was performed as previously reported.Results: BAALC expression levels were heavily scattered in the 339 pts, with a median of 4,716 (range 11–1,120,212). High and low BAALC expression was determined by dichotomizing BAALC at the median. Correlation of BAALC expression levels with FLT3-ITD/TKD, NPM1, CEBPA and MLL-PTD mutation status revealed high BAALC expression to be significantly associated with FLT3-ITDpos (p=0.001), NPM1WT (p<0.0001) and CEBPAmut (p=0.006). In addition, when correlating BAALC expression levels with the combined FLT3-ITD/NPM1 genotypes that recently have been shown to be of prognostic relevance in CN-AML, a significant correlation of low BAALC expression and the favorable FLT3-ITDneg/NPM1mut (p<0.0001) genotype was present. In contrast, high expression levels were significantly correlated with the other unfavorable genotypes. There was a trend towards a lower CR rate in high BAALC expressers (p=0.07). However, in a multivariable logistic regression model, BAALC was not significant (p=0.75); the only significant variables for induction success were the FLT3-ITDneg/NPM1mut (p=0.03) and CEBPAmut genotypes (p=0.04), as well as logarithm of white blood cell count (WBC, p=0.03). In univariable analyses for OS and RFS, high BAALC expressers had significantly inferior clinical outcome (p=0.05 and p=0.02, respectively). Again, multivariable Cox proportional hazard models for RFS and OS revealed no significant impact of BAALC expression (p=0.15 and p=0.96, respectively); significant variables for OS and RFS were the FLT3-ITDneg/NPM1mut (p<0.0001 and p<0.0001) genotype, availability of an HLA-matched family donor (p=0.02 and p=0.001), and WBC (p=0.0002 and p=0.0001); in addition, the CEBPAmut genotype was a significant variable for OS (p=0.002).Conclusions: In our study on a large series of molecularly well characterized CN-AML, BAALC expression levels were significantly correlated with FLT3-ITD/NPM1 genotypes. In the context of these genotypes, BAALC expression lost its independent prognostic impact.

Case-Control Study Using Array-Based Genotyping Produced a Panel of Genes Associated with Risk to Acute Myelogenous Leukemia (AML).

Multiple loci with small genetic effects are thought to be linked to AML pathogenesis. To detect such loci requires systematic screening of large number of single nucleotide polymorphisms (SNPs) within large study population. In this study we tested whether 3104 SNPs in 200 candidate genes were associated with risk to AML. Genes were considered potential candidates for their known or suspected roles in DNA repair system, pharmacogenomics and transcriptional regulation in hematopoiesis or putative pathways related to leukemogenesis. Selection of SNPs was performed using dbSNP and HapMap project databases, with emphasis on non-synonymous SNPs or haplotype tagging SNPs. To discover potential SNPs responsible for AML susceptibility, we conducted a case-control study using Affymetrix targeted genotyping 3K array. We applied this platform to analyze the samples from 309 de novo AML patients and 382 healthy controls. Genotype scorings were processed using GeneChip scanner 3000 TG(Affymetrix) and analyzed with GCOS software(Affymetrix). In total more than 255,000 SNPs were genotyped for this study. Samples with suboptimal call rates were excluded. Statistical testing were carried out using χ2, Cochran-Armitage trend, Fisher's exact, odds ratio, haplotype estimation, LD block definition. Here we report that 23 SNPs in 16 genes are associated with elevated or reduced risk to AML. Some of associated genes were transcription factors such as ZNF23 and ZNF233. While associated genes were distributed evenly among the whole genome, four associated genes were found on chromosome 1. Among those associated SNPs, two were located in coding region, one in exon-intron boundary, while the rest of SNPs were located in introns of associated genes. Among 23 SNPs identified, two intronic SNPs from 1st intron of BAALC(Brain and acute leukemia, cytoplasmic) gene were associated with the reduced risk to AML. Haplotype estimation and linkage disequilibrium pattern of 9 SNPs including two associated SNPs will be presented. In conclusion, we identified SNPs responsible for AML susceptibility by candidate gene-based SNP array approach. These promising data when supported by further molecular validation would greatly enhance the current understanding of AML predisposition and diseases progression. Implication of polymorphic variants in AML etiopathogenesis will be presented and discussed.

Low ERG and BAALC Expression Identifies a New Subgroup of Adult Acute T-Lymphoblastic Leukemia With a Highly Favorable Outcome

Expression of the genes ERG (v-ets erythroblastosis virus E26 oncogene homolog) and BAALC (brain and acute leukemia, cytoplasmic) shows similarity during hematopoietic maturation and predicts outcome in acute myeloid leukemia. We hypothesized that like ERG, BAALC expression might be of prognostic significance in acute T-lymphoblastic leukemia (T-ALL) and that ERG and BAALC expression together would better identify the patient's risk profile. ERG and BAALC mRNA expression were determined by real-time reverse transcriptase polymerase chain reaction in 153 adults with T-ALL. Patients were designated low or high ERG expressers and low or high BAALC expressers. High BAALC expression correlated with a higher frequency of early T-ALL (P < .0001), CD34 positivity (P < .0001), coexpression of myeloid markers (P = .0001), and high ERG expression (P = .03). High BAALC compared with low BAALC patients had an inferior relapse-free survival (RFS; P = .0008) and overall survival (OS; P = .0001). In contrast, patients with low expression of both ERG and BAALC (representing 41% of all T-ALL patients) had the most favorable outcome (P < .0001; 4-year RFS: low ERG/low BAALC 81%; P < .0001; 4-year OS: low ERG/low BAALC 69%). On multivariable analysis, low ERG/low BAALC expression was of independent favorable prognostic significance (RFS, P = .001; OS, P = .003). Low expression of both ERG and BAALC identifies T-ALL patients with a distinctly favorable long-term outcome.

The Significance of BAALC Expression and CEBPa Mutations as New Prognostic Factors in Pediatric Acute Myeloid Leukemia with Normal Karyotype.

Approximately half of children with acute myeloid leukemia (AML) have no chromosomal or genetic abnormalities that can predict outcome. To improve the prognostic stratification on this heterogeneous group of patients, novel markers need to be investigated. Recently, CEBPa (CCAAT/enhancer binding protein alpha) mutations have been described as a favorable prognostic factor and the high expression level of BAALC (brain and acute leukemia, cytoplasmic) was found to be a poor prognostic marker, in adult AML with normal karyotype. To explore their significance in childhood, we studied the prognostic impact of the high expression level of BAALC and CEBPa mutations in pediatric AML with normal karyotype. Samples were available from pediatric patients 3 months to 15 years of age, treated on Japanese Childhood AML Cooperative Study Group Protocol, AML99. BAALC expression was determined by comparative real-time RT-PCR assay in 29 patients. The relative BAALC expression was determined using the comparative cycle threshold method. The median value of healthy volunteers was used as cutoff. The mutational analysis of CEBPa was performed in 49 patients with RT-PCR followed by direct sequencing. For samples carrying mutations, additional subcloning analysis were carried out. 72.4% (21 of 29) of patients had high BAALC expression and 27.6% (8 of 29) had low. High BAALC expression was associated with the FAB subtypes M0, M1, and M2, whereas low BAALC expression correlated with M4 and M5. Overall survival and event-free survival was 52.3%; 49.5% (high BAALC expression group) and 75.0%; 62.5% (low BAALC expression group), respectively. CEBPa mutations were detected in four patients (8.2 %, 4 of 49; two each in M1 and M2). N-terminal frameshift mutations and inframe insertions in the basic-leucine zipper (bZIP) domain were detected. Novel mutations (212 insertion (ins) C, 214–224 deletion CCCCGCACGCG, 720 ins CGCACC, 1074 ins AGA, 1092 ins CAC) were identified. One patient had mutations occurred in both N-terminal part and bZIP domain. Moreover, above patients with CEBPa mutations had not cooperating mutations with FLT3-ITD (fms-like tyrosine kinase 3 internal tandem duplication) and maintain in complete remission for at least 21 months duration. In conclusion, BAALC expression and CEBPa mutations may be prognostic factors in pediatric AML with normal karyotype.

Identification of a PTH regulated gene selectively induced in vivo during PTH‐mediated bone formation

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well-established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH-regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100-fold (maximal) by a PTH 1-34 anabolic treatment regimen in a time-dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR-106 osteoblastic cells show that PTH 1-34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1-34, BAALC expression can also be induced by other bone forming factors including PGE(2) and 1,25 dihydroxy vitamin D(3). We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3-E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1-34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function.

BAALC Expression and FLT3 Internal Tandem Duplication Mutations in Acute Myeloid Leukemia Patients With Normal Cytogenetics: Prognostic Implications

Evaluate the impact of BAALC (brain and acute leukemia, cytoplasmic), a gene whose expression has been associated with adverse outcome in acute myeloid leukemia (AML) with normal cytogenetics, and FLT3 internal tandem duplication (ITD) mutations as independent prognostic factors in a larger study. BAALC expression was determined by real-time reverse transcriptase polymerase chain reaction in pretreatment blood samples of 307 adults <or= 60 years of age with AML with normal cytogenetics. Patients were dichotomized at BAALC's median expression into low and high expressers. The FLT3 ITD mutant:wild-type ratio was determined by fragment analysis. Compared with low-BAALC patients, high-BAALC patients had a higher rate of primary resistant leukemia (16% v 6%; P = .006). High BAALC expression was associated with a higher cumulative incidence of relapse (CIR; P = .018) and an inferior overall survival (OS; 3-year OS, 36% v 54%; P = .001). On multivariable analysis, high BAALC was independently predictive of resistant disease (P = .019), and high BAALC as well as a high FLT3 mutant:wild-type ratio were confirmed as the only factors predicting a high CIR (BAALC, P = .03; FLT3, P = .01) and inferior OS (BAALC, P = .001; FLT3, P = .012). This study strengthens BAALC expression as one of the most important prognostic factors in AML patients with normal cytogenetics. BAALC expression and FLT3 mutation status should assist in tailoring induction and postremission therapies.

Co-Expression of BAALC with ERG, TCF4, and CD133 Indicates a Shared Pathway in Acute Myeloid and Lymphoblastic Leukemia.

The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) is a molecular marker of hematopoietic progenitor cells and is aberrantly expressed in subsets of acute myeloid (AML) and lymphoblastic (ALL) leukemias. High mRNA expression levels of BAALC have been shown to adversely impact outcome in newly diagnosed AML patients (pts) with normal cytogenetics. To gain insight into the functional role of BAALC and its significance to normal hematopoiesis and leukemogenesis we compared gene expression profiles of normal CD34+ progenitors with those of AML and ALL blasts (using oligonucleotide microarrays; HG-U133 plus 2.0, Affymetrix, Santa Clara, CA). First we explored the regulation of BAALC expression during lineage specific maturation of in vitro differentiated human CD34+ bone marrow cells selected from healthy individuals. Microarray analyses were carried out using CD34+ cells stimulated in vitro with EPO, TPO, or G/GM-CSF to induce lineage-specific differentiation. At day 0 of culture and at three different time points during differentiation (days 4, 7, 11) cells were harvested, and if necessary purified by immunomagnetic beads and used for microarray studies. Experiments of all lineages and time points were done in triplicates. A total of 276 genes were identified showing similar changes in expression (with downregulation during differentiation) as BAALC at the three time points in all lineages with a correlation coefficient of R>0.95. This set of 276 BAALC co-expressed genes was investigated in an AML expression dataset generated from 51 adult pts with newly diagnosed de novo AML and normal cytogenetics (Cancer and Leukemia Group B). After exclusion of probesets expressed in fewer than 20% of pt samples, 21 probesets representing 14 named genes 6 of which are known to be involved in AML (BAALC, CD34, CD133, SOX4, ERG, SEPT6) and 4 implicated in lymphoid development (TCF4, SH2D1A, ITM2A, ITM2C) were found to be overexpressed (a significance level of P=0.01 was used) in pts of the highest third compared to pts of the lowest third of BAALC expression values as measured by real-time RT-PCR. We next applied these same 21 BAALC co-expressed probesets to an ALL expression dataset generated from 66 adult pts with newly diagnosed standard risk B-lineage precursor ALL (from the German ALL GMALL study group). A BAALC specific cluster uncovered 7 probesets representing 4 different co-expressed genes: BAALC, CD133, and the transcription factors ERG and TCF4. Thus, applying a BAALC specific expression signature to AML and ALL gene expression profiles revealed 3 genes (CD133, ERG, TCF4), which are highly associated with BAALC in myeloid and lymphoid blasts. Interestingly in non-malignant lymphoid and myeloid cells the oncogeneic ETS transcription factor ERG has shown specificity to immature cells, while its mechanistical role in leukemogenesis remains unknown. ERG and TCF4 may directly regulate BAALC and indicate a specific pathway implicated in leukemogenesis, while co-expression of CD133 and BAALC suggests shared stem cell characteristics. Functional studies are in progress to further explore these findings.