Five Most Common Prognostically Important Fusion Oncogenes are Detected in the Majority of Pakistani Pediatric Acute Lymphoblastic Leukemia Patients and are Strongly Associated with Disease Biology and Treatment Outcome

The incidence of acute lymphocytic leukemia (ALL) is not uniform around the world. It varies from 0.9-4.7 per 100,000 children per year (Zhang et al., 2011).The 1Hematology, Oncology and Pharmacogenetic Engineering Sciences (HOPES) Group, Health Sciences Laboratories, Faculty of Biological Sciences, Department of Zoology, University of the Punjab, 9Centre for Advanced Molecular Biology & National Centre of Excellence in Molecular Biology, 10Post Graduate Medical Institute & Institute of Child Health, 12Department of Oncology, Allama Iqbal Medical College and Jinnah Hospital, Lahore, Pakistan, 13University of Health Sciences, Lahore, 8School of Biological Sciences, University of Sargodha, Sargodha, 14Department of Oncology, Punjab Medical College and Allied Hospital, Faisalabad,15Department of Biotechnology and Informatics, (BUITEMS), Quetta. Pakistan, 2Molecular Clinical Hematology/Oncology Research Laboratory, College of Applied Medical Sciences, King Saud Bin Abdulaziz University of Health Sciences, National Guards Health Affairs, Riyadh, 3Hematology/Oncology Division, Department of Medicine, 4Mircobiology Section, 7Biochemistry Research Section, 11Embryonic Stem Cell Unit, Department of Anatomy, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, 5Centre of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, 6Montefiore Medical Centre, 111 East 210th Street, Bronx, NY, 16Health Centre, University of Texas San Antonio, San Antonio, USA, 17Asian Medical Institute Kant & National Surgical Centre Bishkek, Kyrgyzstan &Equal contributors *For correspondence: mianzafaram@yahoo.com Abstract


Introduction
The incidence of acute lymphocytic leukemia (ALL) is not uniform around the world.It varies from 0.9-4.7 per 100,000 children per year (Zhang et al., 2011).The 5470 is as high as 10 per 100,000 children (Pui et al., 2008).
The genetic lesions in ALL play a key role in the abnormal development of lymphoid cells (Kuiper et al., 2007;Treviño et al., 2009;Iacobucci et al., 2012;Xu et al., 2012).The diagnosis of ALL is based on morphology, immunophenotype and cytogenetic analysis of the leukemic blast cells in the peripheral blood and bone marrow (Bhojwani et al., 2012;McGregor et al., 2012).Molecular analysis of the common genetic alterations in leukemic cells has contributed greatly to our understanding of the pathogenesis and prognosis of ALL (Iqbal et al., 2007;Pui et al., 2008).The most common fusion oncogenes in pediatric ALL are BCR-ABL, ETV6-RUNX1, MLL-AF4, SIL-TAL1 and TCF3-PBX1 (Pui et al., 2008;Schrrape et al., 2012).The frequency of particular genetic subtypes differs in children but the general mechanisms underlying the induction of ALL are likely to be similar to adult ALL (Pui et al., 2004).
Chromosomal translocations are powerful prognostic indicators in pediatric ALL (Kuiper et al., 2007;Iacobucci et al., 2012).The presence of recurrent genetic markers represents subtypes of the disease which may have different etiologies (Iqbal et al., 2007;Kuiper et al., 2007;Treviño et al., 2009;Pui et al., 2011).There is little information available about the molecular epidemiology of pediatric ALL in Pakistan.According to the best of our knowledge this is the first community-based study from Pakistan focused on exploring the prevalence of five common ALL fusion oncogenes in children with ALL and their correlation with the disease biology and treatment outcome.

Materials and Methods
Peripheral blood samples were obtained from 148 pediatric ALL patients admitted to different hospitals of Lahore, Pakistan from January 2009 to December 2011.Patients between the ages of<one to fifteen years, with a confirmed diagnosis of ALL were included.These patients did not have a prior severe physical or psychiatric illness and their renal and hepatic function were adequate.Twenty samples were unfit for further processing while 27 were negative for GAPDH ("housekeeping" genes).The remaining 101 samples were processed for molecular cytogenetics.We studied 5 fusion genes in 101 pediatric ALL patients using Interphase FISH and RT-PCR at the time of diagnosis.The clinical data were recorded at diagnosis and subsequently.

RNA extraction
Total RNA was extracted from leukemic cells by TriZol reagent according to the manufacturer's instruction.

Synthesis of complementary DNA (cDNA)
RNA was reverse-transcribed to cDNA for using as template in PCR reaction.RT reaction protocol and other reaction conditions were adopted from Van Dongen (Van Dongen et al., 1999).Briefly, 10µl of RNA was added to 10 µl of RT-reaction mixture containing 5X RT buffer, 25 mM dNTPS, 10 mM random hexane primers, RiboLockTMRNase inhibitor, M-MuLV reverse transcriptase (Fermentas, USA) and DPCE-treated water.Reaction was carried out by incubating mixture of template, random hexamers and DEPC treated water at 70ºC for 10 min.Then rest of the reagents were added and incubated at 42°C for 60 min, 70°C for 10 min and held at 4°C in the last step.The integrity of cDNA was assessed by amplification of housekeeping genes GAP-DH.

RT-PCR amplifications
PCR primers and nested PCR protocols for the detection of five fusion genes were adopted from Van Dongen et al. (1999).For the first round of nested PCR, a 50 µl PCR reaction was performed containing 5x PCR buffer with KCl, 25 mM MgCl 2 , 25 mM dNTP mix, DEPC water, Taq DNA polymerase primer (forward and reverse) and cDNA as a template.The same test was run in round 2 with the template being the product of round 1. Thermal cycling conditions for nested PCR were preliminary denaturation at 95°C for 3 min followed by 35 cycles of denaturation of double stranded DNA at 95°C for 30 sec, annealing of primers to DNA template at 65°C for 60 sec and extension to form multiple copies of DNA strands at 72°C for 60 sec, followed by a post amplification extension at 72°C for 7 minutes.Round 2 was carried out with the same conditions.The final products were visualized by gel electrophoresis.All recommended precautions were taken to avoid contaminations.Appropriate negative and positive controls were included in each amplification experiment.

Statistical analysis
For this study we used convenient sampling technique to collect the data, and used non-parametric tests, as appropriate, to analyze the data.Chi Square test was used to study the association between different oncogenotypes and clinical and laboratory parameters of leukemia patients.Kaplan and Meier method was used to calculate the median survival times, while Breslow's test was used to study the survival differences between various patient groups.
Frequency of most of the fusion genes was approximately the same as compared to average frequency in Asia (p=0.221),Europe (p=0.462)and America (p=0.917),whereas it was significantly different from

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African countries (p<0.001).However, the frequency of BCR-ABL was much higher as compared to reported from rest of the world.

Clinical features of patients with different fusion oncogenes BCR-ABL:
There was a male preponderance (34 male, 75.5%; 11 females, 24.5%) in BCR-ABL+ patients with a median age of 9 years (Table 5).There were 3 BCR-ABL+ patients in the less than 2 year age group, 16 patients in the 2-7 year age group and 26 patients in the older than 7 group.These figures show that the frequency of occurrence of BCR-ABL positivity is directly proportional to age.The leukocyte count in BCR-ABL+ patients was higher when compared to patients with other oncogenes (Table 5).Organomegaly was not more common in this patients group.There was a significant difference between the survival of patients with BCR-ABL and other genotypes in all age groups (p=0.004)(Figures 6, 7A-C).BCR-ABL positivity was associated with low remission rates and shortened survival.
ETV6-RUNX1: Clinical analysis of 18 ETV6-RUNX1 positive patients is shown in Table 5.This cohort consisted of 11 male and 7 females with a median age of 1.85 years.The gene frequency was highest in patients younger than 2 years.The WBC count in ETV6-RUNX1+ patients was not very high and they had a good prognosis.
MLL-AF4: Seventeen patients had MLL-AF4 gene rearrangement with a median age of 9 years.There were 12 male and 5 female patients (Table 5).Five patients were younger than 2 years, two between 2 and 7 years, and ten patients were in the 7-15 age group.Majority of   TCF3-PBX1: Translocation t (1; 19) occurs in around 2% of patients and involves the fusion of E2A genes on chromosome 19 to the PBX1 gene on chromosome 1.Only two female patients were diagnosed with this translocation.Both the patients were over 2 years of age.This translocation is associated with the inferior outcome in the context of response to chemotherapy with poor prognosis.It was associated with higher risk of CNS relapse although small numbers preclude any firm conclusion (Table 5).
SIL-TAL1: This gene was found in 7 patients, 3 male and 4 females.All the patients were older than 2 years, with the majority falling in the age range 7-15 years.The immunophenotype data were available in all SIL-TAL1 patients showing this fusion gene was associated with T-ALL.Organomegaly was frequently observed in these patients.

Discussion
Acute lymphoblastic leukemia (ALL) is a heterogeneous disease and comprises of many different genetic subgroups as identified by various chromosomal and molecular abnormalities (Treviño et al., 2009;Xu et al., 2012).This heterogeneity is likely to be due to genetic, racial and geographic variations that exist among different populations (Pui et al., 2003;2009;Treviño et al., 2009;Iacobucci et al., 2012;Schmiegelow et al., 2012;Xu et al., 2012).Therefore, the distribution of genetic and molecular subtypes may not be uniform in different parts of the world (Romana., 1995;Ariffin et al., 2007).Moreover, gene-environment interactions, which are critical in leukemogenesis, may differently contribute in defining the relative proportions of molecular subgroups in different geographic regions (Iqbal et al., 2006;Siddique et al., 2010;Faiz et al., 2011;Schmiegelow et al., 2012;Schrappe et al., 2012).The most common oncogenes found in leukemia patients are the fusion genes, which are formed as a result of different genetic abnormalities at the chromosomal level (Zelent et al., 2004).Chromosomal anomalies resulting from fusion oncogenes create hybrid transcripts that usually encode transcription factors (Mesquita et al., 2009).Different oncogenes are believed to be a different molecular entity and they act by different molecular pathways (Iqbal et al., 2012).
Cytogenetic studies of the leukemia patients to identify the presence of fusion oncogenes are extremely important in the prognostication and management planning of this disease (Ariffin et al., 2007).The five major riskstratifying translocations in patients with ALL are BCR-ABL, ETV6-RUNX1, MLL-AF4, SIL-TAL1 and TCF3-PBX1 (Lazic et al., 2010;Iacobucci et al., 2012).The frequency of some of the FO in this study is comparable to the previous studies from Pakistan and other parts of the world (Gaynon et al., 1997;Iacobucci et al., 2012).A strikingly high frequency of BCR-ABL FO was found in this study which is in keeping with the previous reports from Pakistan (Iqbal andTanveer, 2006, Iqbal et al., 2007;Faiz et al., 2011).
The overall prevalence of fusion oncogene shows consistency between population under study and the global reports (Gaynon et al., 1997;Van Dongen et al., 1999;Pui et al., 2008;Iacobucci et al., 2012;Schrrape et al., 2012).Most notable was the high frequency of the BCR-ABL that was 44.5% as compared to population study in Europe where it is not so high (Van Dongen et al., 1999;Lazic et al., 2010;Iacobucci et al., 2012) except in Sudan, Africa (Siddique et al., 2010) while BCR-ABL has been reported to be totally absent in Saudi Arabian pediatric ALL patients (El-sissy et al., 2006;Siraj et al., 2006).The Philadelphia chromosome is present in children with ALL and leads to the production of BCR-ABL fusion protein with tyrosine kinase activity.This subtype of ALL is common in older children than in infants.Pediatric patients are mostly precursor B-Cell ALL and have a high leukocyte count.It is associated with poor prognosis due to poor response to initial therapy especially in case of high leukocyte count.Complete remission in 4 weeks can lead to increase in EFS rate.The Mexican study found highest prevalence of TCF3-PBX1 11.5% (Jimenez et al., 2008.) while this gene is only 1.9% in population under study.The frequency of ETV6-RUNX1 is 17.8% and it is in agreement with the prevalence of this chimerical gene in the affluent societies like France where it is reported 19.7% in pediatric ALL population (DeBraekeleer., 2010;Reichard, 2011) Patients with t (4; 11)/MLL-AF4 are usually infants with high WBC count.They are more likely than other children with ALL to have CNS disease and to have a poor response to therapy and a poor prognosis.They are at high risk of treatment failure.Children with t (4; 11) have better outcome than infants (Raimondi et al., 1996).This reflects ethnic and geographic differences in biology and genetics of pediatric ALL.Similar reports about ethnic differences in the disease biology, genetics and treatment outcome have been reported in adult ALL (Sabir et al., 2012).
A remarkable progress has been made in the treatment of ALL in children with cure rates of around 80% (Bowman et al., 2011;Hunger et al., 2012).Unfortunately, the survival of pediatric ALL patients in Pakistan remain poor which needs urgent attention.There are possibly several factors which contribute to this poor outcome and these include a delay in diagnosis and referral to a specialized centre, lack of understanding and awareness on the part of the parents, poor socio-economic conditions where many patients need to buy the drugs and other themselves which may lead to compromises and suboptimal treatment, and possibly suboptimal supportive care.In addition, the non-availability of tyrosine kinase inhibitors for BCR-ABL+ cases and lack of facilities for hemopoietic stem cell transplantation for high risk patients very likely contributed to the overall and this group's poor survival (Schultz et al., 2009;Leung et al., 2011;Pulsipher et al., 2011;Rives et al., 2011).High incidence of BCR-ABL positive pediatric ALL cases needs urgent attention for further investigation and collaboration of the local and international researchers to study the etio-pathogenesis of this disease entity.
In conclusion, this is the first study from Pakistan correlating molecular markers with disease biology and 5474 treatment outcome in pediatric ALL.Our study revealed the highest reported frequency of BCR-ABL FO in pediatric ALL which, consequently, was associated with poor overall survival.Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.

Figure 1 .
Figure 1.Comparison of Frequency of Fusion Oncogenes in Pediatric ALL Patients between Present Study and the Previous Research Work from Pakistan

Figure
Figure 2. Bar Chart Showing Comparison of Frequencies of Fusion Oncogenes in Different Continents of the World

Figure 3 .Figure 4 .
Figure 3. Overall Survival.Overall 33 Out of 101 Completed Their Study Period, the Average Survival of Peads was 35.71 Months

Figure 7 .Figure 6 .
Figure 7.Comparison of Patient Survival with BCR-ABL and Other Oncogenes.A) Age less than 2 years, B) Age 2-7 years), C) Age 8-15 years.There is a significant difference between the survival of patients with BCR-ABL and other four oncogenes in all age groups (p=0.004)

Table 1 . Comparison of Observed Data with Previous Studies of Pediatric ALL Conducted in Pakistan (Percentages)
Fusion Oncogene Present Iqbal et al.Siddiqui et al.Faiz et al.

Table 2 . Average Percentage of the Fusion Oncogenes in Pediatric ALL from Different Continents Compared with Present Data from Pakistan
*Not detected