High BAALC Research Articles

MiR-3151 interplays with its host gene BAALC and independently affects outcome of patients with cytogenetically normal acute myeloid leukemia

AbstractHigh BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

Abstract 1307: Heritable polymorphism predisposes to high expression of BAALC in cytogenetically normal acute myeloid leukemia (CN-AML)

Abstract Introduction: Risk-adapted therapy of AML patients (pts) is presently based on cytogenetic and molecular findings. However, the outcome remains poor for the majority of pts. Overexpression of BAALC is implicated in leukemogenesis and poor survival of CN-AML pts, but the underlying mechanisms are unknown. We hypothesized that the elevated expression of BAALC might result from a genetic variant in or close to the gene (in cis). Methods and Results: BAALC and RUNX1 expression were measured in pre-therapy samples from a test set of 253 CN-AML pts (Cancer And Leukemia Group B) using Affymetrix U133 plus 2.0 arrays. Pts were grouped as high or low BAALC and high or low RUNX1 expressers using the median expression value as the cut-off and screened for single nucleotide polymorphisms (SNPs) by direct sequencing of the BAALC gene. Nine informative SNPs were identified. By genotyping, marker rs62527607, a non-coding SNP in the promoter region correlated with high BAALC expression (rs62527607G>T: genotypes TT/GT vs. GG: P=2.01E-04). Luciferase reporter constructs (pGL4.11) spanning the respective alleles demonstrated that the T allele (“risk allele”) of rs62527607 increased luciferase activity compared to the G allele (P=1.0E-02). The T allele of rs62527607 is predicted to create a binding site for the transcription factor RUNX1. Indeed, RUNX1-cotransfection showed an increase in luciferase activity for the T allele of rs62527607 (P=2.0E-02) but not for the G allele (P=2.7E-01). An Electrophoretic Mobility Shift Assay comparing the binding activity of the two alleles showed highly increased binding activity of RUNX1 to the T allele vs. the G allele. Testing of the pt samples for a potential correlation of RUNX1 and BAALC expression levels revealed a positive correlation in pts with genotypes TT/GT but not GG when restricting the analysis to the high RUNX1 expressing group (P=1.18E-04). For validation of our findings, a set of 105 CN-AML pts comprising 52 high and 53 low BAALC expressers (AML Study Group [AMLSG], quantitative Real-Time PCR [qPCR]) was genotyped for rs62527607, determined for RUNX1 expresser status (qPCR) and analyzed alone and in combination with the test set. A non-significant association of TT/GT vs. GG with high BAALC expression (31% vs. 19%; P=1.6E-01) was seen. Analyzing both sets combined resulted in a P-value of 9.3E-05 (Odds Ratio [OR]=2.59 [1.62, 4.22; 95%CI]). Combining the results of the CALGB & AMLSG series showed a highly significant association between RUNX1 and BAALC expression in pts with genotypes TT/GT belonging to the high RUNX1 expressing group (P=7.97E-05, OR=4.02 [2.05, 8.21; 95%CI]). Conclusion: We show that the risk allele of rs62527607 creates a RUNX1 binding site, thereby leading to increased transcription of BAALC. The observation that a heritable trait is involved in the leukemogenesis process might initiate a new era of personalized medicine in AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1307. doi:1538-7445.AM2012-1307

Histone post-translational modifications associated to BAALC expression in leukemic cells

BAALC expression is an indicator of aggressiveness in acute myelogenous leukemia (AML). Overexpression of this gene is associated to poor of clinical outcome. It is known that post-translational histone modifications control gene transcription. Thus, here we have investigated BAALC expression and post-translational histone modifications in leukemia cell lines. We show that Kasumi-6 and Kyo cells have high and low BAALC mRNA levels, respectively. Moreover, we demonstrate that these cell lines present distinct profiles in terms of histone post-translational modifications (H3K9K14 acetylation, H3K4 trimethylation and H3K23 trimethylation) at the level of BAALC promoter. These findings, in light of recent data on how histone post-translational modifications control gene expression, indicate that BAALC gene is “paused” and that in leukemia cells its transcription can be activated or repressed by mechanisms acting on epigenetic marks.

Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia

AbstractMutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.

MiR-3151, a Novel MicroRNA Embedded in BAALC, Is Only Weakly Co-Expressed with Its Host Gene and Independently Impacts on the Clinical Outcome of Older Patients (Pts) with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Abstract 1462Despite progress made in understanding the biology and risk-adapted treatment of AML, prognosis of older pts [age ≥60 years (y)] remains poor. We have shown that high expression of the BAALC gene bestows worse outcome in older CN-AML pts. Yet, the mechanism(s) by which BAALC affects response to therapy remain unknown. MicroRNAs (miRs) are small noncoding RNAs that hybridize to target mRNAs and inhibit their translation or promote degradation, resulting in downregulation of the corresponding proteins. Recently, miR-3151 was discovered embedded in intron 1 of BAALC. We thus hypothesized that miR-3151 might be co-expressed with BAALC and contributes alone or in concert with its host to poor prognostic impact in older CN-AML. MiR-3151 and BAALC expression were measured by quantitative real time (RT) PCR in pretreatment blood of 179 older CN-AML pts enrolled on Cancer and Leukemia Group B (CALGB) cytarabine/daunorubicin-based protocols. The expression values of miR-3151 and BAALC were normalized to the internal controls SNORD44 and RN18S1, respectively. MiR-3151 expression correlated only weakly with the expression of BAALC (Spearman correlation r= 0.3). For clinical and prognostic analyses pts were dichotomized into high and low miR-3151 and BAALC expressers at the median miR and gene transcript expression values and analyzed for other molecular prognosticators (FLT3 -ITD, FLT3 -TKD, CEBPA, IDH1, IDH2, NPM1, TET2, WT1 mutations and ERG expression). Gene (GEP) and microRNA (MEP) expression profiles associated with miR-3151 expression were derived, using Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, respectively. At diagnosis, higher miR-3151 expression was associated with lower % blood blasts (P =.02), FAB types M4 and M5 (P =.05), and wild-type NPM1 (P<. 001). High miR-3151 expressers had a lower complete remission (CR) rate (62% v 81%, P=. 005), and, with a median follow-up of 5.1 y for pts alive, shorter disease-free (DFS; hazard ratio [HR]=1.76, P=. 003; 3y rates, 7% v 26%; Figure 1A) and overall survival (OS; HR=1.86, P <.001; 3y rates, 10% v 32%; Figure 1B) than low expressers. In multivariable analyses (MVA), higher miR-3151 expression no longer remained in the model for CR after adjusting for BAALC expression and white blood count (WBC), but higher miR-3151 expression remained associated with shorter DFS (HR=2.32, P <.001), after adjusting for FLT3 -TKD, ERG expression status and WBC, and shorter OS (HR=1.77, P=. 003), after adjusting for ERG and BAALC expression status. To gain biological insights of miR-3151 -associated AML, we derived a GEP comprising 377 annotated genes and a MEP comprising 14 miRs. High miR-3151 expressers exhibited upregulation of genes associated with immature differentiation stage and adverse outcome (eg, MN1, ID1), downregulation of genes involved in hematopoietic differentiation (eg, MEIS1, PDCD4, PBX3) and transcriptional regulators (ZNF s, E2F3), including predicted targets of miR-3151 (eg, MEIS1). In the MEP, high miR-3151 expressers showed downregulation of let-7a/b/c that play crucial roles in cell cycle and proliferation regulation. While miR-3151- associated GEP/MEP shared genes with BAALC expression-associated GEP/MEP (eg, upregulated MN1 and DNTT and downregulated MEIS1, let-7b, miR-10a/b), other genes were unique to miR-3151- associated GEP/MEP (eg, upregulated ID1 and downregulated ZNFs, miR-206 and let-7a/c), suggesting biologic mechanisms distinct from those directly connected with BAALC upregulation operating in miR-3151- associated leukemia. In conclusion, high miR-3151 expression is independently associated with worse survival in older CN-AML pts. The weak correlation between expression of miR-3151 and its host gene BAALC, the independent impact of each on outcome endpoints (BAALC was a strong predictor of CR but did not predict DFS while miR-3151 was not independently associated with CR, but remained in the DFS MVA) and differences in GEP/MEP suggest that miR-3151 and BAALC are deregulated and contribute to disease outcome differently. We surmise that determining the expression levels of miR-3151 at diagnosis might help to improve the risk-stratification of older CN-AML. Development of therapies targeting miR-3151 upregulation with synthetic inhibitors may provide novel, effective strategies for personalized treatment of older CN-AML pts. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Functional role of BAALC in leukemogenesis

High expression of BAALC in cytogenetically normal-acute myeloid leukemia (AML) patients is associated with primary resistant disease, shorter relapse-free, disease-free and overall survival.1, 2, 3, 4 However, there is no published data evaluating the role of BAALC in the hematopoietic system. BAALC is located on human chromosome 8q22.3 and is highly conserved in mammals. High BAALC expression levels were first identified in a study of AML patients with trisomy 8 as a sole abnormality,5 and was shown to correlate highly with MN1 expression,6, 7 a potent oncogene in leukemogenesis.8 Several isoforms of BAALC have been described with isoform 2 (also known as 1-6-8) being the most abundant in the brain and in AML samples.5

Functional Role of BAALC In Leukemogenesis

AbstractAbstract 4194Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known.We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice.In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells.We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients. Disclosures:No relevant conflicts of interest to declare.

Impact of Expression of BAALC, CDKN1B, ERG, EVI1, and MN1 on Prognosis and Their Association with Karyotype, FLT3-ITD, NPM1 and MLL-PTD Status In Adult AML: A Comprehensive Study on 286 Cases

AbstractAbstract 953 Introduction:The WHO classification in 2008 listed for the first time aberrant expression of genes as molecular genetic alterations affecting outcome in AML. High expression of BAALC, ERG and MN1 were shown thus far to be associated with unfavorable outcome in normal karyotype AML (AML-NK). In addition high EVI1 expression was suggested to predict poor outcome. Recently, our group identified low expression of CDKN1B as a favorable prognostic marker. The aim of this study was to evaluate the expression of BAALC, CDKN1B, ERG, EVI1 and MN1 in AML comprising all cytogenetic risk groups with respect to their association with distinct cytogenetic and known molecular genetic subgroups and their impact on prognosis. Patients/Methods::Expression levels of BAALC, CDKN1B, ERG, EVI1 and MN1 were determined by oligonucleotide microarrays (HG-U133 Plus 2.0, Affymetrix) in 286 AML (t(15;17) n=15; t(8;21) n=16; inv(16) n=7; normal karyotype n=99; 11q23/MLL-rearrangements n=10; complex karyotype n=51; other abnormalities n=88). Patients were further analyzed for mutations in NPM1, FLT3-ITD, CEPBA and MLL-PTD. Results:Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varied significantly between genetic subgroups: While t(15;17), t(8;21) and 11q23/MLL-rearrangements were associated with low CDKN1B expression, AML-NK and NPM+ cases showed a higher CDKN1B expression. Lower BAALC expression was observed in AML with t(15;17), 11q23/MLL-rearrangement and AML-NK as well as in FLT3-ITD+ AML and in NPM1+ AML, while in AML with other abnormalities a higher BAALC expression was observed. ERG expression was lower in AML with 11q23/MLL-rearrangement and normal karyotype, while it was higher in AML with complex karyotype. Low EVI1 expression was observed in AML with t(15;17), t(8;21), inv(16) and AML-NK, while it was higher in AML with 11q23/MLL-rearrangements. Low MN1 expression was associated with t(15;17), t(8;21) and AML-NK, while it was increased in cases with inv(16) or other abnormalities. Next, Cox regression analysis was performed with respect to overall survival (OS) and event free survival (EFS). In the total cohort high BAALC and ERG expression as continuous variables were associated with shorter OS and EFS while CDKN1B, EVI1 and MN1 had no impact. Furthermore the cohort was subdivided into quartiles of expression for each gene. After inspection of the survival curves the cut-off for high vs low expression was set as follows: BAALC: 75th percentile, CDKN1B: 25th percentile, ERG and MN1: 50th percentile. For EVI1 expression pts were separated into expressers (n=44) and non-expressers (n=242). Low CDKN1B expression was associated with longer OS and EFS in the total cohort (p=0.005, not reached (n.r.) vs 14.9 months (mo); p=0.013, 31 vs 9.7 mo). High BAALC expression had no impact on OS, but was associated with shorter EFS in the total cohort as well as in AML with intermediate cytogenetics and AML with other abnormalities (p=0.032, 6.2 vs 13.0 mo; p=0.027, 5.1 vs 11.3 mo; p=0.006, 2.3 vs 14.8 mo). High ERG expression was significantly associated with shorter OS and EFS in the total cohort (p=0.002, 12.5 mo vs n.r.; p=0.001, 8.1 vs 15.7 mo) as well as in AML-NK (p=0.001, 11.3 mo vs n.r.; p=0.010, 7.2 vs 22.1 mo). OS was also shorter in AML with unfavorable karyotype (p=0.048, median OS 9.3 mo vs n. r.). With respect to MN1 high expressers had a significantly shorter OS and EFS in the total cohort (p=0.004, 12.3 mo vs. n.r.; p=0.001, 8.1 vs 16.7 mo) as well as in AML-NK (p=0.001, 9.7 mo vs n.r.; p=0.001, 5.1 vs 22.1 mo). In a multivariate analysis including CDKN1B, ERG and MN1 all parameters retained their impact on OS as well as on EFS, while BAALC lost its impact on EFS. Adding MLL-PTD, NPM1+/FLT3-ITD-, favorable and unfavorable karyotype into the model demonstrated an independent significant adverse impact on OS for MLL-PTD (p=0.027, relative risk (RR): 2.38) and ERG expression (p=0.044, RR: 1.59) only. In the respective analysis for EFS only favorable karyotype showed an independent association (p=0.002, RR: 0.261). Conclusion:1) Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varies significantly between cytogenetic subgroups. 2) BAALC as a continuous variable and CDKN1B, ERG and MN1 as dichotomized variables are independently predictive for OS and EFS in AML. 3) ERG expression even retains its independent prediction of shorter OS if cytogenetic and other molecular genetic markers are taken into account. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Treatment Outcome of Acute Myeloid Leukemia Patients From Brazil According to Karyotype, Presence or Not of FLT3-ITD and NPM1 Mutations, and BAALC Gene Expression.

AbstractAbstract 3836Current results regarding AML treatment in developed countries have shown that the estimate five-year overall survival (OS) is around 40–50%. In contrast, the few existing studies concerning the outcome of AML patients in developing countries reported OS values around 18–25%, despite the fact that there is a relative excess of acute promyelocytic leukemia (APL) cases, which are associated with favorable prognosis. In addition, the studies conducted in developing countries have not taken into account recently reported molecular markers, such as mutations in the NPM1 gene and expression levels of the BAALC gene, which were demonstrated to impact on outcome. The present study aimed to evaluate the effects of karyotype, NPM1 and FLT3-ITD mutations, and BAALC gene expression on the outcome of AML Brazilian patients. One hundred and fifty-eight patients were enrolled in two University Hospitals of the Southeast Region of the country. The median age was 43 years (range:12–92 y) with 78 males (49%). Patients were treated with conventional chemotherapy consisting of daunorubicin (60mg/m2/d for 3 days) and cytarabin (200mg/m2/d for 7 days) as induction, followed by two or three cycles of consolidation therapy with high doses of cytarabin (above 1g/m2/d). The following variables were analyzed: age, gender, WBC count, karyotype, NPM1 and FLT3-ITD mutations, and the quantitative evaluation of the BAALC gene expression. Pretreatment bone marrow samples were analyzed by G-banding cytogenetic, of which 143 were successful, and so allowed the stratification of patients in groups according to prognosis, as proposed by the Medical Research Council (MRC) in: favorable (n=48), intermediate (n=77) and adverse (n=18). A combination of ASO-RT-PCR and sequencing was performed to detect NPM1 mutations. FLT3-ITD detection was performed by PCR. The relative expression levels of BAALC gene were measured by RQ-PCR, according to the comparative cycle threshold (Ct) method and patients were classified as presenting high or low expression using the median as the cut-off value. The estimated five-year OS for all patients was of 15.3% (S.D.: ± 3.4%). The median OS according to cytogenetic stratification was of 1234, 593 and 230 days for favorable, intermediate and adverse groups, respectively (p=0.04). Univariate analysis detected as significant the following variables: age, cytogenetic risk group and presence of FLT3-ITD (p=0.03). Indeed, patients harboring FLT3-ITD patients presented a mean OS of only 300 days (range: 116–485 days) in comparison with 770 days (531 – 1010) in patients without FLT3 mutations. Besides, FLT3-ITD was associated with genre (more common in females, p=0.02) and with cytogenetics (more frequent in the favorable and intermediate cytogenetic risk groups). When only cytogenetically normal patients were analyzed (n=55), NPM1 mutations were significantly associated with longer OS (mean: 1058 days, range: 449–1666 days vs. 276 days, range: 145–406 days; p=0.03). Patients expressing high BAALC levels presented shorter OS (p=0.04). The multivariate analysis revealed FLT3-ITD as an independent prognostic factor for OS (p=0.007). Our results provide important insights into the characterization of AML patients in Brazil, suggesting that the frequency of cytogenetic abnormalities and FLT3-ITD mutations are similar to those in developed countries, whereas NPM1 mutations are less frequent. Importantly, the outcome was inferior to that reported in Europe and US, thus suggesting that multicentric collaborative efforts are urgently needed. Disclosures:No relevant conflicts of interest to declare.

High BAALC expression predicts chemoresistance in adult B-precursor acute lymphoblastic leukemia

AbstractOverexpression of BAALC is an adverse prognostic factor in adults with cytogenetically normal acute myeloid leukemia and T-cell acute lymphoblastic leukemia (ALL). Here, we analyzed the prognostic significance of BAALC in B-precursor ALL. BAALC MRNA expression was determined in 368 primary adult B-precursor ALL patients enrolled on the 06/99 and 07/03 GMALL trials. Patients were grouped into tertiles according to BAALC expression (T1-T3). Higher BAALC expression (T3 vs T2 vs T1) was associated with higher age (P < .001), a higher white blood cell count (P = .008), CD34 (P = .001), BCR-ABL (P < .001), and MLL-AF4 (P < .001). Higher BAALC expression predicted primary therapy resistance in the overall cohort (P = .002) and in the BCR-ABL− and MLL-AF4− subgroup (P = .01). In BCR-ABL− and MLL-AF4− patients, higher BAALC expression was associated with a shorter overall survival (OS; 5-year OS: T3, 38%; T2, 52%; T1, 70%; P = .004) and independently predicted OS in multivariate models (P = .03). Gene-expression profiling revealed an up-regulation of stem cell markers and genes involved in chemoresistance (TSPAN7 and LYN) in the high BAALC group. Thus, high BAALC expression is associated with an immature, chemoresistant leukemic phenotype and identifies patients with inferior OS. Determination of BAALC might contribute to risk assessment of molecularly undefined adult B-precursor ALL.

Molecular monitoring of BAALC expression in patients with CD34-positive acute leukemia

Recent studies have shown that high BAALC expression predicts an adverse prognosis and may define an important risk factor in acute myeloid leukemia patients with normal karyotype. We performed, using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR), the molecular analysis of BAALC gene as a possible minimal residual disease (MRD) marker in 45 patients with newly diagnosed acute leukemia. BAALC transcript levels in 32 patients with CD34 expressed in leukemic blasts were 2-3 logs higher than background levels, and the copy number was reduced in patients achieving hematological remission. Comparative monitoring of MRD by RQ-PCR for the Wilms' tumor gene 1(WT1) or specific translocation markers demonstrated that BAALC had similar kinetics as WT1, AML1/ETO and minor BCR/ABL, but not PML/RARA. Quantitation of BAALC gene expression made it possible to assess MRD in patients with CD34-positive acute leukemia. To our knowledge, this is the first report concerning the use of BAALC mRNA expression for MRD monitoring.

Prognostic Significance of CEBPA Mutations and BAALC Expression in Acute Myeloid Leukemia Patients with Normal Karyotype

Aim: The aim of this work is to study the prognostic impact of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/enhancer binding protein-α) and expression of the BAALC gene (for brain and acute leukemia, cytoplasmic), a novel gene involved in leukemia, in 38 adults with AML and normal cytogenetics. Method: Screening for mutations of CEBPA gene was assessed using PCR-single-strand conformation polymorphism (PCR-SSCP), and BAALC expression was determined by real-time reverse transcriptase polymerase chain reaction in blood or bone marrow samples. Result: CEBPA mutations were found in 7 (18.4%) of 38 patients, 36.8 % (14 of 38) had low BAALC expression and 63.2 % (24 of 38) had high BAALC expression. Patients with CEBPA mutations had favorable course of their disease. They had higher rate of complete remission (CR) (85.7 % vs 51.6 %; p= 0.108), lower incidence of relapse (0% vs 41.9%; p= 0.038). Disease free survival (DFS) and overall survival (OS) were significantly longer for patients with CEBPA mutations compared with patients without mutations (mean 13.65±5.41 vs 7.32±4.33 months, p= 0.047; mean 15.32±6.5 vs 8.5±3.21 months, p= 0.039; respectively). Compared to low BAALC expressers, high BAALC expressers had lower incidence of CR (50% vs 71.4%; p= 0.171), higher incidence of relapse (50% vs 14.3%; p= 0.029), and showed significantly shorter DFS (mean 7.5±2.12 vs 11.67±4.6 months, p= 0.038) and inferior overall survival (mean 9.1±3.52 vs 13.22±4.21 months, p= 0.024). Conclusion: From this study, we can conclude that CEBPA mutation status and BAALC expression are important prognostic factors in AML patients with normal cytogenetics and their incorporation into novel risk-adapted therapeutic strategies will improve the currently disappointing cure rate of this group of patients.

Favorable Prognostic Impact of WT1 SNP rs16754 in Cytogenetically Normal AML Independent of MN1, BAALC, and ERG Expression.

Abstract 1622▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-648We have previously shown that the minor allele of single nucleotide polymorphism (SNP) rs16754 located in the mutational hotspot of WT1 predicts a favorable prognosis in patients with cytogenetically normal acute myeloid leukemia (CN-AML) independent of mutations in NPM1, FLT3, CEBPA, or WT1. The minor allele of SNP rs16754 was found in 26% of CN-AML patients without any association to known mutations. In the present study we investigated the prognostic impact of WT1 SNP rs16754 in the context of three additional prognostic markers, MN1, BAALC and ERG, in 249 CN-AML patients. High MN1, BAALC, and ERG expression (defined as expression above the median expression level) has been associated with poor prognosis in CN-AML in previous studies. RNA was extracted from diagnostic bone marrow or peripheral blood cells, reversely transcribed, and amplified by quantitative RT-PCR. All patients were uniformly treated on the AML SHG 0295 or AML SHG 0199 treatment protocols. Statistical analyses were performed using SPSS. In univariate analysis the negative prognostic impact of MN1, BAALC, and ERG could be confirmed in our study population. The complete remission rate was significantly lower in patients with overexpression of MN1 (P=.012), BAALC (P=.001) or ERG (P=.041) compared to low expression of these genes. Furthermore, patients with high expression of MN1 or BAALC had significantly shorter relapse-free survival (RFS, P=.002 and P=.007, respectively), while high expression of ERG did not affect RFS (P=.21). Overall survival (OS) was significantly shorter in patients with high expression of MN1, BAALC, or ERG compared to low expressing patients (P=.005, P <.001, P=.034, respectively). Next we evaluated whether WT1 SNP rs16754 allele status was associated with expression levels of MN1, BAALC, and ERG. No correlation was found by chi-squared analysis (MN1 P=.69, BAALC P=.29 or ERG P=.82). The prognostic impact of WT1 SNP rs16754 was then investigated in the subgroup of patients with high MN1, BAALC, or ERG expression. Interestingly, patients with high MN1 expression (n=123) and at least one minor allele of WT1 SNP rs16754 had a significantly longer RFS (P=.008) and OS (P<.001) than patients with the two major alleles. Similarly, patients with high BAALC (n=122) or ERG expression (n=122) and at least one minor allele of WT1 SNP rs16754 had a longer RFS (P=.05 and P=.055, respectively) and OS (P=.013 and P=.005, respectively) compared to patients with the two major alleles. In multivariate analysis for OS, WT1 SNP rs16754 status independently predicted favorable prognosis (HR 0.47, 95% CI 0.28 to 0.77, P=.003) when considered together with NPM1/FLT3 mutation status, CEBPA mutation status, WT1 expression status, MN1 expression status, BAALC expression status, ERG expression status, age, WBC count, and platelet count. In multivariate analysis for RFS, WT1 SNP rs16754 status was an independent favorable prognostic factor (HR 0.49, 95% CI 0.3 to 0.82, P=.006) when considered together with NPM1/FLT3 mutation status, CEBPA mutation status, MN1 expression status, BAALC expression status, ERG expression status, WBC count, and platelet count. Thus, WT1 SNP allele status appears to be a very powerful prognostic marker that may refine currently available prognostic tools. In summary, we identified the minor allele of SNP rs16754 located in exon 7 of the WT1 gene as an independent favorable risk marker in CN-AML patients, enabling the identification of a significant proportion of patients with favorable prognosis that are neither identified by mutational analysis for NPM1, FLT3-ITD, CEBPA and WT1, nor by expression analysis for MN1, BAALC and ERG. DisclosuresNo relevant conflicts of interest to declare.

Value of Gene Expression Profiling by Taqman® Low Density Arrays in the Diagnosis and Prognosis of Acute Myeloid Leukemia.

Abstract 1620Poster Board I-646 BackgroundSo far, none of the high-density microarray studies in acute myeloid leukemia (AML) have provided a useful approach with relevant diagnostic and prognostic value for the routine clinical practice. We aimed to assess the clinical utility of a small combination of genes and fusion genes included in a microfluidic card by means of real-time quantitative PCR (RQ-PCR) in AML patients. Patients and methodsNinety-six clinically relevant markers were analyzed simultaneously using TaqMan® Low Density Arrays (TLDAs) (Applied Biosystems, Foster City, CA). This plataform includes: 3 control genes (ABL1, GADPH and GUS), 36 AML specific fusion transcripts described, NPM1 mutations and 55 genes which RNA expression levels have been associated with prognosis or pathogenic process in AML. To assess the feasibility of the designed TLDA we evaluated the gene expression profile of 191 leukemia patients at diagnosis: 167 AML (inv(16): n=11, t(8;21): n=9, t(15;17): n=7, with 11q23 abnormalities: n=27, with other cytogenetic abnormalities: n=20 and normal karyotype n=93), 16 pre-B-ALL, 5 T-ALL and 3 CML with rare BCR-ABL fusions (b2a3, b3a3 and e19a2). ResultsWe observed a high correlation between TLDA results for fusion transcripts and those obtained by gold standard techniques (RQ-PCR based on Europe against Cancer protocols). Indeed, we identified 89 patients with different fusion transcripts, including 18 cases previously reported as negative by cytogenetics or FISH approach. Patients with a favorable karyotype showed a distinctive gene expression profile. Thus, inv(16) AML overexpressed: MYH11, CLIP3, CTNNB1, SPARC, SNAI1 and MN1; t(8;21) AML expressed high levels of ETO, POU4F1, CAV1, CD34, FOXO3A, PRAME and BAALC, and t(15;17) AML showed upregulation of HGF, FGF13, WT1 and PRAME. Further, the 51 NPM1 mutated patients (38 A, 5 B, 6 D and 2 DD1, confirmed by sequencing) also showed a specific gene profile, showing high NPM1, CD34, ABCB1, ABCG2, BAALC, PROM1 or BCL2 RNA levels and low HOXA7, HOXA9 and MEIS1 RNA levels. In contrast, AML with 11q23 abnormalities or with FLT3-ITD mutations did not exhibit a characteristic pattern. Several genes (EVI1, BAALC, ERG, PRAME or PIM1) showed prognostic significance in AML with normal karyotype and AML with NPM1 mutations, thus confirming TLDA as a useful approach for risk assessment in AML. Interestingly, PIM1 overexpression was an independent predictor for shorter relapse-free (RFS) and overall survival (OS) in patients with normal karyotype (p=0.012 and p<0.001, respectively) and patients harboring NPM1 mutations (p=0.004 and p=0.002). Moreover, patients with FLT3-ITD mutations and high levels of PIM1 showed an extremely poor prognosis (2-year RFS 26% and OS 30%). These results were confirmed in multivariate analyses. ConclusionsWe showed the diagnostic and prognostic value of the designed TLDA platform for evaluation of AML patients. Furthermore, in our series PIM1 has been identified as the most important prognostic marker and thus this oncogene could be used for a more accurate risk classification of AML patients. DisclosuresGutiérrez:Janssen Cilag: Honoraria; Celgene: Honoraria. Díaz-Mediavilla:Janssen-Cilag: Honoraria; Celgene: Honoraria.

Aberrant Gene Expression of BAALC and ERG in Older [≥60 Years (y)] De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): A Cancer and Leukemia Group B (CALGB) Study.

Abstract 214BAALC &ERG are aberrantly expressed in younger (<60 y) adult CN-AML patients (pts), where expression levels of these genes are also associated with clinical outcome. Whether aberrant BAALC &ERG expression also occurs in older (≥60 y) CN-AML pts is unknown. To assess the BAALC &ERG expression levels in older CN-AML & their impact on outcome we studied pts ≥60 y (median age, 68 y; range 60–83) enrolled on cytarabine/daunorubicin-based protocols [CALGB 10201, 9720, 9420, 8923, 8525], with diagnostic blood samples available for quantitative RT-PCR analysis (n=158), that were also characterized for other molecular prognosticators (FLT3-ITD, FLT3-TKD, NPM1 &WT1 mutations). BAALC &ERG expression values were normalized to an internal control (ABL1), & the median gene expression value was used to define high & low expressers for BAALC &ERG. Gene (GEP) & microRNA (MEP) expression profiling were done using, respectively, Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays. At diagnosis, lower BAALC expression was associated with mutated NPM1 (P<.001) & low ERG expression (P<.001). Lower BAALC expressers had a higher complete remission rate (CR; 86% v 54%, P<.001) & longer disease-free (DFS; P=.006; 3y rates 19% v 12%) & overall survival (OS; P<.001; 3y 29% v 10%) than higher expressers. Lower ERG expression was associated with lower WBC (P=.005), % marrow (BM; P=.001) & blood (P<.001) blasts & absent FLT3-ITD (P<.001) & low BAALC expression (P<.001). Lower ERG expression also associated with longer DFS (P=.001; 3y 18% v 14%) & OS (P<.001; 3y 24% v 15%). In multivariable models (Table 1), low BAALC expression independently associated with CR & longer DFS. BAALC &ERG expression were the only factors associated with OS. Comparison of age-groups (60-69 y v ≥70 y; Table 2) showed BAALC expression had a stronger prognostic impact in ≥70 y pts; lower expression was associated with higher CR rates & longer DFS & OS. ERG expression had instead stronger prognostic impact in 60-69 y pts (Table 2); lower expression was associated with longer DFS & OS. GEP (482 probes) & MEP (22 probes) differentiated low from high BAALC expressers. Low BAALC expressers had down-regulation of stem cell markers (CD34, CD133) & unfavorable outcome predictors (HGF, MN1, CD200), & up-regulation of HOX genes &miR-10a &miR-10b. GEP (1554 probes) & MEP (11 probes) differentiating low from high ERG expressers showed low ERG expressers had down-regulation of DNMT3B & up-regulation of topoisomerase 1 (TOP1), which is associated with enhanced chemotherapy sensitivity. Among up-regulated microRNAs in low ERG expressers was miR-208a, which is predicted to target ERG. In conclusion, lower expression of both BAALC &ERG associated with better outcome in older CN-AML pts even in the context of other established prognostic molecular markers, but have different impact on age-groups. GEP & MEP provided novel information that may elucidate how differential expression levels of these genes contribute to leukemogenesis & aid in developing novel risk-adapted therapies.Table 1:Multivariable analyses for CR, DFS & OSVariables in the final modelCRDFSOSOR*PHR†PHR†PBAALC, low v high (median cut)4.32<.0010.64.030.49<.001‡ERG, low v high (median cut)----0.69.03WBC, each 50 unit increase0.68.02----NPM1, mutated v wild-type2.34.04----FLT3-ITD, any ITD v no ITD--1.79<.001‡--Age, each 10 year increase--0.65.047--*OR, odd ratio, >1 (<1) mean higher (lower) CR rate for the 1st category listed for the dichotomous variables & for the higher values of the continuous variables.†HR, hazard ratio, <1 (>1) indicate lower (higher) risk for an event for the first category listed for the dichotomous variables & for the higher values of the continuous variables. Variables considered in the models were those significant at α=0.20 in univariable analyses.‡Variable did not meet the proportional hazards assumption, a covariate was used to account for time dependence.Table 2:Age-Group comparison for CR, DFS & OSClinical endpointBAALC expressionERG expression60-69 y Pts (n=86)≥70 y Pts (n=72)60-69 y Pts (n=86)≥70 y Pts (n=72)LowHighPLowHighPLowHighPLowHighP% achieving CR8471.198837<.0018076.807153.15DFS, % in CR at 3 y1610.102314.03196<.0011725.48OS, % alive at 3 y2412.01358<.0012412.0062318.03The age-group clinical endpoints significantly associated with BAALC or ERG expression levels are denoted by bold type. Disclosures:No relevant conflicts of interest to declare.

Prognostic Importance of MN1 Transcript Levels, and Biologic Insights From MN1-Associated Gene and MicroRNA Expression Signatures in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in the context of other predictive molecular markers, and to derive MN1 associated gene- and microRNA-expression profiles in cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS MN1 expression was measured in 119 untreated primary CN-AML adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction. Patients were also tested for FLT3, NPM1, CEBPA, and WT1 mutations, MLL partial tandem duplications, and BAALC and ERG expression. Gene- and microRNA-expression profiles were attained by performing genome-wide microarray assays. Patients were intensively treated on two first-line Cancer and Leukemia Group B clinical trials. Results Higher MN1 expression associated with NPM1 wild-type (P < .001), increased BAALC expression (P = .004), and less extramedullary involvement (P = .01). In multivariable analyses, higher MN1 expression associated with a lower complete remission rate (P = .005) after adjustment for WBC; shorter disease-free survival (P = .01) after adjustment for WT1 mutations, FLT3 internal tandem duplications (FLT3-ITD), and high ERG expression; and shorter survival (P = .04) after adjustment for WT1 and NPM1 mutations, FLT3-ITD, and WBC. Gene- and microRNA-expression profiles suggested that high MN1 expressers share features with high BAALC expressers and patients with wild-type NPM1. Higher MN1 expression also appears to be associated with genes and microRNAs that are active in aberrant macrophage/monocytoid function and differentiation. CONCLUSION MN1 expression independently predicts outcome in CN-AML patients. The MN1 gene- and microRNA-expression signatures suggest biologic features that could be exploited as therapeutic targets.

Up‐regulation of BAALC gene may be an important alteration in AML‐M2 patients with t(8;21) translocation

Up‐regulation of <i>BAALC</i> gene may be an important alteration in AML‐M2 patients with t(8;21) translocation

Significance of BAALC, MN1, ERG, CEBPa Gene Expression as New Prognostic Factors, in the Context of Other Molecular Markers in Intermediate Risk AML Group According to Cytogenetics, Including Patients Younger Than 60 Years

Background: High expression of BAALC,MN1, ERG and low CEBPA have been shown to be an adverse risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). However intermediate (IM) risk group of AML includes not only patients with normal karyotype, but also with other aberrations, of unknown clinical significance. That's why it is important to evaluate prognostic impact of the above mentioned markers in the IM group of AML patients as total. So far, the prognostic significance of expression level of the studied markers has not been yet compared with each other, as well as evaluated in the context of other molecular biomarkers, such as FLT3 ITD (internal tandem duplication), MLL-PTD (partial tandem duplication) mutations, and EVI1 expression level.Aims: To evaluate the prognostic impact of BAALC,MN1, ERG, CEBPA expression in the context of FLT3-ITD, MLL-PTD and EVI1 expression on the complete response to induction therapy (CR), and the relapse rate in the patients group representing intermediate risk group of AML according to cytogenetics.Methods: 86 patients (16 to 60 years of age) with untreated primary IM-risk AML were included in this study: 55 with normal karyotype, and 31 with other aberrations. Patients were treated similarly according to the Polish Acute Leukemia Group (PALG) protocol (induction chemotherapy: daunorubicin, cytarabine plus fludarabine or cladribine, and two cycles of consolidation high-dose cytarabine with mitoxantrone and high- dose cytarabine). BAALC, MN1, ERG, CEBPA expressions were evaluated in the bone marrow cells from IM-AML patients by real-time RT-PCR technique, using TaqMan probes, as recently described.Results: High and low BAALC, as well asERGandMN1 expression was determined by dichotomizing delta Ct values at the median (high expression of ERG, MN1 referred further as negative or positive cases). High BAALC correlated significantly with the group positive for MN1 (p=0.01), ERG (p=0.01) as well as MLL-PTD (p=0.045), while no correlation was found between: high BAALC and FLT3-ITD, as well as CEBPA. There was a trend towards significance towards lower CR rates in the groups with high BAALC, positive MN1, positive EVI1 expression or MLL-PTD, while low CEBPA showed negative correlation with CR rate (p=0.02). Next we analysed cumulative presence of the subsequent markers: MLL-PTD, positive ERG, positive MN1, and positive EVI1. Cases negative for all of above markers presented 85 % of CR rate, patients with one or two markers present: 55% CR rate, and a group with more than three of them positive: only 20% CR rate. Further comparing DFS after 2nd and 3rd year of follow up, with each marker separately did not show significant correlation, although a trend towards significance was observed for most of them. However again, cumulative presence of high BAALC, positive MN1, positive ERG, positive EVI1, and MLL-PTD showed significant correlation with 2 and 3 years' DFS (respectively 70% for the cases with 0 –2 markers, while 22% for the group with more than 2 markers present; p=0.035).Conclusions: In our study, we analysed a series of patients with intermediate risk according to the cytogenetics (NC and other aberrations). Cumulative presence of high BAALC, and positiveERG, MN1, EVI1 expression levels as well as MLL-PTD, showed their prognostic significance.

ERG, EVI1 and PRAME Are Relevant Markers of Treatment Response and Survival, and They Could Be Useful for Improving the Risk-Stratification in Citogenetically Normal Acute Myeloid Leukemia (CN-AML)

Several mutations and aberrant gene expression have been proposed as prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML), but few studies have analyzed the clinical value of several genes in a large group of CN-AML patients. In 121 de novo CN-AML included into the Spanish PETHEMA therapeutic protocols, we evaluated FLT3 and NPM1 mutations and BAALC, ERG, EVI1, MLL-PTD, MN1, PRAME, RHAMM and WT1 RNA levels. Ninety-one patients achieved CR with standard induction therapy while 20/111 (16.5%) were refractory to treatment. This latter subgroup showed higher ERG (media 1.0±0.8 vs. 0.6±0.6;p=0.012) and lower PRAME (media 28.8±52.9 vs. 1640.6±6102.0;p=0.014) levels than no-refractory patients. Moreover, high ERG expression (defined by median value) was associated with shorter OS at 2-years (38% vs. 61%;p=0.012) and RFS at 2-years (38% vs. 60%;p=0.005). By contrast, high PRAME levels (defined by 75th percentile) was related to longer OS (61% vs. 48%;p=0.029) and RFS (74% vs. 43%;p=0.016). Additionally, high EVI expression (defined by 75th percentile) was associated with a poorer OS (37% vs. 56%;p=0.049). The same results in OS were observed in patients with high BAALC RNA levels (median value; 41% vs. 62%; p=0.049). Interestingly, ERG, EVI1 and PRAME markers improved the current prognostic classification of CN-AML based on FLT3 and NPM1 status. Therefore, intermediate risk patients (FLT3-ITD/NPM1mut and FLT3wt/NMP1wt) with low ERG, low EVI1 or high PRAME achieved long OS and RFS, similar to the favorable FLT3wt/NPM1mut subgroup. By contrast, the opposite expression subgroup (high ERG, high EVI1 or low PRAME) showed OS and RFS similar to the adverse FLT3-ITD/NPM1wt subset. These results allowed identify clearly two risk-subgroups for OS and RFS, based on three molecular markers (FLT3, NPM and one of the 3 proposed genes). In conclusion, we demonstrated that ERG, EVI1 and PRAME are relevant prognostics markers in CN-AML and they could improve their risk-stratification.

High BAALC expression is indicative of poor survival in acute myeloid leukemia

High BAALC expression is indicative of poor survival in acute myeloid leukemia