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

Background

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to clinical picture and therapeutic outcome, partly reflected by differences in molecular and cyto-genetics. Clonal cytogenetic abnormalities are one of the most important factors predicting clinical outcome in acute myeloid leukemia and are used to guide risk-adapted treatment strategies.

Deregulated expression of genes coding transcription factors involved in cell proliferation, survival or differentiation is known to be implicated in the process of leukomogenesis. Brain and acute leukemia, cytoplasmic gene (BAALC) and ETS-related gene (ERG) are examples of these transcription factors BAALC gene, located on chromosome band 8q22.3, is considered as a marker of early hematopoietic progenitor cells. High levels of BAALC expression were found in AML patients with trisomy 8, as well as in a subset of cytogenetically normal–AML(CN-AML) patients in which it was considered as poor prognostic factor The role of BAALC in leukemogenesis is not fully understood, but it was proposed that BAALC blocks myeloid differentiation ERG gene, located on chromosome band 21q22, belongs to the ETS family of transcription factors required for normal hematopoiesis. It is a transforming proto-oncogene that is expressed in stem cells and endothelial cells. It is frequently overexpressed in AML patients with complex karyotypes and amplification of chromosome 21. The ERG gene has been found to be involved in atypical chromosomal rearrangements with alterations of the transcription factor in several cancers. Aberrant expression of full-length ERG protein has been found in acute myeloid leukemia and acute T-lymphoblastic Leukemia. Chromosomal rearrangements driving the formation of EWS/ERG and FUS/ERG fusion proteins have been described in a subset of Ewing sarcoma and in acute myeloid leukemia.

Most of the previous researches were concerned about levels and prognostic effect of BAALC and ERG in CN-AML patients, while data about their incidence in AML and relation to cases with abnormal karyotype were lacking. The objective of this study was to detect the incidence of BAALC and ERG in a group of AML patients with abnormal karyotype in comparison to normal individuals, their levels and distribution among AML FAB subtypes as well as to study their impact on the disease outcome and reliability in detection of minimal residual disease in relation to the cytogenetic markers.

Methods

The current study was carried out on 44 patients with acute myeloid leukemia (AML), in the period between, July 2011 and July 2012 among cases referred to nuclear medicine and oncology unit of Kasr Alainy School of medicine, Cairo University with follow up period of 18 months, as well as 44 age and sex matched controls. They were patients suspected to have hyperspleenism or idiopathic thrombocytopenia coming for BM aspirate. Cases were diagnosed according to WHO criteria. The diagnosis of AML was based on morphological and phenotypic data. Subtypes according to the French–American–British classification were available for all the patients. Patients were 21 males and 23 females. Age ranged from 21 to 65 years. The control group included 17 male and 27 females with no medical history of any type of cancer. Their ages ranged between 22 and 50 years. All patients and controls were analyzed for clinical and laboratory findings, including full history taking, clinical examination, routine laboratory investigations, LDH, abdominal ultrasound for detection of organomegaly and lymphadenopathy. The patients were subjected as well to cytochemical, immunophenotypic and cytogenetic analysis to confirm diagnosis and to divide the patients into their subtypes. Local institutional research board approval as well as written informed consent was obtained from all the participants before including them in the study. All patients included in the study were treated according to the protocol of the nuclear medicine and oncology department, Cairo University, using ongoing induction and consolidation regimens for treatment of adult AML cases.

Induction of remission

Patients were subjected to 7–3 protocol for induction of remission: Novantrone: 12 mg/m², IV on day 1 and 3. ARA – C: 100 mg/m², continuous IV infusion, from day l–7. If remission is not achieved, this protocol was repeated again. If no or minimal response, patients were shifted to high dose chemotherapy. Induction therapy for acute promyelocytic leukemia (PML) included oral administration of all-trans-retinoic acid (ATRA) 45 mg/m²/day until induces remission.

Consolidation

High dose ARA-C for 4 cycles. ARA-C: 2 g/m², over 2 hours infusions, every 12 hours on days 1–4. Significant association to relapse free survival and overall survival were estimated for studied genes at a median follow-up of 18 months. Complete remission (CR) was defined as recovery of morphologically normal BM and peripheral blood cells with white cell counts ≥ 1,500/L, and platelets ≥ 100,000/L, less than 5% BM blasts and no evidence of extramedullary leukemia. Relapse was defined by ≥ 5% BM or peripheral blood blasts, or development of extramedullary leukemia in patients with previously documented CR. Relapse free survival (RFS) was measured from the date of CR until date of relapse or death. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.

Results

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). The distribution of the positive cases among FAB subtypes did not show any significant differences. The lower values of BAALC and ERG in the cases compared to controls means higher levels in the cases as the numerical value of the CT is inversely related to the amount of amplicon in the reaction. Follow up of the patients revealed 12 cases of CR and 32 with unfavorable outcome; 17 showed partial recovery (PR), 8 cases relapsed and 7 patients died. Highly significant correlation was detected between positive genes expression and presence of bulky tumor and organomegaly. The levels of each gene were expressed as 2-ΔΔ CT i.e. number of fold increased above the mean level of the control group. Patients were divided into two groups according to these levels (high and low) as described previously in statistic paragraph. BAALC levels median and range were 1479.8(220.8–7550.2) for high levels group and 10.1(0.13–54.9) for low levels group. While levels of ERG were 425.5(27–1985) and 2.44(0.021–8.17) for high and low levels groups respectively. There were statistically no significant differences between the 2 studied groups regarding the BAALC gene expression before and after treatment (P value = 0.86) or the ERG gene expression before and after treatment (P value = 0.75). Multivariate regression test had revealed that BAALC and ERG are independent risk factors of acute leukemia, because (table7). Although we abolished the effect of bad prognostic factors (age>60 years, high serum LDH and WBC levels, high bone marrow blast percentage, presence of bulky tumor), still high levels of these genes are associated with lower CR, RFS, and shorter OS.

Conclusion

As regard ERG, the levels in AML cases did not differ significantly from theirs in normal BM. So determination of cut off value to detect MRD will not be applicable. 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 that could be promising due to their high incidence of expression in AML.

Keywords

BAALC, ERG, gene expression, Quantitative real-time RT-PCR, AML.