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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

Authors:
  • King Saud bin Abdulaziz University for Health Sciences, Al-Ahsa, Saudi Arabia
  • Government post graduate Islamia college for women Cantt

Abstract and Figures

Background and objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. Method: We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival (43.7±4.24 weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). Conclusions: This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, 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.
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Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5469
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5469
Fusion Oncogenes in Pakistani Pediatric Acute Lymphoblastic Leukemia Patients
Asian Pacic J Cancer Prev, 13 (11), 5469-5475
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
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, 11 Embryonic 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, 6Monteore 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
Background and Objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving
many fusion oncogenes (FO) having prognostic signicance. The frequency of various FO can vary in different
ethnic groups, with important implications for prognosis, drug selection and treatment outcome. Method: We
studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations
with clinical features and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL t (22;
9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found
in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a
signicantly lower survival (43.7±4.24 weeks) and higher white cell count as compared to others, except patients
with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed
closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1
and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). Conclusions: This is the rst
study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric
ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, 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.
Keywords: Acute lymphoblastic leukemia - pediatric ALL - fusion oncogenes - Pakistan
RESEARCH ARTICLE
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
Tashfeen Awan1&, Zafar Iqbal1,2,8&*, Aamer Aleem1,3& Noreen Sabir1&, Muhammad
Absar1,4&, Mahmood Rasool5, Ammara H Tahir1,4,6, Sulman Basit7, Ahmad
Mukhtar Khalid8, Muhammad Farooq Sabar9, Sultan Asad9, Agha Shabbir Ali10,
Amer Mahmood11, Muhammad Akram12, Tariq Saeed12, Arsalan Saleem13, Danish
Mohsin13, Ijaz Hussain Shah14, Muhammad Khalid14, Muhammad Asif15, Riazul
Haq16, Mudassar Iqbal1,17, Tanveer Akhtar1
infant mortality rate in Pakistan is 71% which is one of
the highest in the world (Ashraf, 2012). ALL comprises
of eighty percent of childhood acute leukemias (Cheok
and Evans, 2006) and there is a striking incidence peak
during the ages two to seven years where the incidence
Tashfeen Awan et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
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). Briey, 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 amplications
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 MgCl2, 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.
Results
Patients’ characteristics
A total of 101 patients were included in the final
analysis. A total of 101 patients were included in the
final analysis. There were 70 (69.3%) male and 31
(3o.6%) females with a median age of 7 (range1-15)
years. Eighteen patients (20.2%) were<2 years old.
There were 33 (32.6%) patients between the ages of 2-7
years and fifty (49.5%) patients were in the age range of
7-15 years. Median WBC count was 16.3×109/l (range
0.4-465), median platelet count was 54.5×109/l (range
7-525), and median Hb level was 6.5 g/dl (range 3.5-13.7).
Sixty five (64.3%) patients had WBC counts<30×109/l
while 36 (35.6%) had WBC counts >30×109/l. Sixty five
(64.3%) patients had a fever before starting chemotherapy.
Twenty one (21.7%) patients had a mediastinal mass, 30
(29.7%) had a splenomegaly, and 43 (42.5%) patients had
hepatomegaly. Palpable lymphadenopathy was present
in 39 (38.6%) patients. CNS disease, as confirmed by
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5471
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5469
Fusion Oncogenes in Pakistani Pediatric Acute Lymphoblastic Leukemia Patients
spinal cytology, was found in 7 (9.3%) patients. Seventy
nine patients could be classified by the pathology review
committee using the FAB criteria; 36 (35.6%) cases
were L1, 48 (47.5%) were L2. Immunopphenotyping
was reported in 77/101 cases. Majority of the patients
expressed B-lineage antigens (70 cases) and 7 patients
expressed T-lineage antigens (Tables 4, 5).
Molecular cytogenetic analysis
Of the 101 samples processed for molecular
cytogenetics, BCR-ABL FO was detected in 45/101
(44.5%) patients, ETV6-RUNX1 in 18/101 (17.8%)
patients, MLL-AF4 in 17/101 (16.8%) patients and SIL-
TAL1was found in 7/101 (6.9%) patients. Only 2/101
(2.0%) ALL patients were positive for TCF3-PBX1 (Table
1, Figure 1). This study found a low prevalence of ETV6-
RUNX1, MLL-AF4, SIL-Tal1 and TCF3-PBX1 fusion
oncogenes in Pakistani children with ALL but there was
a relatively high occurrence of BCR-ABL rearrangement.
Table 2 shows the comparison of BCR-ABL frequency in
pediatric ALL from different parts of the world.
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
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.
study (2006) (2010) (2011)
BCR-ABL 44.5 49.0 3.5 24.0
ETV6-RUNX1 17.8 12.6 3.5 9.7
MLL-AF4 16.8 15.5 5.0 14.0
SIL-TAL 6.9 5.8 * *
TCF3-PBX1 1.9 2.0 0 2.0
*Not detected. Present data is not significantly different as compared
to Iqbal (p-value 0.754), Siddique et. al, (p-value 0.085) and Faiz et al
(p-value 0.806)
Figure 1. Comparison of Frequency of Fusion
Oncogenes in Pediatric ALL Patients between Present
Study and the Previous Research Work from Pakistan
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
6.3
51.7
75.0
51.1
30.0
31.3
54.2
56.3
27.6
25.0
33.1
30.0
31.3
23.7
31.3
Table 2. Average Percentage of the Fusion Oncogenes
in Pediatric ALL from Different Continents Compared
with Present Data from Pakistan
Fusion Gene Present study Asia Africa Europe America
BCR-ABL 44.5 4.00 22.00 4.30 3.90
ETV6-RUNX1 17.8 16.59 8.55 22.65 17.85
MLL-AF4 16.8 3.00 17.00 6.80 2.00
SIL-TAL1 6.9 * 5.50 5.00 20.80
TCF3-PBX1 1.9 5.00 * * 8.25
*Not detected
Figure 2. Bar Chart Showing Comparison of
Frequencies of Fusion Oncogenes in Different
Continents of the World
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
Table 3. Overall Frequency, Median, 95%CI and
Statistical Signicant Differences of Numeric Variables
(Age Groups, TLC, Platelets and Complete Remission)
in Patients with Leukemia
Variable Frequency Median 95%CI P-Value
Age <2 18 1.5 1.11-2.3 <0.001
2-7 33 5 4.7-5.9
8-15 50 12 10.7-12.7
TLC <30,000 65 5210 6213.35-10175.42
>30,000 36 70300 52100-131000 <0.001
Platelets <50,000 40 16000 15100-24900 <0.001
>50,000 61 109000 75700-14200
CR <4 69 4 1.22-1.84 <0.001
(Weeks) >4 20 17 16.33-33.99
Table 4. Overall Frequency, Percentage and Statistical
Signicance of Categorical Variables of Leukemia
Patients
Variable No. of patients % P-Value
FAB L1 36 35.60 <.001
L2 48 47.50
unknown 17 16.80
Mediastinal Mass Present 21 20.70 <.001
Absent 80 79.20
Hepatomegaly Present 43 42.50 <.001
Absent 58 57.40
Splenomegaly Present 30 29.70 <.001
Absent 71 70.00
Lymphadenopathy Present 39 38.60 <.001
Absent 62 61.30
CNS involvement Present 7 6.90 <.001
Absent 94 93.00
Testicular involvement Present 3 1.98 <.001
Absent 98 97.00
Figure 3. Overall Survival. Overall 33 Out of 101
Completed Their Study Period, the Average Survival of Peads
was 35.71 Months
Figure 4. Survival of the Patient with Respect to Age.
Overall survival of patients in age group ≤2 is less than other
age groups but it is not significantly different p-value 0.145
Tashfeen Awan et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
5472
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
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
Table 5. Comparison of Clinical Characteristic of Pediatric ALL Patients with Different Fusion Oncogenes
Clinical and laboratory BCR-ABL ETV6-RUNX1 MLL-AF4 SIL-TAL1 TCF3-PBX1 FO not detected
parameters No (%) No (%) No (%) No (%) No (%) No (%)
N=45 N=18 N=17 N=7 N=2 N=12
Sex Male 34 (75.0) 11 (61.0) 12 (70.5) 3 (42.8) 0 9 (75.0)
Female 11 (24.4) 7 (38.8) 5 (29.4) 4 (57.1) 2 (100) 3 (25.0)
Age <2 3 (6.7) 10 (55.5) 5 (29.4) 0 0 0
2-7 16 (35.6) 7 (38.8) 2 (11.8) 2 (28.6) 1 (50) 5 (41.6)
8-15 26 (57.8) 1 (5.5) 10 (58.8) 5 (71.4) 1 (50) 7 (58.4)
WBC <30,000 24 (53.3) 17 (94.4) 10 (58.8) 4 (57.1) 1 (50) 9 (75.0)
>30,000 21 (46.6) 1 (5.5) 7 (41.1) 3 (42.8) 1 (50) 3 (25.0)
Hepatomegaly No 23 (51.1) 15 (83.3) 8 (47.1) 3 (42.9) 0 9 (12.0)
Yes 22 (48.9) 3 (16.7) 9 (53) 4 (57.1) 2 (100) 3 (25.0)
Splenomegaly No 35 (77.8) 15 (83.3) 9 (53) 5 (71.4) 0 7 (58.3)
Yes 10 (23.3) 3 (17.7) 8 (47.1) 2 (28.6) 2 (100) 5 (41.7)
Lymphadenopathy No 30 (66.7) 11 (61.1) 5 (29.4) 5 (21.0) 2 (100) 0
Yes 15 (33.3) 7 (38.9) 12 (70.6) 2 (28.0) 0 2 (16.7)
Platelets <50,000 14 (31.1) 6 (33.3) 9 (52.9) 6 (85.7) 2 (100) 3 (25.0)
>50,000 31 (68.9) 12 (66.7) 8 (47.1) 1 (14.7) 0 9 (75.0)
CR <4weeks 13 (28.9) 16 (94.4) 4 (23.5) 5 (71.4) 1 (50) 8 (66.7)
>4weeks 29 (64.4) 1 (5.6) 10 (82.4) 1 (14.2) 1 (50) 2 (16.6)
No remission 3 (6.7) 1 (5.6) 3 (13.6) 1 (14.2) 0 2 (16.6)
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)
A) B) C)
Figure 6. Comparison of Number of Patients with
BCR-ABL and Other Oncogenes According to Age
Groups. BCR-ABL is detected in comparatively older pediatric
ALL patients
Figure 5. Overall Survival with Respect to Fusion
Oncogenes. The highest relapse-free survival was documented
in ETV6-RUNX1 (14.167 months) followed closely by those
cases in which no gene was detected (13.100). Survival in BCR-
ABL, MLL-ASF4, TCF-PBX4 and SIL-TAL1 was less than 10
months (7.994, 3.559, 5.500 and 8.080 months respectively)
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012 5473
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.11.5469
Fusion Oncogenes in Pakistani Pediatric Acute Lymphoblastic Leukemia Patients
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
our patients were older unlike the usual occurrence where
most of the patients are infants.
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 risk-
stratifying 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 and Tanveer, 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 afuent 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 reects 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
Tashfeen Awan et al
Asian Pacic Journal of Cancer Prevention, Vol 13, 2012
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.
Acknowledgements
This work was partially supported by the College
of Medicine Research Center, Deanship of Scientific
Research, King Saud University, Riyadh, Saudi Arabia.
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