Brain And Acute Leukemia, Cytoplasmic Research Articles

Relation of BAALC and ERG Gene Expression with Overall Survival in Acute Myeloid Leukemia Cases

The objectives of this study were to evaluate the expression of brain and acute leukemia, cytoplasmic (BAALC) gene and erythroblast transformation-specific related gene (ERG) in de novo cases of acute myeloid leukemia (AML) and identify roles in disease progression and outcome. This study included 50 newly diagnosed AML patients, along with 10 apparently healthy normal controls. BAALC and ERG expression was detected in the bone marrow of both patients and controls using real-time RT-PCR. BAALC and ERG expression was detected in 52% of cases but not in any controls. There was a statistically significant correlation between BAALC and ERG gene expression and age (p- value=0.004 and 0.019, respectively). No statistical significance was noted for sex, lymphadenopathy, hepatomegaly, splenomegaly, other hematological findings, immunophenotyping and FAB sub-classification except for ERG gene and FAB (p-value=0.058). A statistical significant correlation was found between response to treatment with ERG expression (p-value=0.028) and age (p-value=0.014). A statistically significant variation in overall survival was evident with patient age, BM blast cells, FAB subgroups, BAALC and ERG expression (p-value= <0.001, 0.045, 0.041, <0.008 and 0.025 respectively). Our results suggest that BAALC and ERG genes are specific significant molecular markers in AML disease progression, response to treatment and survival.

BAALC Expression Is a Feasible Marker for Risk Stratification and Detection of Minimal Residual Disease in Cytogenetically Normal Acute Myeloid Leukemia

IntroductionDuring the last years it has been shown that PCR-based assessment of minimal residual disease (MRD) is of great importance for risk stratification and early detection of relapse in acute myeloid leukemia (AML). Most frequently, monitoring of MRD was restricted to patients carrying specific genetic markers such as fusion genes and gene mutations. However, many patients lack such markers amenable to sensitive detection by PCR. For this reason, it is still crucial to identify molecular targets that are applicable for MRD assessment in patients with AML. High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal AML (CN-AML) and has been proposed to represent a clinically useful biomarker to monitor response to therapy. A significant correlation of BAALC expression levels with the mutation ratio of internal tandem duplications in FLT3 (FLT3-ITD), partial tandem duplications in MLL (MLL-PTD) and NPM1 mutations has been shown previously (Weber et al., BCJ 2014). The objective of this study was to further validate the applicability of BAALC expression as a marker for detection of MRD.Patients and MethodsWe investigated BAALC mRNA expression levels in 550 diagnostic and follow-up samples from 116 de novo CN-AML patients showing high BAALC expression at initial diagnosis. BAALC mRNA expression was assessed by quantitative real-time PCR and normalized against the expression of the control gene ABL1. To identify high BAALC expressers the previously defined cutoff ratio of 33.1% BAALC/ABL1 (Weber et al., BCJ 2014) was used. The cohort was composed of 55 females (47%) and 61 males (53%). Age ranged from 18 to 85 years (median: 55). The median number of follow-up samples per patient was 3 (range: 1-21) with a median follow-up time of 32 months (range: 16-48 months). All patients included in this study were treated according to standard AML protocols in curative intent. In addition to BAALC expression, a full molecular characterization for the presence of FLT3 -ITD (42/116, 36%), MLL -PTD (10/116, 9%) and mutations in NPM1 (38/116, 33%) was performed.ResultsAt diagnosis, BAALC expression ranged from 33.2 to 5655.7% BAALC/ABL1 with a median of 163.9%. Of the 116 patients, 62 patients (53%) harbored at least one of the previously established MRD markers (FLT3-ITD, MLL-PTD and NPM1), while 54 patients (47%) lacked these MRD targets. To validate stability of BAALC expression we analyzed 25 patients with paired diagnostic and relapsed samples available. The respective BAALC expression levels at both time points showed good correlation (Mean % BAALC/ABL1 of diagnosis vs. relapse: 343.1 vs. 297.4, r=0.642, p=0.001). Three of the 25 patients relapsed after allogeneic stem cell transplantation. Also in these patients BAALC expression levels were similar between diagnosis and relapse (Mean %BAALC/ABL1: 686 vs. 684), supporting the use of BAALC expression for follow-up evaluation. Moreover, in 5 cases with cytomorphologic complete remission during follow-up a molecular relapse based on elevated BAALC expression levels (32.4-315.2% BAALC/ABL1) could be detected 21-42 days before cytomorphologic relapse. To investigate the predictive value of BAALC expression levels during follow-up, BAALC expression was analyzed in 58 patients after the second course of induction chemotherapy, but before start of consolidation chemotherapy. To separate low from high BAALC expression in this follow-up analysis, the cutoff established for diagnostic samples (33.1% BAALC/ABL1) was used. According to this cutoff, 29% (17/58) of the patients had high BAALC expression levels, while 71% (41/58) exhibited low BAALC expression levels. High BAALC expression was associated with a shorter EFS (median: 5 month vs. not reached, 1-year-EFS: 61% vs. 21%, p<0.001)ConclusionThis data validates the applicability of BAALC expression as a target for MRD monitoring in CN-AML. A significant number of CN-AML would benefit from molecular monitoring using a quantitative BAALC expression assay aimed at following disease course and early detection of relapse. DisclosuresWeber:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 2782: Risk loci for a breast-colon cancer phenotype: results from a genome-wide association study

Abstract Purpose: Clustering of breast and colorectal cancer has been observed within families in population-based studies giving rise to speculation that there are “breast-colon” cancer susceptibility genes. We performed a genome-wide association study (GWAS) to identify genetic markers associated with a potential breast-colon cancer phenotype. Methods: Cases and controls were ascertained from the Breast and Colon Cancer Family Registries’ (CFR) GWAS subjects. “Breast-colon phenotype” definition was based on 2 or more breast and colon cancer cases diagnosed in first- or second-degree relatives within a family. Cases (n = 985) were women with a history of breast cancer and a family history of colorectal cancer, or persons with colorectal cancer with a family history of breast cancer. Unrelated controls (n = 1769) were frequency matched to cases for age and gender. Following standard quality control measures, 6,220,060 directly measured and imputed single nucleotide polymorphisms (SNPs) were included in the discovery set. SNPs associated at p&amp;lt;1×10−5 were analyzed in a replication dataset of cases (n = 293) and controls (n = 2103) from the Genetics and Epidemiology of Colorectal Cancer Consortium. Results: In a regression model that included gender, age, CFR center and 6 principal components, the top association in the discovery set was in the chromosome 8q22.3 region overlying the BAALC gene (top SNP rs12548629, P = 3.63×10−7). In the replication dataset, of 341 SNPs tested, the top signal was found in multiple correlated SNPs overlying the ROBO1 gene on chromosome 3p12 (rs7429100, P = 2.8×10−3), however, the signal on chromosome 8, the BAALC gene did not replicate. In the discovery set, P values for the SNPs overlaying ROBO1 gene ranged from 2.2×10−5 to 9.7×10−5 (rs7429100, P = 3.9×10−5). The combined meta-analysis showed strongest association at the ROBO1 gene (rs7429100, Pfixed effects 1.84×10−6, Pheterogeneity &amp;gt;0.05 for testing for heterogeneity between the overall discovery set and the replication set). ROBO1 (roundabout, axon guidance receptor, homolog 1) is a transmembrane receptor of the immunoglobulin family and is differentially expressed in human cancers, with a possible role as a tumor suppressor gene. Low ROBO1 expression has been shown to be an adverse prognostic factor for invasive ductal breast cancer and may also play a role in pathogenesis of colorectal cancer. BAALC (brain and acute leukemia, cytoplasmic) is predominantly expressed by neural and blood cells and is largely implicated in acute leukemia. Conclusion: In this exploratory analysis, to elucidate genes/regions associated with pleiotropic effect for breast and colorectal cancer risk we identified germline variation in the region of ROBO1 and BAALC. Validation in a larger dataset and functional characterization of the loci is warranted to elucidate mechanisms by which these genes/SNPs may contribute to the development of breast and colorectal cancer. Citation Format: Mala Pande, Aron Joon, Sanjay Shete, Abenaa M. Brewster, Cathy Eng, Wei V. Chen, Habibul Ahsan, Irene L. Andrulis, Esther M. John, Yi Lin, Polly A. Newcomb, Noralane M. Lindor, Christopher I. Amos, John Hopper, Patrick M. Lynch. Risk loci for a breast-colon cancer phenotype: results from a genome-wide association study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2782. doi:10.1158/1538-7445.AM2015-2782

BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4.

Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell-cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by upregulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Krüppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.

Prognostic significance of the BAALC gene expression in adult patients with acute myeloid leukemia: A meta-analysis.

Numerous studies have investigated the prognostic role of brain and acute leukemia, cytoplasmic (BAALC) gene expression in adult patients with acute myeloid leukemia (AML); however, the results are inconclusive. A meta-analysis was conducted to provide a comprehensive evaluation of the prognostic role of BAALC gene expression in AML. Eligible studies were searched through PubMed, Embase, the Cochrane Library, the China National Knowledge Infrastructure and the China Biology Medicine Disc. Correlations between the BAALC gene expression and clinicopathological features and prognosis were analyzed. A total of 15 studies were examined. The pooled results suggest that high BAALC expression had an unfavorable outcome in AML. The combined hazard ratio (HR) for overall survival (OS) was 1.53 and the summary HR for the disease-free survival rate was 1.64. In addition, subgroup analyses considering cytogenetic and survival analysis were also conducted. High BAALC gene expression appeared to be an adverse prognostic indicator in patients with cytogenetically normal AML (HR for OS, 1.43) and in subgroups of survival analysis with multivariate analysis (HR for OS, 2.35). These results indicate that high BAALC gene expression served as an independent poor prognostic indicator in adult patients with AML.

Prognostic value of brain and acute leukemia cytoplasmic gene expression in egyptian children with acute myeloid leukemia.

BackgroundAcute myeloid leukemia (AML) accounts for 25%–35% of acute leukemia in children. BAALC gene (Brain and Acute Leukemia Cytoplasmic gene) is a recently identified gene on chromosome 8q22.3 that has prognostic significance in AML. The aim of this work was to study the impact of BAALC gene expression on prognosis of AML in Egyptian children.Patients and methodsThis study was conducted on 40 Egyptian children with newly diagnosed AML who were subjected to full history taking, clinical examination and laboratory investigations including: complete blood count, LDH, bone marrow aspiration, cytochemistry, immunophenotyping and assessment of BAALC Gene by real time PCR in bone marrow aspirate mononuclear cells before the start of chemotherapy.ResultsPositive BAALC gene expression was found in 24 cases (60%) and negative expression in 16 cases (40%). Positive BAALC gene expression group includes 14 males and 10 females with mean age at presentation of 8.35±2.63 while negative BAALC gene expression includes 10 males and 6 females with mean age at presentation of 7.74±3.23 with no statistically significant differences between patients with positive and negative BAALC gene expression regarding age, sex and clinical presentations at time of diagnosis including pallor, purpura, splenomegaly, hepatomegaly and lymphadenopathy and laboratory investigations including WBCs and platelets counts, hemoglobin and LDH levels, and peripheral blood and bone marrow blast cell counts. There was significant association between positive BAALC gene expression and M1 and M2 compared with negative BAALC gene expression which is significantly associated with M4. There were statistically significant differences in disease outcome between positive and negative BAALC gene expression groups with higher rate of relapse and death and lower rate of complete remission and disease free survival in positive BAALC gene expression group compared with negative BAALC gene expression group. (p = 0.017).Conclusion and RecommendationBAALC expression is an important bad prognostic factor in AML patients with normal karyotype and therefore we recommend its incorporation into novel risk-adapted therapeutic strategies to improve the currently disappointing cure rate of patients with AML.

Combined Assessment of WT1 and BAALC Expression Levels Improves Risk Stratification in Myelodysplastic Syndromes

BACKGROUND AND AIMSIn patients with myelodysplastic syndromes (MDS) several validated prognostic scores, such as IPSS and R-IPSS, are available to assess the risk of AML progression and predict overall survival (OS) as well as leukemia-free survival (LFS).A number of molecular aberrations can be identified in MDS. However, differently from AML, none of the current prognostic indexes takes into account molecular profile at diagnosis.WT1 expression has often been evaluated in acute leukemias and MDS. High WT1 expression levels on bone marrow at diagnosis have been reported to identify MDS patients who are at high risk of progression to AML. BAALC (Brain And Acute Leukemia Cytoplasmic) hyper-expression has been associated with a poor prognosis in AML patients, whereas its prognostic value in MDS is not yet clearly defined. The aim of our study was to determine if combined assessment of WT1 and BAALC expression levels at diagnosis could be predictive of leukemic evolution.MATERIALS AND METHODSWe selected 86 patients with available WT1 and BAALC expression levels on BM samples at diagnosis.According to IPSS score, 22 patient were considered low-risk, 27 intermediate-1 and 28 intermediate-2 or high risk. Patients underwent different treatment schedules including supportive care, erythropoietin, hypomethylating and immunomodulating agents, according to their risk group. Median follow-up was 36 months (range 4 -121 months).Leukemia-free survival (LFS) was calculated from the diagnosis until last follow-up or documented leukemic progression as defined in literature. LFS was estimated using the Kaplan–Meier method.All Real-Time PCR were performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 1000x104 was used as cut-off value for high WT1 expression, a level of 1000x104 BAALC copy number/Abl copy number was set as cut-off for BAALC hyper-expression.RESULTSAfter a median time of 32 months, 43 patients died. The main cause of death was leukemic evolution (accounting for 31/43 deaths, 72%), other causes were cardiovascular events and infections (data not shown).The risk of death by any cause was significantly affected by leukemic evolution, diagnosis according to WHO classification and molecular expression profile at diagnosis. Multivariate analysis showed that leukemic evolution was an independent predictor of death (p <0.001).Twenty-nine leukemic evolutions were observed. Median LFS was 34 months. The probability of leukemic evolution was significantly affected by karyotype, IPSS and R-IPSS scores, diagnosis according to WHO classification, and molecular profile at diagnosis.According to our data WT1 and BAALC combined expression levels further enhanced prognostic stratification. In IPSS Int-1, Int-2/high and in R-IPSS high risk groups, low levels of expression resulted in significantly lower probability of leukemic progression, whereas high levels predicted poor outcome. Furthermore, in patients assigned to IPSS unfavorable prognostic groups, low levels of WT1 and BAALC seemed to predict a significantly longer LFS.In the univariate analysis LFS duration was significantly affected by WT1 and BAALC expression levels (fig. 1), IPSS and R-IPSS scores, karyotype and WHO classification at diagnosis. A multivariate Cox Regression model showed that LFS duration was significantly influenced only by molecular profile at diagnosis and R-IPSS risk group (p <0.001 and p <0.01, respectively).Median OS was 32 months. In univariate analysis OS was significantly influenced by diagnosis according to WHO classification, karyotype, R-IPSS score, leukemic evolution and molecular profile expression at diagnosis. The multivariate model disclosed molecular expression profile, R-IPSS score and leukemic evolution as independent predictor of OS (p <0.02, <0.03 and <0.01, respectively).CONCLUSIONSIn MDS patients combined WT1 and BAALC expression levels on bone marrow samples at diagnosis is a reliable predictor of risk of AML progression, LFS and OS. This can improve risk stratification especially in intermediate and high risk groups and may lead to a risk tailored therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Optimized Risk Stratification Model in Acute Promyelocytic Leukemia Identifies Patients with an Exceptionally Good Prognosis Regarding the Incidence of Relapse

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on the easily accessible Sanz-Score, which combines leukocyte and platelet counts at initial diagnosis. This score showed significant differences in relapse-free survival (RFS) of APL patients in various studies and is currently used to determine whether a patient can be treated with ATRA and ATO alone or needs additional chemotherapy. However, to make therapeutic decisions based on a risk stratification system derived from the endpoint RFS bears the drawback that relapses are rare in APL and most events are deaths in complete remission (CR), which can be therapy related (e.g. toxicity). The cumulative incidence of relapse (CIR) therefore seems to be a better parameter for decision making with regard to therapy intensity. In this study, we optimized a risk score combining data on gene expression of BAALC (brain and acute leukemia, cytoplasmic), ERG (ets’ related gene) and WT1 (Wilms’ tumor 1) to retrospectively predict the CIR of APL patients.Methods: Data on BAALC, ERG and WT1 expression levels of 79 patients with newly diagnosed APL were obtained from bone marrow mononuclear cells using quantitative real-time RT-PCR in preceding studies. The following gene expression levels were identified as negative risk factors: BAALC expression ≥25th percentile (BAALChigh), ERG expression >75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). As ERGhigh was the only independent predictor for relapse in multivariate analysis with a hazard ratio (HR) of 11.6, its predictive weight was regarded superior, respectively . Cut-off analyses were performed to determine the optimal ERG expression level cut-off for risk of relapse. Accordingly, the new cut-off for high ERG expression was set at ≥62nd percentile (optimized ERGhigh: optERGhigh; Sensitivity: 1.0, Specificity: 0.71). A combined risk score was developed as follows: For the presence of one of the mentioned risk factors, one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, optERGhigh and WT1low or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into two risk groups: 34 patients scored 0-1 points and 45 patients scored 2-3 points. CIR, overall survival (OS) and RFS were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the two risk groups (p<0.05).Results: Patients with 2-3 points had a CIR of 18% at 10 years of follow-up whereas none of the patients with 0-1 points suffered a relapse (CIR: 0%; p=0.02; Fig. 1). All relapses occurred between 8.4 months and 3.5 years after first CR. Moreover, OS and RFS also differed significantly between the two risk groups: OS was 53% for patients with 2-3 points vs. 85% for patients with 0-1 points (p=0.004); RFS was 49% vs. 93%, respectively (p<0.0001). In multivariate analysis the optimized combined risk score was the strongest independent risk factor for every endpoint.Conclusion: The combination of expression levels of BAALC, ERG and WT1 into a risk score identified a group of patients at high risk for relapse which could benefit from close monitoring resulting possibly in an early intervention when molecular relapse is detected. On the other hand, it identified a low risk group with very good outcome and no APL-related events after patients had achieved first CR. A molecular risk score focusing on relapse risk might be a promising approach to guide therapeutic decisions in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overexpression of BAALC: clinical significance in Chinese de novo acute myeloid leukemia.

To investigate the expression of brain and acute leukemia, cytoplasmic (BAALC) and analyze its clinical significance in Chinese de novo acute myeloid leukemia (AML). Real-time quantitative PCR (RQ-PCR) was carried out to detect BAALC transcript level in 121 de novo AML patients and 41 normal controls. BAALC transcript level in AML patients was significantly up-regulated compared with normal controls (P<0.001). Patients with high BAALC expression had significantly older age than those with low BAALC expression (P=0.021). The percentage of blasts in bone marrow of the BAALC high-expressed patients was significantly higher than that in the low-expressed patients (P<0.001). The incidence of BAALC overexpression was significantly higher in M0/M1 (8/9, 89%) and M2 subtypes (33/48, 68%) than in M3 subtype (6/27, 22%) (P<0.001). The frequency of IDH1/2 wild type in CN-AML patients with high BAALC expression was significantly higher than those with low BAALC expression (P=0.031). BAALC high-expressed patients had a significantly lower complete remission than low-expressed patients in both entire AML cohort and CN-AML (P=0.013 and 0.029, respectively). Furthermore, both whole AML cohort and CN-AML patients with high BAALC expression showed a shorter overall survival than those with low BAALC expression (P=0.002 and 0.008, respectively). Multivariate analysis confirmed high BAALC expression as an independent adverse prognostic factor in both AML and CN-AML patients. Our study indicates that overexpression of BAALC serves as an independent prognostic biomarker in both whole AML cohort and CN-AML patients.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

BAALC Gene Expression in Adult B-precursor Acute Lymphoblastic Leukemia: Impact on Prognosis

Background: Adult B-precursor acute lymphoblastic leukemia (ALL) remains a major therapeutic challenge. Various molecular markers have extensively been investigated to improve its risk profile characterization, disease progression and resistance to treatment. Aim: To analyze the brain and acute leukemia, cytoplasmic (BAALC) gene expression and to assess its prognostic impact in B-precursor ALL. Subjects and Methods: BAALC mRNA expression was analyzed using real time PCR in 200 primary adult B-precursor ALL patients. Patients were grouped into 2 groups according to median BAALC expression. Results: High BAALC expression was associated with older age (P=0.037), higher white blood cell count (P =0.019), LDH concentration (p=0.007), higher incidence of positive CD34 (P =0.011) and positive BCR-ABL (P=0.011). High BAALC expression was associated with primary therapy resistance in the overall cohort (P = 0.001), in BCR-ABL− and BCR-ABL+ subgroups (P = 0.039, 0.003 respectively). Multivariate analysis showed that BAALC expression was an independent risk factor for chemotherapy resistance in the overall cohort ( =0.003, OR=3.133, 95% CI=1.482-6.623) and in both BCR-ABL- (p=0.049, OR=2.359, 95%CI=1.004-5.538), and BCRABL+ subgroups (p=0.014, OR=2.672, 95%CI=1.824-3.326). Higher BAALC expression was associated with a shorter overall survival (OS) (p= 0.010) and disease free survival (DFS) (p<0.001) in the overall cohort, and DFS in BCR-ABL- subgroup (p<0.001). Multivariate analysis showed that higher BAALC expression independently predicted OS and DFS in the overall cohort (P = 0.039, 0.002 respectively) and DFS in BCR-ABL- subgroup (p=0.001). Conclusion: High BAALC expression is associated with refractory disease in adult B-precursor ALL, and predicts shorter OS and DFS. Determination of BAALC expression may contribute to risk stratification of adult B-precursor ALL, and may improve the currently disappointing cure rate.

BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia

High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of ⩾0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OSTXcens). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.

BAALC Acts As a Scaffold-Like Protein In The ERK Signaling Pathway and Promotes Leukemogenesis By Facilitating MEK–mediated ERK Activation

Significance of BAALC (brain and acute leukemia, cytoplasmic) as a poor prognostic factor for patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute lymphoblastic leukemia (ALL) has now been widely established, however, its molecular mechanisms are still mostly unknown. Available evidence suggests that BAALC is a cytoplasmic protein. Based on this finding, we carried out phosphoprotein array of cytoplasmic signal transduction-related proteins and sought to find out the deregulated signaling underlying BAALC-mediated leukemogenesis. Using BAALC-overexpressed HEK293T cells, we found enhanced phosphorylation of Extracellular signal-regulated kinase (ERK), and its downstream target p90RSK. Besides, Jurkat cells, a human ALL cell line, showed sustained phosphorylation of ERK when BAALC is overexpressed. These results suggest that BAALC acts as a scaffold-like molecule for the cytoplasmic mitogen-activated protein kinase kinase (MEK), upstream ERK activator and activates ERK signaling pathway. Immunoprecipitation assay revealed that BAALC binds to MEK in HEK293T cells. Co-localization of BAALC and MEK in the cytoplasm of HEK293T was also detected by immunofluorescence experiment. The proportion of the phosphorylated form in the MEK-bound ERK was increased compared to its control, indicating that BAALC binds to MEK and directly up-regulates its kinase activity to phosphorylate ERK. BAALC accelerated cell growth and increased the phosphorylation of ERK when overexpressed in Jurkat or HEL cells. Those cells showed accelerated cell cycling with no change of apoptotic status. On the other hand, BAALC knockdown decreased cellular proliferation as well as phosphorylation of ERK in the human leukemia cell line expressing a high level of BAALC, such as Kasumi-1 cells. Decelerated cell cycling was also observed in the BAALC-knocked down cells. Notably, the proportion of the phosphorylated ERK bound to MEK was decreased when BAALC was knocked down, which is consistent with BAALC-overexpressed experiments. Interestingly, in AML cell lines without active Ras mutations, the expression of BAALC was positively correlated to its sensitivity to MEK inhibitor (U0126). Moreover, growth advantage of BAALC overexpression in leukemic cell lines was canceled by the treatment with U0126, implying that BAALC-induced growth advantage depends on MEK-ERK signaling activity. Analysis of deletion mutants of BAALC revealed that binding ability of BAALC to MEK resides in its N-terminal region, which is highly conserved among mammals. This N-terminal region of BAALC was necessary for phosphorylation of ERK and the mutant lacking this region failed to promote proliferation of the leukemic cell lines. Taken together, BAALC acts as a scaffold-like protein that binds to MEK in the cytoplasm and directly up-regulates MEK-ERK signal transduction pathway. The fact that overexpression of BAALC significantly promotes proliferation of leukemic cells is consistent with the clinical observation that BAALC-high leukemia has a poor prognosis. The novel function of BAALC as a scaffold-like protein that activates MEK-ERK signal transduction pathway may account for the underlying mechanism for the generation of aggressive form of leukemia and would be a promising therapeutic target of BAALC-high leukemia. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

A Risk Score Integrating BAALC, ERG and WT1 Expression Can Be Used To Improve Risk Stratification In Acute Promyelocytic Leukemia

IntroductionWith current therapy regimens over 75% of patients with de novo acute promyelocytic leukemia (APL) can be cured. Approaches to further improve patient outcome by stratifying patients at the time of initial diagnosis according to their individual risk and to adjust therapy accordingly have been based on clinical features only. Molecular markers have not been established for risk stratification as yet. Recently, we have shown that high expression levels of the genes brain and acute leukemia, cytoplasmic (BAALC) and ets related gene (ERG) are associated with inferior outcome in APL patients. In addition, data indicate that aberrant expression of the gene Wilms' tumor 1 (WT1) is a negative prognostic factor with regard to overall survival (OS) after complete remission (CR) and relapse free survival (RFS) in APL. In this study we evaluated the prognostic relevance of a combined score integrating the expression levels of the above mentioned genes to further improve risk stratification in APL patients. MethodsExpression levels of BAALC, ERG and WT1 of 62 patients with newly diagnosed APL were retrospectively analyzed in bone marrow mononuclear cells using multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR). Median age of patients was 47 years (range: 19 to 82y). All patients gave informed consent. Patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study with a treatment of simultaneous ATRA and double induction chemotherapy including high-dose ara-C, consolidation and maintenance chemotherapy. The following gene expression levels were identified as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression >75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low/high). A risk score was developed as follows: for the presence of one of the mentioned risk factors one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low/high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into four risk groups: 7 patients scored 0 points (= low risk), 27 patients scored 1 point (= intermediate 1 risk), 19 patients scored 2 points (= intermediate 2 risk) and 9 patients scored 3 points (= high risk). Subsequently, OS, RFS and relapse free interval (RFI) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the four risk groups (p<0.05). ResultsThe integrative risk score divided patients into four groups with significantly different outcome. The low risk group showed a RFS of 100% at 10 years of follow-up compared to the intermediate 1 risk group with 81%, the intermediate 2 risk group with 58% and the high risk group with a RFS of 42% only (median survival: 4.6y) (p=0.02). In accordance, the RFI differed significantly between the four groups: low risk 100%, intermediate 1 risk 100%, intermediate 2 risk 89% and high risk 71% (p=0.049). There was no statistically significant difference between the 4 groups with regard to OS in the entire patient cohort. However, there was a clear trend towards a difference in OS in patients who achieved a CR after induction therapy: low risk 100%, intermediate 1 risk 81%, intermediate 2 risk 68% and high risk 53% survival at 10 years of follow-up (p=0.09). ConclusionIntegration of expression levels of the genes BAALC, ERG and WT1 into a scoring system identifies 4 risk groups with significantly different outcome with regard to RFS and RFI. It might be a promising approach to guide therapeutic decisions in patients with APL. However, multivariate analyses and validation of these data in an independent patient cohort is warranted. Disclosures:No relevant conflicts of interest to declare.

ITRAQ-based proteomics reveals novel biomarkers of osteoarthritis

Objective: We performed comprehensive proteomic analyses of articular cartilage by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and searched for candidate biomarkers for osteoarthritis (OA).Methods: Articular cartilage was collected from patients with OA or femoral neck fracture for the control group. Molecular variations were detected by the iTRAQ method, and quantitative analyses were performed by western blot.Results: Using the iTRAQ method, we identified 76 proteins with different expression levels in OA patients and the control group. Among these proteins, we selected LECT2 (leukocyte cell-derived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6 (peroxiredoxin-6), which had not been reported as biomarkers for OA.Conclusions: Use of these proteins in combination with conventional OA biomarkers may better reflect the grade and prognosis of OA.

BAALC overexpression retains its negative prognostic role across all cytogenetic risk groups in acute myeloid leukemia patients

Overexpression of brain and acute leukemia cytoplasmic (BAALC) gene confers poor prognosis in cytogenetically normal acute myeloid leukemia (AML) patients, while less defined is its role in AML with abnormal karyotype. We evaluated the effect of BAALC overexpression on outcome of 175 adult AML patients with different cytogenetic risks. Karyotype was favorable in 13, intermediate in 117 and unfavorable in 45 patients, respectively. Quantitative BAALC expression was determined by real-time PCR, with cut off value set at 50th percentile. BAALC was overexpressed in 87/175 (50%) patients, without association with cytogenetic status. High BAALC was associated with unmutated NPM (P = 0.006) and CD34 positivity (P < 0.0001). Complete remission (CR) was attained in 111 patients (63%), and was maintained at 5 years in 52 ± 7%. BAALC overexpression had a negative impact on CR achievement (P = 0.04), while did not influence relapse probability. Median survival was 22 months with a 5-years overall survival (OS) of 35%. Factors with a negative impact on OS were older age (P = 0.0001), unfavorable cytogenetic (P = 0.005), ABCG2 overexpression (P = 0.03) and high BAALC levels (P = 0.01). We observed a worse outcome in patients with high BAALC expression through all cytogenetic risk categories: 5-years OS was 100% vs. 71% in patients with favorable cytogenetics (P = 0.05), 55% vs. 40% in cases with intermediate karyotype (P = 0.04) and 34% vs. 23% in unfavorable cytogenetic subgroup (P = 0.02). BAALC overexpression identified AML patients with poor prognosis in all cytogenetic groups. Though relatively rare, BAALC positivity in patients with favorable or unfavorable karyotype significantly worsened survival.

Prognostic Implication of BAALC Gene Expression in Adult Acute Myeloid Leukemia

Recently various molecular markers and global gene expression profiling have been investigated to improve risk profile characterization of AML with normal cytogenetics. Our main objective is to investigate the prognostic impact of brain and acute leukemia, cytoplasmic (BAALC) expression in AML with normal karyotype. BAALC expression was analysed using quantitative real time (QRT) PCR. High expression was detected in 22 of 45 patients (48.9%) and its expression did not correlate with the clinical parameters of patients. High BAALC expressers had significantly lower incidence of CR (22.7% vs. 73.9%; p = 0.001), higher mortality rate (72.1% vs. 39.1%; p = 0.023), showed significantly shorter DFS (mean 4.5 vs. 13.21 months, p < 0.001), and inferior overall survival (7.02 vs. 15.02 months, p < 0.001). Multivariable analysis confirmed high BAALC expression as an independent risk factor for DFS and OS. BAALC expression is an important prognostic factor in AML patients with normal karyotype and its incorporation into novel risk-adapted therapeutic strategies will improve the currently disappointing cure rate of this group of patients.

Elevated BAALC Gene Expression Lacks Clinical Significance in Pediatric AML- A Report From the Children's Oncology Group.

AbstractAbstract 2553▪▪This icon denotes a clinically relevant abstract Background:High expression of the brain and acute leukemia, cytoplasmic (BAALC) gene is associated with inferior outcomes in adult AML patients with normal cytogenetics. Studies regarding the significance of BAALC expression in pediatric AML are limited. We studied BAALC expression in a cohort of children with de novo AML to evaluate its clinical significance in pediatric AML, particularly in patients with standard-risk (SR) disease. Methods:BAALC mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 206 patients enrolled on COG AAML03P1. Expression levels were normalized against pooled normal blood controls (PNB). To evaluate the impact of BAALC expression values on clinical outcome, samples were dichotomized at the median expression level (14.9) into a BAALClow (N= 103, median expression 2.9; range 0.0089–14.897) and BAALChigh (N=103; median expression 65; range 15.064–369.53) subset. Baseline clinical features were compared for BAALClow vs. BAALChigh patients and categorical variables compared using the chi-square test and Fisher exact test when data were sparse. The Kaplan-Meier method was used to estimate OS, EFS and DFS for the 2 groups. Estimates of RR were obtained using methods that accounted for competing events. Similar methods were used for sub-analysis of n=99 SR patients. Results:Compared to expression in PNB, BAALC expression varied over 5 log fold (range 0.0089–369.53); the median BAALC expression was 14.98. There were no differences between BAALClow and BAALChigh patients with respect to disease presentation (WBC, BM blast%, Hb, platelet count) though patients with BAALChigh expression were older (median age 12.2 vs. 7.8; years; p=0.001). NPM1 mutations were more frequent in the BAALClow subset (12% vs 0%, p=0.002) however CEBPA and FLT3/ITD mutations were not associated with BAALC expression. Favorable cytogenetics were associated with BAALChigh expression [t(8;21): 1% vs. 23%, p=<0.001; inv (16) 5% vs. 26%;p=<0.001] whereas normal cytogenetics and 11q23abnormalities were more frequent in the BAALClow cohort [normal cytogenetics 32% vs 13%; p=0.002; 11q23: 33% vs. 6%; p=<0.001]. High-risk cytogenetic features were not associated with BAALC expression in limited analysis (n=6 patients). BAALC expression was also correlated with disease-risk, a classification derived from prognostic cytogenetic and molecular features. BAALChigh was more common in patients with low-risk disease (19% vs. 54%, p=<0.001) whereas BAALClow was more frequently seen in SR patients (69% vs 32% p=<0.001). There was no association between high-risk patients and BAALC expression in a limited number of samples analyzed (n=26). Induction response was independent of BAALC expression (77% vs. 82%, p=0.442) although minimal residual disease (MRD) at end of induction was marginally associated with high expression (20% vs. 35%, p=0.058). There were no differences in OS and EFS from study entry for BAALClow vs. BAALChigh patients (5 year OS: 64% vs. 63%, p=0.832; EFS 49% vs. 48%, p=0.868) Corresponding DFS and RR from CR were also comparable (DFS: 58% vs. 56%, p=0.608; RR 34% vs. 31%, p=0.735).We further evaluated the association of BAALC expression with outcome in patients with SR disease. SR BAALClow and SR BAALChigh patients had similar disease presentation (WBC, BM blast %, platelet count, Hb) and were similar in age. Abnormalities of 11q23 were associated with SR BAALClow expression (51% vs. 24%; p=0.006) but other SR cytogenetic abnormalities including normal cytogenetics were not. Rates of induction CR were similar for SR BAALClow versus SR BAALChigh patients (77% vs. 66%, p=0.225) as were rates of end induction MRD (21% vs. 37%, p=0.178). Importantly, there were no differences in OS and EFS from study entry for SR BAALClow vs. SR BAALChigh patients (5 year OS: 56% vs. 59%, p=0.464; EFS 42% vs. 37%, p=0.899). Corresponding DFS and RR from CR were also comparable (DFS: 56% vs. 47%, p=0.756; RR 36% vs. 41%, p=0.990). Conclusions:In this study, elevated BAALC expression was not associated with clinical outcome, within our total study cohort and in those patients with SR disease. This suggests that BAALC expression may lack independent prognostic significance within pediatric AML, despite its association with specific disease characteristics. Validation of our findings in a larger pediatric cohort, and in all disease-risk groups, is ongoing within COG protocol AAML0531. Disclosures:Franklin:Amgen: Employment.

Detection of an Unbalanced Ratio Between WT1 Isoforms in Acute Myeloid Leukemia and Its Correlation with Molecular Abnormalities.

AbstractAbstract 2500Wilms’ tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity.The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results:KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion:Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures:No relevant conflicts of interest to declare.

The clinical relevance of BAALC and ERG expression levels in pediatric AML

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease,1 for which survival rates have increased up to 70% over the past years.2 Approximately 45% of adult AML patients and 20% of pediatric AML patients are diagnosed with cytogenetically normal AML (CN-AML), which is currently classified and treated as a homogeneous intermediate-risk group.3 For future stratification of pediatric CN-AML, it is important to unravel the underlying molecular aberrations. For example, single-gene mutations such as NPM1 and CEBPA double mutations appeared to predict favorable outcome, whereas WT1 mutations and the cryptic NUP98/NSD1 translocation4 are of poor prognostic significance. In adult AML patients, the expression levels of specific genes have been associated with prognosis, mainly in CN-AML. For instance, high expression levels of BAALC (brain and acute leukemia cytoplasmic; 8q22) or ERG (E26 transformation-specific-related; 21q22) genes predict a poor prognosis in most adult AML studies, confined to CN-AML, as described by Schwind et al.5