Brain And Acute Leukemia, Cytoplasmic Expression Research Articles

Over-Expression of ERG Predicts Poor Outcome in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) treatment aims at the eradication of all leukemic cells to achieve cure. Available treatments produce complete remission (CR) in up-to 80% of patients. However, ~60% of them will relapse. Various prognostic factors such as baseline white blood cell count (WBC), cytogenetics and mutations of FLT3 , NPM1 , CEBPA have been shown to affect the survival. The high expression of brain and acute leukemia cytoplasmic (BAALC) and ETS- related gene (ERG) has been associated with poor prognosis but due to limited prospective studies, their role as prognostic factors is still unclear. We aim to substantiate the prognostic value of BAALC and ERG expression in adult AML patients.Materials and methods: All patients included in this prospective study (N=149) between March 2012 and February 2015 underwent marrow for following investigations: (i) Cytogenetic analyses- by standard techniques of chromosome banding and FISH (ii) Mutation profiling of NPM1 , FLT3 -ITD and CEBPA by capillary electrophoresis and direct DNA sequencing. Long range PCR was done to detect KMT2A-PTD (MLL -PTD) (iii) BAALC and ERG expressions by quantitative real time PCR assays. Abelson (ABL ) gene was used as an internal control. According to the baseline median values of BAALC and ERG expression, patients were divided into high and low expressers.Patients received standard induction chemotherapy with daunorubicin 60 mg/m2 for 3 days and cytosine arabinoside 100 mg/m2 for 7 days. Marrow was done 21-28 days after start of chemotherapy. If marrow was in remission, then patients received 3 courses of consolidation therapy with 12-18 gm/m2 of cytosine arabinoside (HiDAC). Patients underwent allogeneic transplant if feasible according to molecular and cytogenetic risk.Statistical analysis was done using SPSS-21. Overall survival (OS) was defined as time from start of induction therapy to time to last follow up or death. Relapse free survival (RFS) was defined as time from achieving CR to time to relapse, death or last follow up if in CR. Results of the expression studies, cytogenetic and molecular groups were analyzed for their impact on OS and RFS. Variables known to have prognostic implications such as baseline WBC, cytogenetics risk groups and molecular markers were grouped according to high/low BAALC and ERG expressers. Comparison of these prognostic factors within high/low expressers was carried out by Chi-square method. Survival was calculated by Kaplan-Meier method and prognostic factors were compared by log-rank test. Multivariate analysis was carried out using the Cox proportional hazards model. All covariates whose univariate models reflected a P value less than 0.2 were considered for inclusion in the model.Results: A total of 149 patients were accrued. The median age of the cohort was 27 years (range 15-58 ) with male predominance (M:F =98:51). Of 149 patients, 105 achieved CR after 1 or 2 cycles of chemotherapy. Twenty-seven patients were refractory of which 18 were lost to follow up and hence excluded from OS analysis. Seventeen patients died during induction. The OS of 131 patients was 36.5% at 2 years. The OS and RFS of 105 patients who achieved CR was 54% and 43.8% respectively. The median value (60 BAALC copies /ABL and 58 ERG copies /ABL ) was used to distinguish high expressers from low expressers. At baseline, copy numbers of 149 patients ranged from 11.58 to 242.23 for BAALC and 16.2 to 328.2 for ERG. For BAALC , 69/149(46.3%) were high expressers as compared to 80/149 (53.7%) as low expressers. Concerning ERG , 76/149(51%) were high and 73/149(49%) were low expressers. The OS and RFS of high BAALC versus low BAALC expressers at 2 years was 13.8% vs 59.8% (p<0001) and 17.6% vs 72.1% (p<0001) respectively. Similarly OS and RFS of high ERG vs low ERG expressers at 2 years was 5.3% vs 66.4% (p<0001) and 5% vs 71.2% (p<0001) respectively (Figure).The median baseline WBC count was significantly higher in high BAALC/ERG expressers. There was no difference in distribution of FLT3 -ITD, NPM1, CEBPA, MLL -PTD and cytogenetic risk groups in high versus low BAALC and ERG expressers. Multivariate analysis showed that ERG overexpression was only prognostic factor affecting OS and RFS compared to high BAALC expressers, MLL -PTD mutation, cytogenetic risk groups and baseline WBC.Conclusion: This study shows that high ERG expression adversely affects prognosis of AML patients independent of other well known prognostic factors [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Role for Brain and Acute Leukemia Cytoplasmic (BAALC) in Human Breast Cancer Metastasis.

Brain and Acute Leukemia, Cytoplasmic (BAALC) is a protein that controls leukemia cell proliferation, differentiation, and survival and is overexpressed in several cancer types. The gene is located in the chromosomal region 8q22.3, an area commonly amplified in breast cancer and associated with poor prognosis. However, the expression and potential role of BAALC in breast cancer has not widely been examined. This study investigates BAALC expression in human breast cancers with the aim of determining if it plays a role in the pathogenesis of the disease. BAALC protein expression was examined by immunohistochemistry in breast cancer, and matched lymph node and normal breast tissue samples. The effect of gene expression on overall survival (OS), disease-free and distant metastasis free survival (DMFS) was assessed in silico using the Kaplan-Meier Plotter (n=3,935), the TCGA invasive breast carcinoma (n=960) and GOBO (n=821) data sets. Functional effects of BAALC expression on breast cancer proliferation, migration and invasion were determined in vitro. We demonstrate herein that BAALC expression is progressively increased in primary and breast cancer metastases when compared to normal breast tissue. Increased BAALC mRNA is associated with a reduction in DMFS and disease-free survival, but not OS, in breast cancer patients, even when corrected for tumor grade. We show that overexpression of BAALC in MCF-7 breast cancer cells increases the proliferation, anchorage-independent growth, invasion, and migration capacity of these cells. Conversely, siRNA knockdown of BAALC expression in Hs578T breast cancer cells decreases proliferation, invasion and migration. We identify that this BAALC associated migration and invasion is mediated by focal adhesion kinase (FAK)-dependent signaling and is accompanied by an increase in matrix metalloproteinase (MMP)-9 but not MMP-2 activity in vitro. Our data demonstrate a novel function for BAALC in the control of breast cancer metastasis, offering a potential target for the generation of anti-cancer drugs to prevent breast cancer metastasis.

Prognostic significance of combined BAALC and MN1 gene expression level in acute myeloid leukemia with normal karyotype.

Acute myeloid leukemia with normal karyotype (AML-NK) is the largest group of AML patients with very heterogeneous disease outcome. In order to ensure more precise risk stratification new molecular markers have been introduced, like expression level for BAALC (Brain and Acute Leukemia, Cytoplasmic) and MN1 (Meningioma 1) genes. In this study, we investigated expression level of both genes in 111 adult AML-NK at diagnosis and examined their prognostic potential. BAALC and MN1 expression were detected in about one third of the patients, and positive correlation between these two genes was found. The BAALC+ /or MN1+ status was not associated with the presence of FLT3-ITD mutations, but exhibited strong correlation with NPM1wt status (P<.001). Therefore, among BAALC+ /or MN1+ patients the most frequent ones were FLT3-ITD- /NPM1- double negative patients with intermediate prognosis. When BAALC+ /or MN1+ patients were divided into BAALChigh /BAALClow (21/21) and MN1high /MN1low (21/22) groups, we detected that BAALChigh /or MN1high patients had a tendency toward lower complete remission rate. Also, survival analysis showed that BAALChigh /or MN1high patients had shorter disease-free survival and overall survival (OS). The most pronounced influence on prognosis was detected in FLT3-ITD- /NPM1- group of patients that are lacking reliable prognostic markers, where OS in BAALChigh /or MN1high was only 5months vs 25months in BAALClow /or MN1low . These findings indicate that BAALC and MN1 expression level could be used for more precise risk stratification of AML-NK patients and especially FLT3-ITD- /NPM1- patients, transforming this intermediate-risk group, into a group with an adverse prognosis.

Brain and Acute Leukemia, Cytoplasmic Gene Overexpression as a Prognostic Factor in Egyptian De novo Adult Acute Myeloid Leukemia Patients

Abstract Background: Brain and acute leukemia, cytoplasmic (BAALC) gene is identified on chromosome 8q22.3 and implicated in normal hematopoiesis. BAALC gene overexpression is associated with poor outcome. Methods: We aimed to evaluate BAALC expression in de novo Egyptian acute myeloid leukemia (AML) cases and determine its prognostic value. We recruited 70 patients with de novo AML diagnosed and treated at clinical pathology and medical oncology departments, fulfilling inclusion criteria in our prospective study and evaluated BAALC expression level. Patients received induction therapy. The Institutional Review Board approved our study. Results: The mean age was 39.2 years ± 11.87, (18–60) with a male/female ratio of 3/2. The cutoff value of BAALC as a prognostic factor was 2.11 with sensitivity (86.1%), specificity (80%), positive predictive value (88.6%), and negative predictive value (76.2%.) (P &lt; 0.001), 43 (61.4%) patients had high BAALC expression. Seventy-two percent of patients in the low BAALC group achieved complete remission (CR) compared to 42.1% in high BAALC expression group (P = 0.03). Patients with low BAALC (123.1 ± 4.9) had longer mean survival time than high BAALC group (45.85 ± 5.1) (P = 0.000). Conclusion: High-BAALC expression is an adverse prognostic factor, with a higher risk of relapse, lower CR rates, and lower survival in Egyptian de novo AML patients.

Over expression of brain and acute leukemia, cytoplasmic and ETS-related gene is associated with poor outcome in acute myeloid leukemia.

The high expression of brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) has been reported to influence the outcome in acute myeloid leukemia (AML), but due to limited prospective studies, their role as prognostic factors is unclear. At diagnosis, the prognostic value of BAALC and ERG expression with respect to other cytogenetic and molecular markers was analyzed in 149 AML patients. Patients were divided into quartiles which resulted in the formation of four groups (G1-G4) based on expression values of BAALC and ERG and clinical response defined across groups. Groups with similar survival probabilities were merged together and categorized subsequently as high versus low expressers. Patients with high BAALC and ERG expression had significantly lower overall survival (OS; BAALC: p = 0.001 at 5 years 29.4% vs. 69.8%; ERG: p < 0.0001 at 5 years 4% vs. 50.4%) and disease-free survival (BAALC: p = 0.001 at 5 years 19.5% vs. 69.8%; ERG: p < 0.0001 at 5 years 4.2% vs. 47%). Patients were further stratified combining BAALC and ERG expression in an integrative prognostic risk score (IPRS). After a median follow-up of 54 months (95% CI 45-63 months) among survivors, IPRS for high versus low expressers was a significant predictor for OS (BAALC + ERG: 4% vs. 71.6%, p < 0.0001) and DFS (BAALC + ERG: 4.5% vs. 74.1%, p < 0.0001). In a multivariate model, IPRS of BAALC + ERG expression retained prognostic significance for OS (hazard ratio [HR] 2.96, 95%CI 1.91-4.59, p < 0.001) and DFS (HR 3.61, 95%CI 2.26-5.76, p < 0.001).

Allogeneic stem cell transplantation mitigates the adverse prognostic impact of high diagnostic BAALC and MN1 expression in AML

For most acute myeloid leukemia (AML) patients, an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of sustained remissions and long-term survival. At diagnosis, high expression of the AML-associated genes BAALC (brain and acute leukemia, cytoplasmic) and MN1 (meningioma-1) were repeatedly linked to inferior outcomes in patients consolidated with chemotherapy while data for patients receiving HSCT remain limited. Using clinically applicable digital droplet PCR assays, we analyzed the diagnostic BAALC/ABL1 and MN1/ABL1 copy numbers in 302 AML patients. High BAALC/ABL1 and MN1/ABL1 copy numbers associated with common adverse prognostic factors at diagnosis. However, while high diagnostic copy numbers of both genes associated with shorter event free survival (EFS) and overall survival (OS) in patients receiving chemotherapy, there was no prognostic impact in patients undergoing HSCT. Our data suggests that the adverse prognostic impact of high BAALC and MN1 expression are mitigated by allogeneic HSCT. But preHSCT BAALC/ABL1 and MN1/ABL1 assessed in remission prior to HSCT remained prognosticators for EFS and OS independent of the diagnostic expression status. Whether allogeneic HSCT may improve survival for AML patients with high diagnostic BAALC or MN1 expression should be investigated prospectively and may improve informed decisions towards individualized consolidation options in AML.

The Prognostic Significance of the BMI-1 and BAALC Genes in Adult Patients with Acute Myeloid Leukemia.

The aim of this work is to investigate the different expression patterns of B cell-specifics moloney murine Leukemia virus integration site-1 (BMI-1) and brain and acute leukemia, cytoplasmic (BAALC) genes, their prognostic and clinical significance in newly diagnosed cytogenetically heterogenous adult acute myeloid leukemia patients. BMI-1 and BAALC expression was detected in the bone marrow of patients using quantitative real-time reverse transcription polymerase chain reaction with cut off value set at 50th percentile for both genes. BMI-1 and BAALC overexpression was detected in 50% of cases which suggest their potential as molecular markers. A statistical significant correlation was found between BMI-1 expression with hepatomegaly (P value = 0.007), hemoglobin level-grouped (P value = 0.047) and cytogenetic risk groups (P value = 0.036). There was a statistically significant correlation between BAALC and age (P value = 0.015), lymphadenopathy (P value = 0.043), CD34 expression (P value = 0.003) and near statistical significance with FAB sub-groups (P value = 0.054). No statistical significance was noted for other hematological findings and response to treatment except for BAALC gene and treatment response (P value = 0.014). No statistical significance in overall survival and disease free survival for both genes was found. Their prospective screening in combination with other molecular markers can help refine myeloid leukemia staging and prognosis toward optimizing therapeutic interventions.

Mechanism of BAALC-Mediated Leukemogenesis Downstream of RUNX1-Mutations in Severe Congenital Neutropenia

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. We recently reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. To study the mechanism of leukemia development in CN, we established a model for step-wise leukemia progression in CN using iPSC-based hematopoietic differentiation in combination with CRISPR/Cas9-mediated gene editing of iPSCs. Using this model, we confirmed that co-acquisition of CSF3R and RUNX1 mutations is necessary and sufficient to induce leukemia in CN. We also identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event downstream of RUNX1 and CSF3R mutations. BAALC mRNA was upregulated in CN/AML blasts (n = 5) and in CD34+ HSPCs generated from CN/AML iPSCs of two patients. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs reversed defective myeloid differentiation of CN/AML blasts to the levels observed in healthy donor hematopoietic stem cells.We further investigate the mechanism of BAALC up-regulation. In silico analysis of the BAALC gene promoter in combination to publicly available ChIP-Seq data revealed three putative RUNX1 binding sites that were validated using ChIP assay in lysates of NB4 cells. Interestingly, transduction of healthy donor CD34+ cells with lentiviral constructs expressing WT RUNX1 led to inhibition of BAALC mRNA expression, whereas transduction with two RUNX1 RUNT domain mutants resulted in the marked BAALC up-regulation, as compared to the control BFP transduced cells. These data suggest that mutated RUNX1 failed to inhibit BAALC expression in CD34+ HSPCs.To evaluate the mechanism of leukemogenic transformation in CN, we performed RNA-Seq analysis of CD34+ cells derived from CN and CN/AML iPSC clones. GSEA revealed that changes in gene expression between CN- and CN/AML-HSPCs were strongly correlated with gene expression signatures of “Wierenga STAT5 targets” and “reactome ATF4 targets”, an observation in line with the markedly elevated levels of STAT5 and ATF4 in CN/AML-HSPCs. Importantly, gene expression differences between CN/AML-HSPCs and CN-HSPCs were correlated to “Valk AML” targets in GSEA, suggesting that HSPCs generated from CN/AML-iPSCs possess characteristics of AML cells. Strong support for the leukemogenic role of upregulated BAALC in CN/AML was provided by further GSEA analysis of the BAALC KO CN/AML-HSPCs. We observed a reversal in the expression of a majority of genes in the studied leukemia-associated pathways in CN/AML-HSPCs after BAALC knockout compared with CN/AML-HSPCs.Since there are no direct inhibitors for BAALC available and protein structure is not solved yet, BAALC effects can be targeted only indirectly. Morita et al., Leukemia, 2015 showed that BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and Klf4. We treated CD45+ cells generated from CN/AML or healthy donor (HD) iPSCs with MEK1/2- or MEK1-specific inhibitors or vehicle control and evaluated cell proliferation and differentiation (CFU assay). We were able to induce ~ 40-60 % cell death of CN/AML cells upon treatment with each of inhibitors, whereas HD CD45+ cells were unaffected. Moreover, treatment of CN/AML cells with MEK1/2 inhibitor led to an increase in CFU-G formation, as compared to vehicle control cells.Using connectivity Map analysis of RNA-Seq data of CD34+ cells generated from CN/AML iPSCs vs CN/AML BAALC KO iPSCs, we identified small molecule p38/MAPK14 inhibitor that could possibly reverse BAALC-mediated leukemogenic gene expression signature. We treated CN/AML iPSC-generated CD34+ cells for 7 days with this inhibitor and subsequently performed CFU assay. We found an increase in CFU-GM formation.In summary, using CN/AML-iPSC-model, we confirmed the major role of BAALC in leukemia development downstream of CSF3R and RUNX1 mutations in CN. Inhibition of MAPK/ERK-pathway downstream of BAALC reduced proliferation and partially induced myeloid differentiation of CN/AML-derived hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

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.

Validation of a Molecular Risk Score for Prognosis of Patients with Acute Promyelocytic Leukemia

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on clinical parameters, namely leukocyte and platelet counts at initial diagnosis as combined in the Sanz Score. However, during the last years the influence of additional molecular genetic markers on prognosis of APL patients has been postulated. In 2015 we published the results of a molecular risk score integrating expression data of the genes brain and acute leukemia, cytoplasmic (BAALC), ets' related gene (ERG) and Wilms' Tumor 1 (WT1) with strong independent influence on outcome and relapse risk of APL patients treated in the German AMLCG studies (Hecht et al., Leuk Res 2015). The aim of our study was to validate our data in an independent patient cohort.Methods: In cooperation with the German SAL (Study Alliance Leukemia) group we obtained a validation set of samples of mononuclear cells derived from the bone marrow of 76 patients with confirmed diagnosis of APL prior to therapy. The validation cohort consisted of 37 female and 39 male patients with a median age of 50 years (range 20-82 years; Table 1). Patients were diagnosed and treated between 2000 and 2014 mostly with an ATRA plus Idarubicin based induction therapy followed by Sanz score-dependent consolidation and maintenance chemotherapy. RNA was extracted using the AllPrep DNA/RNA Mini Kit and cDNA was synthesized using 500µg of RNA with a QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Expression levels of BAALC, ERG and WT1 were then analyzed using quantitative real-time RT-PCR with the same conditions and primers as used in the original test cohort and the same calibrator cDNA from cell lines was used for the analysis. The following gene expression levels were defined 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 or high). The integrative risk score was calculated as described before: 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 or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors). Overall survival (OS), relapse free survival (RFS) and cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method.Results: The expression of the three genes BAALC, ERG and WT1 compared to healthy controls were exactly the same as in the original cohort. Application of the molecular risk score on APL patients of the validation cohort clearly discriminated patients according to their risk comparable to the original APL cohort (Figure 1). Patients with 0 points had a very good prognosis with no deaths or relapses, whereas patients with 3 points (i.e. concurrent presence of all three risk factors) had a very poor outcome with an OS of 51%, a RFS of 50% and a CIR of 38%. The differences between patients with 1 and 2 points were less pronounced in the validation cohort. However, the statistical analyses in the validation cohort did not yield significance. As the main reason we assume that the validation cohort altogether showed a significantly improved OS and RFS compared to the original cohort which was treated a decade earlier, so differences between the subgroups could not be that pronounced. For both cohorts stratification by the Sanz Score did not show significant differences in outcome (analysis for RFS p=0.46 in original cohort and p=0.44 in validation cohort).Conclusion: A validated molecular-based integrative risk score was able to define subgroups of APL patients with different outcome independently of the Sanz score. The validation of the strong influence of the combination of molecular risk factors is particularly interesting as the new analyses of the three genes BAALC, ERG and WT1 separately did not confirm the strong results of the single analyses of each gene. The discrimination of patients according to the risk score is evident though. This argues for their integration in future studies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BAALC and ERG Expression in Egyptian Patients with Acute Myeloid Leukemia, Relation to Survival and Response to Treatment

AIM:Aim was to detect Brain and Acute Leukemia, Cytoplasmic (BAALC) and ETS-related gene (ERG) expression in patients with acute myeloid leukemia (AML) as well as to study their biologic and prognostic impact on the disease outcome and survival.PATIENTS AND METHODS:The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.RESULTS:The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression. BAALC was expressed in 36 (81.82%) of AML cases versus 10 (22.72%) of the control group which was highly statistically significant (P < 0.001). While ERG was positive in 39(88.64%) of cases and 8(18.18 %) of controls and that was also highly statistically significant (P < 0.001).CONCLUSION:Further researches still needed to clarify the role of BAALC and ERG in the pathogenesis of leukemia and their importance as targets for treatment of AML.

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.

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.

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.

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.

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.

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.

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 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.