Brain And Acute Leukemia, Cytoplasmic Expression Levels Research Articles

BAALC and ERG expression levels at diagnosis have no prognosis impact on acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell transplantation.

Brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) expression levels are independent prognostic factors for acute myeloid leukemia (AML); however, their prognostic impacts on AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) require further investigation. We studied 71 de novo AML patients treated with allo-HSCT and defined low and high expressers according to the median expression levels of BAALC and ERG at diagnosis respectively. High BAALC expression was associated with wild-type NPM1 (P = 0.000) and RUNX1 mutations (P = 0.027). High ERG expression was associated with FLT3-ITD absence (P = 0.003) and wild-type NPM1 (P = 0.001). BAALC and ERG expression levels were significantly correlated with each other (P = 0.001). Survival analyses including Kaplan-Meier curves and univariate and multivariate analysis consistently reported that there were no significant differences for both event-free survival (EFS) and overall survival (OS) (all P > 0.1), between high versus low BAALC and ERG expressers. Our study suggested that despite of their well-known adverse role in prognosis of AML, neither BAALC nor ERG expression levels at diagnosis had effect on survival of AML patients who underwent allo-HSCT.

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.

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.

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

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

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

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

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

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

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

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

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

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

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

Significant Down-Regulation of BAALC during Lineage Specific Hematopoietic Differentiation.

The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) implicated in normal hematopoiesis and leukemia is a marker of immature hematopoietic progenitor cells (PNAS 2001; 98:13901). To explore its role in hematopoiesis we investigated the regulation of BAALC during lineage specific differentiation. Semi-quantitative RT-PCR was performed on subsets of in vitro differentiated bone marrow CD34+ cells from healthy individuals using the cytokines G-CSF, M-CSF or EPO. Cultures were harvested on day 4, 8, 12, and 16. In addition, oligonucleotide microarray analyses (HG-U133A, Affymetrix) of in vitro stimulated CD34+ cells were independently carried out using EPO, TPO, G/GM-CSF to induce lineage-specific differentiation. For each of the lineages, cells were harvested at days 4, 7 and 11 for expression profiling. All experiments were done in triplicates for each time point and condition. RT-PCR revealed down-regulation of BAALC and CD34 as early as day 4 in cultures with G-CSF, M-CSF or EPO. In concordance, gene expression profiling showed significant down-regulation of BAALC and CD34 on day 4 of culture (BAALC: EPO p=0.04, G/GM-CSF p=0.17, TPO p=0.03; CD34: EPO p=0.01, G/GM-CSF p=0.003, TPO p=0.01). 275 genes showing a significant correlation (correlation coefficient r>0.95) to BAALC expression levels (at the 4 time points and using 3 different cytokines) were identified. Of these CD34 ranked second (r=0.99), and genes related to leukemia and neuronal cells included: Hepatic leukemia factor (HLF; r=0.99) and Paired box gene 5 (PAX5; r=0.99). It has been speculated that BAALC might be involved in cell movement or cell-cell interaction; we therefore investigated the association to genes implicated in cytoskeletal function. Expression of Myosin 5C (MYO5C; r=0.99) and Synaptic nulcei expressed gene 2 (SYNE2; r=0.95) were highly correlated with BAALC expression across all conditions (EPO, TPO, G/GM-CSF). In conclusion, using two independent approaches we show the significant down-regulation of BAALC during hematopoietic differentiation. This underscores the potential role of BAALC in early hematopoiesis and the possible contribution of aberrant BAALC expression in leukemogenesis. We postulate that similar to CD34, PAX, and HLF implicated in hematopoiesis and leukemia, down-regulation of BAALC and structural genes including Myosin 5C are critical steps during hematopoietic differentiation.

High BAALC Expression as an Independent Adverse Risk Factor in 309 Patients with Acute Myeloid Leukemia (AML) and Normal Cytogenetics: Results of the SHG'96 Study.

High mRNA expression levels of the human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) have been shown to be an adverse risk factor in newly diagnosed AML patients (pts; Blood 2003; 102:1613). The first study included 86 de novo AML pts under the age of 60 with normal cytogenetics and a more favorable FLT3 mutation status, which were uniformly treated on the Cancer And Leukemia Group B protocol 9621. An independent confirmation of these data is necessary, to confirm the impact of BAALC expression in intermediate risk AML pts. Here we present the results of a Süddeutschen Hämoblastosegruppe (SHG) study including 309 adult pts diagnosed with de novo (n=280) and secondary AML (n=29), normal cytogenetics, age <60 years that were uniformly treated on the SHG'96 protocol. Treatment consisted of 2 cycles of induction therapy followed by intensive consolidation including autologous as well as allogeneic stem cell transplantation. Median follow-up for patients alive was 28.7 months (range: 0–86 months). BAALC expression was measured in pretreatment peripheral blood blasts by comparative real-time RT-PCR. Pts having BAALC expression values within the lower 50% were classified as low BAALC and pts having BAALC expression values within the upper 50% were classified as high BAALC. Whereas no correlation was seen between BAALC expression and the prevalence of FLT3 internal tandem duplications (ITD; P=0.16), high BAALC patients had a significantly higher FLT3 mutant/wild-type (wt) ratio as determined by Genescan analysis (mean mutant/wt ratio: 0.59 vs. 0.17; P=0.012). There was no significant difference in the prevalence of MLL partial tandem duplications between the high and the low BAALC group. High BAALC expression was significantly more frequent in FAB groups M0/M1 as compared to the other subtypes (P=0.002). Pts with high BAALC expression levels had a significantly lower CR rate (62.3% vs. 73.5%; P=0.039) and a higher relapse rate (43.8% vs. 28.9%; P=0.030). The overall survival (OS) was significantly shorter for pts with high BAALC expression (OS at 4 years: 29.8% vs. 53.3%; P=0.0018), event-free survival (EFS, at 4 years: 21.6% vs. 45.8%; P=0.0001), and disease-free survival (DFS, at 4 years: 30.4% vs. 57.6%; P=0.0018). Pts with a high FLT3 mutant/wt ratio (greater than 0.8) had a significantly shorter OS and DFS. For patients with a low FLT3 mutant/wt ratio, high BAALC expression allowed identification of pts with a high risk of treatment failure. A multivariate analysis confirmed high BAALC expression and a high mutant/wt FLT3 ratio as the only independent adverse risk factors. For pts with high BAALC expression the hazard ratio of death was 1.7 (95% CI 1.18–2.48; P=0.005), and the hazard ratios for EFS and DFS were 1.8 (95% CI 1.3–2.6; P=0.0003) and 2.0 (95% CI 1.2–3.3; P=0.004), respectively. In conclusion, this independent and larger study strengthens high BAALC expression as one of the most relevant adverse prognostic risk factors in intermediate risk AML pts with normal cytogenetics. Thus, determination of BAALC expression should be considered for risk adaptive treatment strategies.