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

en el presente estudio es analizar la expresión de BAALC y evaluar su impacto pronóstico en el resultado clínico de la LLA de precursores B en adultos. Métodos Pacientes Doscientos recién diagnosticados como LLA de precursores B adultos se inscribieron entre 2005 y 2014 en el Centro de Oncología Mansoura, Mansoura, Egipto. Las muestras se seleccionaron de forma consecutiva

section_content: Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease with distinct manifestations and prognostic and therapeutic implications [1]. ALL in adults is a rare disease. The results of therapy remain unsatisfactory, and progress has been relatively slow [2]. B-cell ALL (B-ALL) is a clonal malignant disease originated in a single cell and characterized by the accumulation of blast cells that are phenotypically reminiscent of normal stages of B-cell differentiation [3]. 

Outcome of adult B-precursor ALL has considerably improved because of identification of clinical and genetic risk factors stratifying patients to different treatment groups [4]. Commonly accepted risk factors in B-precursor ALL include age, performance status, white blood cell (WBC) count, lactate dehydrogenase concentration, the immunophenotype, response to induction therapy, level of minimal residual disease, cytogenetics and genetic aberrations [5, 6]. Patients lacking clinical and molecular risk factors are considered standard risk (SR). Outcome for SR patients is still unsatisfactory [5, 7] indicating the clinical and biologic heterogeneity of these patients. Therefore, the identification of novel predictive molecular markers in adult B-precursor ALL may improve treatment stratification of this subgroup. 

The Brain And Acute Leukemia, Cytoplasmic (BAALC) gene is highly expressed in normal uncommitted progenitor cells and downregulated with the onset of differentiation [8]. An elevated expression of the BAALC gene was originally discovered in a gene expression profiling study of acute myeloid leukemia (AML) with trisomy 8, but was later also found in other AML and in ALL [9, 10]. Over expression of BAALC predicts an inferior outcome in AML and ALL patients [9] [10] [11].
section_name: Introduction

section_name: Molecular analyses

section_content: To assess molecular responses, total RNA was extracted from PB or BM blood cells. BCR-ABL and internal control transcript levels were quantified using real-time PCR analysis (TaqMan) on an ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Specific PCR products were amplified and detected using dual-fluorescent non-extendable probes labeled with 6-carboxyfluorescein (FAM), reporter and 6-carboxytetramethylrhodamine (TAMRA), quencher at 5`-end and 3`-end, respectively. The relative mRNA expression of BCR-ABL transcript was calculated using the comparative cycle threshold (Ct) method [12].
section_name: Real-time quantitative polymerase chain reaction (RTQ-PCR) for BCR-ABL

section_content: From each patient and healthy subject 3 ml of PB or BM samples were collected in sterile EDTA vacutainers. BAALC mRNA expression was normalized to the simultaneously analyzed glucose phosphate isomerase (GPI) gene. The relative BAALC expression was determined using the comparative cycle threshold (CT) method. Glucose phosphate isomerase (GPI) and BAALC were coamplified in the same tube using 1 μL cDNA, 1× master mix (IQ Mix; BioRad, Munich, Germany), GPI probe (5'-HEX-TTCAGCTTGACCCTCAACACCAAC-TAM-RA-3') with GPI forward (5'-TCTTCGATGCCAACAAGGAC-3') and reverse (5'-GCATCACGTCCTCCGTCAC-3') primers, and BAALC probe (5'-FAMCTCTTTTAGCCTCTGTGGTCTGAAGGCCAT-TAMRA-3') with BAALC forward (5'-GCCCTCTGACCCAGAAA-CAG-3') and reverse (5'-CTTTTGCAGGCATTCTCTTAGCA-3') primers. Reactions were performed using real-time PCR 7000 sequence detection system (Applied Biosystems, Foster City, USA). Positive and negative controls were included in all assays.
section_name: Real-time quantitative PCR analysis for BAALC

section_content: The statistical analysis of data was done by using excel program and SPSS (statistical package for social science) program (SPSS, Inc, Chicago, IL) version 16. Qualitative data were presented as frequency and percentage. Chi square test was used to compare groups. Quantitative data were presented as mean and standard deviation. For comparison between two groups; student t-test and Mann-whitney test (for non-parametric data) were used. Kaplan-Meier test was used for survival analysis and the statistical significance of differences among curves was determined by Log-Rank test. Prediction of survival was done using multivariate analysis. For gene expression quantification, we used the comparative Ct method. First, gene expression levels for each sample were normalized to the expression level of the housekeeping gene encoding GPI within a given sample (∆Ct). Results were evaluated by using 2-∆∆CT method as relative gene expression values. N. B: p is significant if ≤ 0. 05 at confidence interval 95%.
section_name: Statistical analysis

section_name: Results

section_content: ALL Patients were divided into 2 BAALC expression groups; high BAALC expression (above median) and low BAALC expression (below median). The clinicohematological features differed in patients with high BAALC and those with low BAALC mRNA expression levels. Patients expressing high BAALC gene were significantly older (P =0. 037), had higher WBC (P =0. 019), LDH concentration (p=0. 007) patients who had sufficient material available. They were 106 males (53%) and 94 females (47%) with mean age 42. 32 ± 10. 675 years. Patients were diagnosed according to standard diagnostic methods including clinical, cytomorphological, cytochemical, immunophenotypic methods. In addition, 20 healthy subjects, of matched age (39 ± 11. 202 years) and sex (12 males (60%) and 8 females (40%)); with normal laboratory findings; were selected as a control group. Informed consent was obtained from all patients. Risk groups were assigned as follows: SR (no risk factor), high risk (≥ 1 risk factor), and very high risk (presence of t (9;22)/BCR-ABL).
section_name: BAALC expression and relationship with clinical, laboratory and molecular features

section_content: ALL cases were treated according to our risk adapted chemotherapy protocol: The patients were stratified according to their prognostic factors into standard, high, and very high risk groups. The treatment plan included: Prephase for patients with high WBC and/or organomegaly. Induction phase I: Four drugs: Vincristine, Doxorubicin, L-Asparaginase and prednisone with intrathecal MTX. Patients that attained complete remission (CR) were subjected to cranial irradiation with 24 Gy and intrathecal MTX for four injections. Phase II induction with Cyclophosphamide and Cytarabine. Consolidation phase I: Vincristine, Doxorubicine and prednisone with Triple intrathecal. Phase II consolidation: Cyclophosphamide, Cytarabine and Etoposide with triple intrathecal. Maintenance therapy: two years with 6 mercaptopurine and methotrexate. For patients with high and very high risks who were not planned to stem cell transplantation, one cycle of high dose cytarabine and mitoxantrone (HAM regimen) was added between induction and consolidation. Bone marrow aspirate was done to evaluate response to chemotherapy (status post induction). Cases who died before treatment or who didn't receive treatment due to poor performance status or elderly cases kept on supportive treatment were excluded, as well as patients who received SCT in first CR.
section_name: Treatment protocol

section_content: Complete remission (CR) was assessed after completion of induction chemotherapy. CR was defined as follows: granulocyte count of at least 1. 5 × 10 9 /L, platelet count of at least 100 × 10 9 /L, no peripheral blood (PB) blasts, bone marrow (BM) cellularity of at least 20% with maturation of all cell lines and less than 5% blasts, and no extramedullary leukemia. Primary therapy failure (refractory disease) was defined as persistence of PB blasts or at least 25% blasts in BM after induction therapy. Relapse was defined as reappearance of PB blasts, more than 5% blasts in BM, or appearance of extramedullary manifestations after CR was achieved. OS was defined as the time from diagnosis to date of death. For patients achieving CR, DFS was the time from the date of first CR to an event (death in first CR or relapse).
section_name: Criteria of response and survival definitions

section_content: Immunophenotypic analyses were performed by flow cytometry on fresh pretreatment BM and PB samples. A wide panel of monoclonal antibodies (Mo Abs) was used. Lymphoid markers included CD19, CD22 for B Lineage and CD1, CD2, CD3, CD4, CD5 and CD7, for exclusion of T-lineage and other markers used included CD45, HLA-DR, CD10, and CD34. All the monoclonal antibodies were obtained from Coulter Hialeah, FL. A cell-surface antigen was considered positive when at least 20% of cells showed fluorescence intensity greater than the negative control. and CD34 positivity (p=0. 011) compared with patients with low BAALC expression group. In addition, higher BAALC expression was associated with the presence of BCR-ABL (P=0. 011). There was no significant association between BAALC expression level and sex, hemoglobin concentration, platelet count, peripheral or marrow blasts, or immunophenotypic subgroups of B-precursor ALL. No other association was seen between BAALC expression and clinical features (Table 1 ).
section_name: Immunophenotypic analysis

section_content: Complete remission rate did not differ significantly between high and low BAALC expression groups (p=0. 120), although patients with high BAALC expression showed a marginally significant lower CCR rate than low expression group (30% versus 43% respectively; p=0. 056) and a higher incidence of primary resistant disease (34% versus 14% respectively; p=0. 001). No influence on the relapse rate (P =0. 674) was observed with respect to BAALC expression. There were no significant differences regarding deaths in induction therapy (P =0. 203) between the two BAALC expression groups. Total mortality rates during the entire period of the study were higher in high BAALC expression group when compared to low expression group, but did not reach significant level (P =0. 063) (Table 2 ). 

When applying BAALC expression, age, marrow blasts, immunophenotypes and BCRABL as covariates for prediction of resistant disease, BAALC remained a predictive factor for primary therapy resistance (P = 0. 003; OR= 3. 133; 95% confidence interval [CI], 1. 482-6. 623), as well as immunophenotype (common versus pre B-ALL) (P = 0. 015; OR= 2. 326; 95%CI, 1. 431-7. 956) (Table 3 ). 

Overall survival (OS) was significantly shorter in patients with higher BAALC expression compared with those with lower BAALC expression (34. 39 versus 50. 11 months; 4-year cumulative OS %, 39. 6%, 53. 6% respectively; p=0. 010). In addition, high BAALC expression group showed significantly shorter DFS than those with low expression (27. 93 versus 58. 82 months; 51. 7% versus 64. 9% respectively; p<0. 001) (Figure 1 ). BAALC expression was independently predictive for OS and DFS in multivariate analysis (p=0. 039, HR=1. 652, 95%CI=1. 025-2. 663; p=0. 002, HR=1. 214, 95%CI=1. 013-4. 368 respectively). In addition, BCR-ABL is an independent unfavourable prognostic factor for OS (p<0. 001, HR=2. 749, 95%CI=1. 124-4. 940), as well as common B-ALL immunophenotype for DFS (p<0. 001, HR=0. 254, 95%CI= 0. 187-2. 872) (Table 4 ).
section_name: Outcome in B-precursor ALL patients with respect to BAALC expression

section_content: All studied patients were stratified according to BCR-ABL into BCR-ABL-and BCR-ABL+ groups (n =133, 67 respectively). We analyzed the prognostic impact of BAALC expression in these low-and high-risk subgroups of BCR-ABL B-precursor ALL. In the BCR-ABLgroup, no significant differences were obtained regarding CR, CCR, relapse, induction death and total mortality rates. Refractory disease was significantly higher in patients with high BAALC expression versus those with low BAALC expression (p=0. 039). In addition, in the BCR-ABL+ group, no significant differences were obtained regarding CR, CCR, relapse and total mortality rates. Resistance to chemotherapy was higher in patients expression high BAALC expression versus those with low BAALC expression group (p=0. 003). Death during induction therapy was significantly higher in low versus high BAALC expression groups (p=0. 025) (Table 2 ). Multivariate analysis showed that BAALC gene expression was independent risk factor for resistance to chemotherapy in BCR-ABL-and BCR-ABL+ groups (OR=2. 359, 95% CI=1. 004-5. 538, p=0. 049; OR= 2. 672, 95% CI=1. 824-3. 326, p=0. 014 respectively) (Table 5 ). 

OS in BCR-ABL -patients was shorter in high versus low BAALC expression group, although did not reach significant level (4-year OS: 58. 5%, 66. 2%; 47. 63, 60. 48 months; p=0. 102). Higher BAALC expression was significantly associated with an inferior DFS when compared to low BAALC expression group for BCR-ABL -patients (4year DFS: 69. 1%, 51. 2%; 60. 36, 28. 21 months P <0. 001). BCR-ABL+ patients showed no significant differences between BAALC expression groups for OS and DFS (p=0. 991, 0. 671 respectively) (Figure 2 ). In the Cox regression analysis, BAALC expression was an independent adverse factor regarding DFS in BCR-ABL-group (p=0. 001, HR=3. 774, 95% CI= 1. 831-5. 448) (Table 6 ).
section_name: BAALC expression and outcome according to BCR-ABL in B-precursor ALL patients

section_content: Acute lymphoblastic leukemia (ALL) remains one of the most challenging adult malignancies, especially with respect to therapy. Immunophenotyping, cytogenetic-molecular studies [12]. However, most of the studies focused on children and therefore a deep molecular characterization of adults is still challenging, especially for those cases lacking high-risk markers (eg, BCR-ABL). In this study, we have evaluated the prognostic significance of BAALC expression in B-precursor ALL. 

In B-precursor ALL, higher BAALC expression was associated with older age, higher WBC, LDH concentration and BCR-ABL, which are known risk factors in accordance with others [13]. However, no significant differences were found between low and high BAALC expression groups regarding clinical presentations, sex, hemoglobin concentration, platelet count, peripheral or marrow blasts, or immunophenotypic subgroups of B-precursor ALL. 

Patients with low BAALC gene expression continued CR at a rate marginally significantly higher than those with high BAALC gene expression. High BAALC gene expression group confer higher resistance to chemotherapy than those with low BAALC gene expression in agreement with Kohnl [13]. Total mortality rate was marginally significantly higher in high versus low BAALC gene expression groups. This may be due to lower CCR rate, higher resistance to chemotherapy, more aggressive disease having more immature cells. In multivariate analysis, high BAALC expression retains its high risk for primary therapy failure, in accordance with other reports [13]. 

When stratifying all patients according to BCR-ABL, 133 cases were BCR-ABL negative and 67 patients were BCR-ABL+. High BAALC gene expression group had significantly higher resistance to chemotherapy in BCR-ABL and BCR-ABL + groups. In multivariate analysis, high BAALC gene expression is still independent prognostic factor for resistance to chemotherapy in BCR-ABL and BCR-ABL + groups. 

In addition, higher BAALC expression was independently predictive for DFS and OS all patients and for DFS in BCR-ABL -patients. 

In previous studies, high BAALC expression was associated with an unfavorable outcome and inferior long-term survival in adult CN-AML and T-ALL [9, 14, 15]. Thus, BAALC characterizes a more aggressive, immature, highly proliferative, and chemoresistant leukemic phenotype. 

Our data suggest that BAALC may identify patients with an immature, chemoresistant leukemic phenotype associated with an unfavorable outcome and shorter survival of adult B-precursor ALL. 

BAALC expression could better discriminate patients into various prognostic groups and identify patients who might benefit from doseintensified induction chemotherapy. 

Stratification of adult B-precursor ALL into 2 distinctive groups of patients with outcome and survival characteristics, might in the future facilitate treatment stratification for adult B-precursor ALL.
section_name: Discussion