|Year : 2016 | Volume
| Issue : 2 | Page : 237-241
Frequency and clinical impact of ETV6/RUNX1, AF4-MLL, and BCR/ABL fusion genes on features of acute lymphoblastic leukemia at presentation
IC Ajuba1, AJ Madu2, C Okocha3, OG Ibegbulam2, I Okpala1, OE Nna4
1 Department of Haematology and Immunology, University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu State, Nigeria
2 Department of Haematology and Immunology, University of Nigeria, Enugu Campus, PMB 01129, Enugu 400001, Nigeria
3 Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria
4 Safety Molecular Laboratory Pathology Laboratory, Faculty of Health Sciences and Technology, University of Nigeria, Enugu Campus, Nigeria
|Date of Acceptance||12-May-2015|
|Date of Web Publication||9-Feb-2016|
Dr. A J Madu
Department of Haematology and Immunology, University of Nigeria, Enugu Campus, PMB 01129, Enugu 400001
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Variations in disease presentation and outcome of leukemia treatment has been associated with the presence of certain mutant genes. Three major translocations (ETV6-RUNX1, BCR-ABL, and AF4-MLL) in acute lymphoblastic leukemia (ALL) have been shown to affect treatment outcome. This study is aimed at assessing the relationship between these translocations and the presence of other indicators of disease severity (white cell count, hemoglobin concentration, platelet count, and hematocrit) in ALL.
Patients and Methods: Forty chemotherapy naïve patients aged between 9 months and 54 years had their marrow samples analyzed for the prevalent mutations. Their clinical and laboratory details on presentation were also obtained.
Results: Abnormal genes detected were BCR/ABL1 major transcript in 5 (12.5%), ETV6/RUNX1 in 2 (5.0%), MLL/AF4 none and none of the patients had more than one fusion gene. There was no relationship between the presence of these fusion genes and the clinical and laboratory features of ALL. An association exists between the fusion genes and ethnic origin of the patients (P = 0.005). There is no significant association between the abnormal fusion genes detected and some laboratory features of prognostic importance, which include total white blood cell count (P = 0.416) and FAB subtype (P = 0.576).
Conclusion: Presence of fusion the genes BCR/ABL1, ETV6/RUNX1, and MLL/AF4 does not have any impact on the clinical and laboratory features of ALL at presentation.
Keywords: Acute lymphoblastic leukemia, BCR/ABL1, ETV6/RUNX1, fusion genes, MLL/AF4
|How to cite this article:|
Ajuba I C, Madu A J, Okocha C, Ibegbulam O G, Okpala I, Nna O E. Frequency and clinical impact of ETV6/RUNX1, AF4-MLL, and BCR/ABL fusion genes on features of acute lymphoblastic leukemia at presentation. Niger J Clin Pract 2016;19:237-41
|How to cite this URL:|
Ajuba I C, Madu A J, Okocha C, Ibegbulam O G, Okpala I, Nna O E. Frequency and clinical impact of ETV6/RUNX1, AF4-MLL, and BCR/ABL fusion genes on features of acute lymphoblastic leukemia at presentation. Niger J Clin Pract [serial online] 2016 [cited 2020 Aug 4];19:237-41. Available from: http://www.njcponline.com/text.asp?2016/19/2/237/164351
| Introduction|| |
Acute lymphoblastic leukemia (ALL) is a neoplastic proliferation of lymphoid cells arrested at a premature stage of differentiation. It can occur in both children and adults and accounts for 80% of all cases of childhood leukemia , and 20% of adult cases., In a study done at University of Benin Teaching Hospital, Benin, to determine the pattern of leukemia incidence in Benin city, the incidence of ALL was found to be 10.3% of 253 cases of leukemia seen between 1990 and 2004 and showing increasing trend at 5 years intervals. Similarly, in a preliminary report of a prospective study on the neoplastic diseases of the hematopoietic system carried out at University College Hospital Ibadan, 19.5% of the cancers were found to be ALL.
Accurate characterization of the lymphoid malignant cell requires not only morphology but also, immunologic, cytogenetic, and molecular diagnostic methods. These methods can identify the different subtypes requiring different treatment approaches in terms of either conventional or more intensive therapy according to risk-based stratification and prognosis. This has led to improved treatment outcome over the years with event-free survival rates over 80% being achieved. The situation is not the same in developing countries, where achieving remission is a major challenge, and the relapse rate is very high; because the different subtypes are not accurately identified to inform the choice of chemotherapeutic treatment.
The clinical and laboratory findings in ALL are varied and have been associated with specific underlying chromosomal abnormalities., The prognostic clinical and laboratory factors include age at diagnosis, initial white blood cell (WBC) count, central nervous system (CNS) involvement at presentation, testicular involvement, gender, and race. Molecular studies in recent times have availed science the knowledge of genetic abnormalities and correlation with the clinical presentation of the disease.,,
Four major chromosomal translocations found in ALL define distinct clinic-pathological subgroups and have been used in risk stratification for treatment purposes. They include t(12;21)/ETV6-RUNX1 also known as TEL-AML1, t(1;19)/E2A-PBX1, t(9;22)/BCR-ABL, and t(4;11)/AF4-MLL. Patients with t(1;19), t(9;22) or t(4;11) respond poorly to chemotherapy and have a poor prognosis. The cryptic t(12;21)(p13;q22) results in the ETV6-RUNX1 fusion gene formerly called TEL/AML1 and is known to confer excellent prognosis.
In this study, the fusion gene transcripts ETV6-RUNX1, BCR-ABL, and AF4-MLL was analyzed. These transcripts are known to occur more frequently in ALL and influence behavior of the leukemic blasts hence are valuable for risk stratification of treatment as they are predictors of prognosis. They are also useful in the evaluation of minimal residual disease. This study was aimed at determining the frequency of the fusion gene transcripts ETV6-RUNX1, BCR-ABL, and AF4-MLL and their effect on the pattern of presentation of ALL.
| Patients and Methods|| |
This was a cross-sectional study carried out on newly diagnosed ALL patients in centers from South East Nigeria. Forty chemotherapy naïve patients were recruited, over 6 months (from November 2012 to July 2013) for the study. These were patient whose bone marrow aspiration cytology had more than 20% lymphoblasts. Only patients who gave informed oral and written consent were recruited. Ethical approval was obtained from the University of Nigeria Teaching Hospital Health Research and Ethics review Board, as well as from other centers, from where patients were recruited. This was done in accordance with the Helsinki declaration of 1964. Sociodemographic details of patients were obtained using a questionnaire, which was completed by the investigator.
From each patient, 10 ml of venous blood was obtained and used for full blood count using Sysmex 2000 auto-analyzer (Sysmex Corporation, UK). The RNA extraction was done using guanidine isothiocyanate solution containing ethylenediaminetetraacetic acid 5 mM, guanidine thiocynate (GTC) 4.0 M, tris citrate 25 mM, sarcosyl 0.5%, and β-mercaptethanol 25 ml to activate the GTC. RNA was extracted with Thermo scientific GeneJet RNA purification kit (Lot NO 00118481) according to manufacturer's guideline. The purity of the RNA was assessed with BioRad Spectrophotometer (Bio-Rad Laboratories Inc.) read off at A260/A280. The RNA was immediately converted to cDNA for stability by adding 21 µl of cDNA cocktail to the RNA extracted. The cDNA was tested for quality by running standard polymerase chain reaction (PCR) and agarose gel electrophoresis. TaqMan reverse transcription-PCR was then used for amplification of the target fusion genes using Applied Biosystems thermal cycler Step One software version 2. 0 (Life Technologies, Thermo Fisher Scientific Corporation, UK). Each patient sample was run in duplicate and tests were repeated on separate occasions to ensure reliability and reproducibility of the results. Positive standard controls and nuclease-free water as negative controls were also included in the run.
A sample size of 40 has been chosen for this study and has 80% clinical power to detect a difference of 16.67% with a significance level (alpha error) of 0.05 (two-tailed analysis). The sample size was calculated using GraphPad StatMate version 2. 0 (GraphPad Software Inc., UK). The calculation is based on study design for two proportions. Factors considered in sample size calculation include:
- Mean frequency of the fusion genes from previous studies
- Clinical power of 80% (conventional)
- Alpha level of 0.05 (conventional)
- Effect size (the level of difference to be detected = 16.67%, informed by the average frequency of 15%, and an additional margin of 1.67%).
The expected pattern of distribution of the fusion genes is unknown. The Kendaull Tau-b correlation coefficient was calculated using SPSS 17.0 (IBM Corporation, Armonk NY, USA) and expressed in tables.
| Results|| |
A total of 40 patients were included in the study, age ranged from 9 months to 54 years with a median of 16 years and mean 17.8 ± 14.4 years. Most of the patients 42.5% (17/40) were <10 years old. There were 27 (67.5%) males and 13 (32.5%) females with male-to-female ratio of 2.1:1. The patients presented with a constellation of clinical signs and symptoms with fever as the most common presenting symptom and lymphadenopathy as the most common presenting sign [Figure 1]. None of the patients had any feature suggestive of involvement of the CNS; however, one patient (2.5%) presented in an unconscious state and died within 72 h of admission.
|Figure 1: (a) Frequency of symptoms of acute lymphoblastic leukemia (ALL) at presentation. (b) Frequency of symptoms of ALL at presentation|
Click here to view
The mean hemoglobin concentration at diagnosis was found to be 6.1 ± 2.1 g/dl; mean platelet count 39 ± 25 × 109/L while the mean total WBC count was 55 ± 71.7 × 109/L. The mean platelet count was 39 ± 25 × 109/L and blast percentage of 85.3% ±9.8%. The FAB L2 subtype was the most common seen in the study. Of the 40 samples analyzed, L2 subtype constituted 67.5% of ALL while L1 subtype was observed in 32.5% of patients. No L3 subtype was seen in the series.
The abnormal genes detected were BCR/ABL1 major transcript in 5 (12.5%) patients and ETV6/RUNX1 in 2 (5.0%) patients. Of the patients who had the BCR/ABL1 transcript, 4 were <18 years of age while only one patient was more than 18 (54 years). Both patients who had the ETV6/RUNX1 were children, <10 years of age. MLL/AF4 fusion gene abnormality was not found among the patients; neither were there any combinations of abnormal fusion observed. There was no relationship between the fusion genes detected and clinical manifestations of the patients [Table 1], however, there appear to be an association between the fusion genes and ethnic origin of the patients (P = 0.005). There is no significant association between the abnormal fusion genes detected and some laboratory features of prognostic importance which include total WBC count (P = 0.416) and FAB subtype (P = 0.576).
|Table 1: Relationship between the fusion genes and clinical/laboratory features at presentation|
Click here to view
| Discussion|| |
Acute lymphoblastic leukemia is invariably fatal and affects predominantly the younger patient population as seen in this study. The mean age of the study group was found to be similar to what was obtained by Olaniyi etal. in an audit of acute leukemia in UCH, Ibadan where a mean age of 28 ALL patients they studied was 19 years, but higher than the study by Nwannadi et al. in South-South Nigeria that reported 4 years. The male-to-female ratio in this study was similar to findings by Okpala et al. in Ibadan in their study of 30 patients with ALL to determine prognostic factors.
Males had a worse outcome than females, and this has been attributed to the involvement of sanctuary sites like testis, which can be a source of relapse. In addition, patients seen in this study appear to have a high tumor burden and invariably poor outcome as shown by the majority of the patients presenting with high leukocyte count (WBC = 55.9 ± 71.6 × 109/L), tissue infiltration, and L2 subtype similar to outcome of the study reported by Williams in Ibadan, South-West Nigeria.
The clinical importance of determining the underlying aberrant genetic mutation is to characterize adequately the leukemic cells for risk stratification and prognostication, targeted therapy, and modification to achieve improved survival. The majority of the patients in this study did not have any of the mutant genes being screened for, the BCR-ABL1 translocation was the most common type of mutation observed. Furthermore, all the BCR-ABL1 positive patients were of the major transcript type e13a2 and e14a2, as seen in chronic myeloid leukemia (CML). However, the possibility of CML in blastic transformation in these patients needs to be ruled out. Disease outcome for these patients is poor but would benefit from treatment with tyrosine kinase inhibitors and allogeneic hematopoietic stem cell transplant as noted in some studies.,
Half of the childhood ALL patients who screened positively for these fusion genes had the ETV6-RUNX1, while the other half had the BCR-ABL1. This is similar to findings reported by Romana etal. where a high frequency of the fusion gene was detected in childhood B-lineage ALL. The relatively lower frequency in children may be partly due to the fact that some adult ALLs may be the terminal event of a CML with a rather short chronic phase. The BCR-ABL1 was found in all the adults who screened positively for the fusion genes being studied.
There were some differences in the frequency of the gene rearrangements detected in this study when compared to reports from other parts of the world. This might be due to genetic diversity that exists among diverse races. In the study done by Noreen et al. in Pakistan, (studying 5 fusion genes in adult patients),MLL-AF4 was found in 10 (9.7%) patients as compared to none in this study. BCR-ABL1 was seen in 21 (20.3%) patients as compared to 5 (12.5%) in this study, which included both adults and children. However, some similarity exists in the frequency of ETV6-RUNX1; 4.8% in their study versus 5% in this index study though in their study majority of the patients who had this fusion gene were adults. In 33 patients, no fusion gene was detected, and MLL-AF4 fusion gene was undetectable in this study. This may be explained by the presence of other partner gene rearrangements involving MLL, which could have been present and include AF9, AF10, and the over 60 partner genes recorded in literature.
Limitations of the study
The low sample size though representative of the study population may not be completely adequate to assess some minor clinical outcomes.
| Conclusion|| |
The fusion genes detected had no impact on the clinical and laboratory features at presentations of the disease. There was no difference in the pattern of presentation of ALL in this study when compared to other previous studies. However, there seems to be some variability in the frequency of fusion genes observed in most ALL patient groups across various geographical and racial populations. This observation deserves a multi-racial analysis as this may indicate varied etiological factors, which may be peculiar to different populations.
Authors duly acknowledge the unalloyed help given by Prof. M. Durosinmi and the doctors working in the hematology team of the University of Nigeria Teaching Hospital, Enugu.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
McNeil DE, Coté TR, Clegg L, Mauer A. SEER update of incidence and trends in pediatric malignancies: Acute lymphoblastic leukemia. Med Pediatr Oncol 2002;39:554-7.
Smith MA, Ries LAG. Cancer Incidence and Survival among Children and Adolescents. United States SEER Program 1975-1995. Cancer Statistics Branch, Cancer Surveillance Research Program, Division of Cancer Control and Population Sciences. Bethesda, MD: National Cancer Institute; 1999. p. 17-34.
Pui CH, Crist WM. Biology and treatment of acute lymphoblastic leukemia. J Pediatr 1994;124:491-503.
Margolin JF, Steuber CP, Poplack DG. Acute lymphoblastic leukemia. Principles and Practice of Pediatric Oncology. 15th
Omoti CE, Imiere EO Trends in the pattern of leukaemia incidence in a tertiary health center in Nigeria (1990-2004). J Med Biomed Res 2006;5:44-9.
Williams CK. Neoplastic diseases of the haemopoietic system in Ibadan: Preliminary report of a prospective study. Afr J Med Med Sci 1985;14:89-94.
Möricke A, Zimmermann M, Reiter A, Henze G, Schrauder A, Gadner H, et al.
Long-term results of five consecutive trials in childhood acute lymphoblastic leukemia performed by the ALL-BFM study group from 1981 to 2000. Leukemia 2010;24:265-84.
Zhao C, Li X, Li J, Zhang Y, Ren S, Chen X, et al.
Detecting ALK, ROS1 and RET fusion genes in cell block samples. Transl Oncol 2014;7:363-7.
Vrooman LM, Silverman LB. Childhood acute lymphoblastic leukemia: Update on prognostic factors. Curr Opin Pediatr 2009;21:1-8.
Cimino G, Elia L, Mancini M, Annino L, Anaclerico B, Fazi P, et al.
Clinico-biologic features and treatment outcome of adult pro-B-ALL patients enrolled in the GIMEMA 0496 study: Absence of the ALL1/AF4 and of the BCR/ABL fusion genes correlates with a significantly better clinical outcome. Blood 2003;102:2014-20.
Emerenciano M, Bungaro S, Cazzaniga G, Dorea MD, Coser VM, Magalhães IQ, et al.
ETV6-RUNX1 fusion gene and additional genetic changes in infant leukemia: A genome-wide analysis. Cancer Genet Cytogenet 2009;193:86-92.
Harrison CJ, Moorman AV, Barber KE, Broadfield ZJ, Cheung KL, Harris RL, et al.
Interphase molecular cytogenetic screening for chromosomal abnormalities of prognostic significance in childhood acute lymphoblastic leukaemia: A UK Cancer Cytogenetics Group Study. Br J Haematol 2005;129:520-30.
Pui CH, Chessells JM, Camitta B, Baruchel A, Biondi A, Boyett JM, et al.
Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003;17:700-6.
Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC, Hancock ML, et al.
TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia 1995;9:1985-9.
Nwannadi AO, Bazuaye G, Nwagu M, Borke M. Clinical and Laboratory Characteristics of Patients with Leukaemia in South-South Nigeria. The Internet Journal of Oncology. 2011;7:2.
Okpala IE, Olatunji PO, Okunade MA, Ogunsanwo BA, Jeje OM, Shokunbi WA, et al.
Prognosis of acute lymphoblastic leukaemia in Ibadan, Nigeria. Afr J Med Med Sci 1990;19:313-7.
Williams CK. Neoplastic diseases of the haemopoietic system in Ibadan: Preliminary report of a prospective study. Afr J Med Sci 1985;14:89-94.
Schrauder A, Reiter A, Gadner H, Niethammer D, Klingebiel T, Kremens B, et al.
Superiority of allogeneic hematopoietic stem-cell transplantation compared with chemotherapy alone in high-risk childhood T-cell acute lymphoblastic leukemia: Results from ALL-BFM 90 and 95. J Clin Oncol 2006;24:5742-9.
Silverman LB. Acute lymphoblastic leukemia in infancy. Pediatr Blood Cancer 2007;49:1070-3.
Romana SP, Poirel H, Leconiat M: High frequency of t(12;21) in childhood B-lineage acute lymphoblastic leukemia. Blood. 1995;86:4263-69.
Noreen S, Zafar I, Aamer A, Tashfeen A, Tahir N, Sultan A, et al
. Prognostically significant fusion oncogenes in Pakistani patients with adult acute lymphoblastic leukaemia and their association with disease biology and outcome. Asian Pacific J Cancer Prev 2012;13.7:3349-55.