Frequency of Bcr-Abl Fusion Oncogene Splice Variants Associated with Chronic Myeloid Leukemia (CML)

doi:10.4236/jct.2011.22022

Paper Menu >>

Journal Menu >>

Journal of Cancer Therapy, 2011, 2, 176-180

doi:10.4236/jct.2011.22022 Published Online June 2011 (http://www.SciRP.org/journal/jct)

Frequency of Bcr-Abl Fusion Oncogene Splice

Variants Associated with Chronic Myeloid

Leukemia (CML)

Zafar Iqbal1,2,3#, Fatima Manzoor1*, Mudassar Iqbal1,4, Shahid Ali5, Nadeem Sheikh6, Mahwish Khan1,3,

Aamer Aleem7, Tanveer Akhtar1

1Molecular Genetic Pathology (MGP) Unit, Department of Pathology, College of Medicine and King Khalid University Hospital,

King Saud University, Riyadh, Saudi Arabia; 2Hematology, Oncology and Pharmacogenetic Engineering Sciences (HOPES) Group,

Health Sciences Research Laboratories, Faculty of Biological Sciences, Department of Zoology, University of the Punjab, Lahore,

Pakistan; 3Functional Molecular Biology/Hematology and Oncology Group, Centre for Research in Molecular Medicine and Institute

for Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan; 4Department of Medicine, Kyrgyz State

Medical Academy, Bishkek, Kyrgyzstan;5National Genetics Laboratory Lahore, Pakistan; 6Molecular Biology Laboratory, Depart-

ment of Zoology, University of the Punjab, Lahore, Pakistan; 7Division of Hematology and Oncology, Department of Medicine,

College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia.

Email: #[email protected], #[email protected]

Received April 9th, 2010; revised April 2nd, 2011; accepted April 15th, 2011.

ABSTRACT

BCR-ABL fusion oncogene originates from the reciprocal translocation of chromosome 9 and 22 t(9;22) (q34;q11). It

translates a chimeric protein, p210, characterized by constitutive activation of its tyrosine kinase, which triggers leu-

kemogenic pathways resulting in onset of chronic myeloid leukemia (CML). In CML, the classic fusion is b2a2 or b3a2

fusing exon 13 (b2) or exon 14 (b3) of BCR to exon 2 (a2) of ABL. The type of bcr/abl transcripts may be associated

with different prognosis and hence useful in therapeutic plan. This study was conducted to calculate the frequency of

these splice variants as the frequencies of different fusion oncogenes associated with leukaemia can vary in different

geographica l regions du e to interplay of genetic varia tion in different ethnic popu lation s, diverse environ men tal factors

and living style. A very sensitive nested RT-PCR wa s established to detect BCR-ABL splice variants in CML. Sensitivity

of RT-PCR assay was of the order of 10–6. Thirty CML patients were subjected to BCR-ABL analysis. Out of 30 Paki-

stani patients, 19 (64%) expressed b3a2 while 11 (36%) expressed b2a2 transcript. This shows that BCR-ABL splice

variants differ in their frequencies which may have an effect on biology and implications for prognosis and manage-

ment of BCR-ABL posi t i ve Le ukemias.

Keywords: BCR-ABL Positive Leukemia, Leukemia Cytogenetics , Philadelphia Chromosome, Chronic Myeloid

Leukemia, BCR-ABL Alternative Splicing, BCR-ABL Splice Variants, Leukemia Alternative Splicing,

Pharmacogenetics

1. Introduction

Cytogenetically chronic myeloid leukemia (CML) is

characterized by the presence of Philadelphia (Ph) chro-

mosome, the diagnostic hallmark of CML, which is pre-

sent in majority of CML patients. It originates from the

reciprocal translocation of chromosome 9 and 22 t(9;22)

(q34;q11) [1]. This reciprocal translocation gives rise to

BCR-ABL fusion oncogene, which translates a chimeric

protein, p210BCR-ABL that is characterized by constitu-

tive activation of its tyrosine kinase activity. In CML, the

classic fusion is b2a2 or b3a2 fusing exon 13 (b2) or

exon 14 (b3) of BCR to exon 2 (a2) of ABL [2,3]. Both

b3a2 and b2a2 transcripts can be formed as a result of

alternative splicing. These transcripts lead to the produc-

tion of an 8.5 kb transcript coding for a 210-KDa (p210)

chimeric protein [4,5]. This constitutively active cyto-

plasmic tyrosine kinase does not block differentiation,

but enhances proliferation and viability of myeloid line-

age cells and leads to development of CML [6].

*Authors contributed equally to this work and thus share first author-

ship.

Frequency of Bcr-Abl Fusion Oncogene Splice Variants Associated with Chronic Myeloid Leukemia (CML) 177

Genetic abnormalities lead to formation of fusion on-

cogenes and this phenomenon is driven by the environ-

mental factors and living style which differ in different

geographical regions, the frequencies of different fusion

oncogenes associated with leukaemia can vary in differ-

ent ethnic groups [7], which can have a significant effect

on the management and prognosis of this type of leuka-

emia [8,9]. This study was conducted to calculate the

frequency of BCR-ABL gene splice variants associated

with CML. RT-PCR (Reverse Transcriptase Polymerase

Chain Reaction) was chosen as the method of choice to

detect BCR-ABL gene as this technique is one of the

most sensitive methods for this purpose.

2. Materials and Methods

2.1. Sample Collection

Blood samples of 30 CML patients were collected from

different hospitals of Lahore. Clinical features of the pa-

tients are given in Table 1. Samples were collected in

EDTA vacutainer tubes as per manufacturer’s instruction

with patient number, age and time/date of collection

written on the tubes. Sample collection and processing

for analysis was always started within 24 hours to mini-

mize mRNA degradation [10]. All the samples were

stored at 20˚C. The procedure for isolation of RNA was

adapted from Chomczynski and Sacchi with slight modi-

fication [11,12].

2.2. RNA Extraction and cDNA Synthesis

Quantity of RNA was estimated spectrophotometrically

[13], while the quality of RNA was checked by native

Table 1. Clinical features of CML patients.

Characteristics Patient Number (n = 30)

Age (years) Mean = 35

Range = 12-70

Gender

Male

Female

Splenomegaly 26

Hemoglobin (< 10 g/dL) 16

WBC Count (/mm3)

50,000 - 100,000

> 100,000

Platelet Count (/mm3)

100,000 - 450,000

> 450,000

Mode of Diagnosis

Ph+

BCR-ABL+

agarose gel electrophoresis and formaldehyde denaturing

gel electrophoresis [14]. RNA was reverse-transcribed to

cDNA for using as template in PCR reaction. RT-reaction

protocol and other reaction conditions were adapted from

Van-Dongen [15]. Briefly, 10 µL of RNA was added to

10 µL of RT-reaction mixture containing 5X RT buffer

(20 mM Tris HCl, 50 mM KCl, pH 8.3, 10 mM DTT), 10

mM dNTPs, 10 mM random hexamer primers, RiboLockTM

RNase inhibitor (20 units), M-MuLV Reverse Transcrip-

tase (40 units) (Fermentas, USA) and DPCE-treated wa-

ter. Reaction was carried out by incubating at room tem-

perature for 10 min, 37˚C for 60 min, 99˚C for 3 min and

held at 4˚C in the last step in the PCR machine.

2.3. RT-PCR Amplifications

PCR primers and nested PCR protocols for the detection

of BCR-ABL fusion gene in CML patients were adopted

from Radich [16]. The sequences of primers are given in

Table 2.

The cDNA product was PCR amplified in two rounds

with 1 U/μL of Taq DNA Polymerase, 125 mM primers,

10x PCR buffer with KCl, 25 mM MgCl2 and 10 mM

dNTP Mix. Sterile water was used as negative control.

Thermal cycling conditions for nested PCR were: Pre-

liminary denaturation at 95˚C for 2 min followed by 35

cycles of denaturation of double stranded DNA at 95˚C

for 5 sec, annealing at 55˚C for 10 sec and extension at

72˚C for 15 sec, followed by a final extension at 72˚C for

3 minutes. Round 2 was carried out with the same condi-

tions. The PCR products were run on a 2% agarose gel

with ethidium bromide to analyze the size of the ampli-

cons.

3. Results

Primer combinations used in amplification allowed to

amplify both types of transcripts in a single reaction.

Quality of RNA was analyzed on formaldehyde gel elec-

trophoresis (Figure 1) and efficiency of cDNA synthesis

on agarose gel electrophoresis. Two types of PCR products

were detected as 305bp and 234bp for b3a2 and b2a2

respectively (Figure 2). The frequencies of both fusion

transcripts were found to be 63.33% and 36.66% for

b3a2 and b2a2 respectively. The sensitivity assay was

Table 2. Primers used for detec tion of fusion gene BCR- ABL

in CML by RT-PCR.

PCR

Round PrimerPrimer Sequence (5/-3/)

CML C5/-GAAGTGTTTCAGAAGClTCTCC-3/

1st RoundCML D5/- TGATTATAGCCTAAGACCCGGA-3/

CML A5/-TGGAGCTGCAGATGCTGACCAACTCG-3/

2nd RoundCML B5/-ATCTCCACTGGCCACAAAATCATACA-3/

Frequency of Bcr-Abl Fusion Oncogene Splice Variants Associated with Chronic Myeloid Leukemia (CML)

178

Figure 1. Formaldehyde gel showing bands of RNA from 3

different samples of CML patients.

Figure 2. 2% agarose gel containing Ethidium bromide

showing two types of characteristics BCR-ABL transcripts

b2a2 (1,4) and b3a2 (2,3,6) (234bp and 305bp respectively),

M: 50bp Ladder, NC: Negative Control.

also performed by generating 10-fold serial dilutions to

evaluate the sensitivity of RT-PCR for detection of these

fusion transcripts. Using these dilutions, detection limit

of the nested PCR was found upto 10–6 of cDNA for each

sample (Figure 3).

4. Discussion

RT-PCR is one of the most sensitive techniques to detect

BCR-ABL transcripts associated with CML [17]. Real

time quantitative PCR is increasingly used to assess

treatment response in patients with CML. When the level

of residual leukaemia falls below the level of detection

by bone marrow cytogenetic analysis, PCR based assays

like the real time quantitative PCR or nested reverse tran-

scriptase PCR are the methods of choice for further

monitoring [18]. In this study nested PCR was opted as

Figure 3. Amplification of BCR-ABL fusion transcript b3a2

(305 bp) from serial dilutions of cDNA (10–0 - 10–7) showing

a very good sensitivity of nested PCR for the detection of

this transcript. M; 50 bp ladder.

the method of choice due to its sensitivity to detect both

b3a2 and b2a2 fusion transcripts in a single reaction. In

the sensitivity assay of nested PCR, very high-quality

results were obtained and this technique showed sensi-

tivity upto 10–6.

In this study, the frequency of b3a2 and b2a2 was

found to be 63.33% and 36.66% respectively. The fre-

quency of b3a2 is two times higher than b2a2 with the

ratio of 2:1 in our population. The coexpression of tran-

scripts was not found in any of the patients. Spleen was

grossly enlarged in most of the patients and mildly

enlarged in some but without any correlation to transcript

type. Data has been published about the frequency of

different types of fusion oncogenes associated with acute

lymphocytic leukemia (ALL) in Pakistan [19]. The fre-

quency of BCR-ABL fusion oncogene in Pakistani child-

hood ALL patients was reported to be 49%, which is

higher as compared to other reports from around the

world [19]. Frequency of different fusion oncogenes in

Pakistani ALL patients is different from other geographic

regions [9]. The frequency of fusion oncogenes associ-

ated with different leukemia types was compared with

western populations and significant differences were

observed due to geographical, racial and ethnic differ-

ences [7]. In a study carried out by Verschraegen, the

frequencies of b2a2 and b3a2 transcripts were 30.2% and

67.9% respectively [20]. Reiter found the incidence of

b2a2 and b3a2 transcripts in CML patients to be 31.6%

and 68.4% respectively [21]. In Korean CML patients,

frequency of b2a2 and b3a2 was collectively found to be

98.18%, corresponding to 67.66% b3a2, and 32.34%

b2a2 transcripts [22] where the number of patients with

b3a2 was twice the number of patients with b2a2. Fre-

quencies of fusion transcripts in Iranian CML patients

were found to be 21% and 62% for b2a2 and b3a2 re-

spectively [23]. The frequency of b3a2 transcripts was

found to be almost three times higher than that of b2a2

[23].

Several authors have reported that CML patients with

b2a2 BCR-ABL splice variants have a better prognosis

and response to imatinib. De-Lemos et al. [24] reported

that statistically significant difference was found for the

levels of expression of transcripts b2a2 and b3a2 at six

months of imatinib treatment, which shows that b2a2

may have a better molecular response than b3a2. Verma

et al. [25] reported that rare variants like e1a2 have an

inferior response to imatinib. Sharma et al. [26] found

that 59% patients with b2a2 type achieved complete cy-

togenetic response (CGR) as compared to 28% patients

with b3a2 (p = 0.04) while in 24 patients with minor or

no CGR, 25% had b2a2 compared to 75% b3a2 type (p =

0.04). Moreover, expression of BCR-ABL/ABL% was

higher in b3a2 patients compared to b2a2 (p = 0.120).

Frequency of Bcr-Abl Fusion Oncogene Splice Variants Associated with Chronic Myeloid Leukemia (CML) 179

They also found that pre-treatment characteristics like

mean spleen size, mean hemoglobin, mean, and mean

platelets counts were not significantly different in the

b3a2 vs. b2a2 transcripts groups [26], which supports our

findings. These observations highlight the need for more

extensive studies in different ethnic groups on the role of

different BCR-ABL splice variants in biology [27] and

treatment response of the BCR-ABL positive leukemia

patients, as acquired BCR-ABL point mutations and

other factors associated with imatinib resistance do not

explain the reason of imatinib resistance in all BCR-ABL

positive patients [28-30]. Studies related to the differ-

rences in clinical features and response to treatment in

CML and philadelphia positive acute leukemia patients

are specially required with the advent and FDA approval

of other tyrosine kinase inhibitors (TKIs) like nilotinib

and dasatinib for front-line treatment of BCR-ABL posi-

tive CML and ALL.

In conclusion, frequencies of BCR-ABL splice vari-

ants can vary in different geographical regions due to

interplay of natural genetic variations in different ethnic

populations, diverse environmental factors and living

style. Moreover, the knowledge about the rate of occur-

rence of these transcripts associated with CML can be of

very significance as it can lend a hand to further under-

stand the pathobiology of t(9;22)-positive leukemic cells.

Moreover, it will also assist in prognosis, treatment and

management of these CML transcripts types.

5. Acknowledgements

This work was partially supported by the College of

Medicine Research Center, Deanship of Scientific Re-

search, King Saud University, Riyadh, Saudi Arabia,

Research funding provided by Higher Education Com-

mission Pakistan is also acknowledged.

6. Conflict of Interest Statement

The authors have no potential conflict of interest.

REFERENCES

[1] Q. Cardama and J. E. Cortes, “Chronic Myeloid Leuke-

mia: Diagnosis and Treatment,” Mayo Clinical Proceed-

ings Journal, Vol. 81, No. 7, 2006, pp. 973-988.

[2] Y. Ben-Neriah, G. Q. Daley, A. M. Mes-Masson, O. N.

Witte and D. Baltimore, “The Chronic Myelogenous Leu-

kemia Specific P210 Protein is the Product of the Bcrabl

Hybrid Gene,” Science, Vol. 233, No. 4760, 1986, pp.

212-214. doi:10.1126/science.3460176

[3] E. Shtivelman, B. Lifshitz, R. P. Gale and E. Canaani,

“Fused Transcript of Abl and Bcr Genes in Chronic Mye-

logenous Leukaemia,” Nature, Vol. 315, No. 6020, 1985,

pp. 550-554. doi:10.1038/315550a0

[4] R. M. Arana-Trejo, E. R. Sanchez, G. Ignacio-Ibarra, E. B.

Fuente, O. Garces, E. G. Morales, M. C. Granados, R. O.

Martinez, M. E. Rubio-Borja, L. S. Anaya, P. Herrera, J.

D. Llamas and S. Kofman, “BCR/ABL P210, P190 and

P230 Fusion Genes in 250 Mexican Patients with Chronic

Myeloid Leukaemia,” (CML) Clinical Labortary Haem-

otolgy, Vol. 24, No. 3, 2002, pp. 145-150.

doi:10.1046/j.1365-2257.2002.00413.x

[5] B. D. Lichty, A. Keating, J. Callum, K. Yee, R. Croxford,

G. Corpus, B. Nwachukwu, P. Kim, J. Guo and S.

Kamel-Reid, “Expression of P210 and P190 BCR-ABL

due to Alternative Splicing in Chronic Myelogenous

Leukaemia,” British Journal of Haematology, Vol. 103,

No. 3, 1998, pp. 711-715.

doi:10.1046/j.1365-2141.1998.01033.x

[6] R. Ren, “Mechanisms of BCR-ABL in the Pathogenesis

of Chronic Myelogenous Leukaemia,” Nature Review

Cancer, Vol. 5, No. 3, 2005, pp. 172-183.

doi:10.1038/nrc1567

[7] Z. Iqbal, A. Tanveer, M. Iqbal, M. I. Naqvi, Z. Aziz, T. J.

Gill, A.-U. Qayyam, A. S. Taj, M. Khalid, I. H. Shah, A.

Jameel, M. Naeem, N. U. Rehman, M. Baig, M. Ferhan

and A. M. Khalid, “First Comprehensive Report of Strong

Interplay of Genetic and Environmental factors as well as

High Degree of Ethnic and Geographical Variations in

Biology of Leukemia as Manifested by Frequencies of

Common Fusion Oncogenes of Prognostic Significance

associated with Different Leukemic Subtypes in Pakistani

population,” In: Online Proceedings of 100th Annual

Meeting, American Association of Cancer Research,

Colorado. October 2009.

[8] H. Ariffin, S. P. Chen, C. S. Kwok, T. C. Quah, H. P. Lin

and A. E. Yeoh, “Ethnic Differences in the Frequency of

Subtypes of Childhood Acute Lymphoblastic Leukemia:

Results of the Malaysia-Singapore Leukemia Study

Group,” Journal of Pediatric Hematology oncology, Vol.

29, No. 1, 2007, pp. 27-31.

doi:10.1097/MPH.0b013e318030ac4c

[9] Z. Iqbal, “Frequency of Chromosomal Abnormalities and

Corrresponding Fusion Oncogenes in Acute Lympoblas-

tic Leukemia (ALL) Patients of Pakistan and Its Implica-

tion in Differential Diagnosis and Prognosis of Leukae-

mia,” Haematologica, Vol. 91, No. S3, 2006, p. 65.

[10] T. Hughes, M. Deininger, A. Hochhaus, S. Branford, J.

Radich, J. Kaeda, M. Baccarani, J. Cortes, N. C. Cross, B.

J. Druker, J. Gabert, D. Grimwade, R. Hehlmann, S.

Kamel-Reid, J. H. Lipton, J. Longtine, G. Martinelli, G.

Saglio, S. Soverini, W. Stock and J. M. Goldman, “Moni-

toring CML Patients Responding to Treatment with Ty-

rosine Kinase Inhibitors: Review and Recommendations

for Harmonizing Current Methodology for Detecting

BCR-ABL Transcripts and Kinase Domain Mutations and

for Expressing Results,” Blood, Vol. 108, No. 1, 2006, pp.

28-37. doi:10.1182/blood-2006-01-0092

[11] P. Chomczynski and N. Sacchi, “Single Step Method of

RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-

Chloroform Extraction,” Analytical of Biochemistry, Vol.

162, No. 1, 1987, pp. 156-159.

doi:10.1016/0003-2697(87)90021-2

Frequency of Bcr-Abl Fusion Oncogene Splice Variants Associated with Chronic Myeloid Leukemia (CML)

180

[12] P. Chomczynski, “A Reagent for the Single-Step Simul-

taneous Isolation of RNA, DNA and Proteins from Cell

and Tissue Samples,” Biotechniques, Vol. 15, No. 3, 1993,

pp. 532-537.

[13] J. A. Glasel, “Validity of Nucleic Acid Purities Monitored

by A260/A280 Absorbance Ratios,” Biotechniques, Vol.

18, No. 1, 1995, pp. 62-63.

[14] J. Sambrook and W. D. Russel, “Molecular Cloning,” A

Laboratory Manual, Cold Spring Harbor Laboratory

Press, New York, 2001.

[15] J. J. Van-Dongen, E. A. Macintyre, J. A. Gabert, E. De-

labesse, V. Rossi, G. Saglio and E. Gottardi, “Standard-

ized RT-PCR Analysis of Fusion Genes Transcripts from

Chromosome Aberrations in Acute Leukemia for Detec-

tion of Minimal Residual Disease,” Report of the BIO-

MED-I Concerted Action: Investigation of Minimal Re-

sidual Disease in Acute Leukemia, Leukemia, Vol. 13, No.

12, 1999, pp. 1901-1928.

[16] J. P. Radich, G. Gehly, T. Gooley, E. Bryant, R. A. Clift,

S. Collins, S. Edmands, J. Kirk, A. Lee and P. Kessler,

“Polymerase Chain Reaction Detection of the BCR-ABL

Fusion Transcript After Allogeneic Marrow Transplan-

tation for Chronic Myeloid Leukemia: Results and Impli-

cations in 346 Patients,” Blood, Vol. 85, No. 9, 1995, pp.

2632-2638.

[17] J. Q. Guo, H. Lin, H. Kantarjian, M. Talpaz, R. Champlin,

M. Andreeff, A. Glassman and R. B. Arlinghaus, “Com-

parison of Competitive-Nested PCR and Real-Time PCR

in Detecting BCR-ABL Fusion Transcripts in Chronic

Myeloid Leukemia Patients,” Leukemia, Vol. 16, No. 12,

2002, pp. 2447-2453. doi:10.1038/sj.leu.2402730

[18] S. Menif, S. Zarrouki, R. Jeddi, N. ben Alaya, Z. B. Ali,

H. B. Abid, S. Hdeiji, M. Elloumi, A. Khlif, B. Meddeb

and K. Dellagi, “A Quantitative Detection of Bcr-Abl

Transcripts in Chronic Myeloid Leukemia,” Pathologie

Biologie, Vol. 57, No, 5, 2009, pp. 388-391.

doi:10.1016/j.patbio.2007.12.010

[19] Z. Iqbal and A. Tanveer, “High Incidence of Bcr-Abl

Fusion Oncogene in Pakistani Childhood Acute Lymp-

hoid Leukaemia (ALL) Patients Reflects Ethnic Differ-

ences in Molecular Genetics of ALL,” Haematologica,

Vol. 91, No. S3, 2006, p. 65.

[20] C. F. Verschraegen, H. M. Kantarjian, C. Hirsch-Ginsberg,

M. S. Lee, S. O’Brien, M. B. Rios, S. A. Stass, M. Keating

and M. Talpaz, “The Breakpoint Cluster Region Site in Pa-

tients with Philadelphia Chromosome-Positive Chronic

Myelogenous Leukemia,” Clinical, Laboratory, and Prog-

nostic Correlations, Cancer, Vol. 76, No. 6, 1995, pp.

992-997.

doi:10.1002/1097-0142(19950915)76:6<992::AID-CNCR

2820760612>3.0.CO;2-L

[21] E. Reiter, H. T. Greinix, S. Brugger, F. Keil, W. Rabitsch,

C. Mannhalter, I. Schwarzinger, P. Höcker, G. Fischer, K.

Dieckmann, W. Hinterberger, W. Linkesch, B. Schneider,

K. Lechner and P. Kalhs, “Long-Term Follow-Up after

allogeneic Stem Cell Transplantation for Chronic Mye-

logenous Leukemia,” Bone Marrow Transplant, Vol. 22,

No. S4, 1998, pp. S86-S88.

[22] H. G. Goh, J. Y. Hwang, S. H. Kim, Y. H. Lee, Y. L. Kim

and D. W. Kim, “Comprehensive Analysis of BCR-ABL

Transcript Types in Korean CML Patients Using a Newly

Developed Multiplex RT-PCR,” Translational Research,

Vol. 148, No. 1, 2006, pp. 249-256.

[23] M. Yaghmaie, S. H. Ghaffari, A. Ghavamzadeh, K. Ali-

moghaddam, M. Jahani, S. A. Mousavi, M. Irvani, B.

Bahar and I. Bibordi, “Frequency of BCR-ABL Fusion

Transcripts in Iranian Patients with Chronic Myeloid

Leukemia,” Archives of Iranian Medicine, Vol. 11, No. 3,

2008, pp. 247-251.

[24] J. A. de Lemos, C. M. de Oliveira, A. C. Scerni, A. Q.

Bentes, A. C. Beltrão, I. R. Bentes, T. C. Azevedo, L. M.

Maradei-Pereira, “Differential Molecular Response of the

Transcripts B2A2 and B3A2 to Imatinib Mesylate in

Chronic Myeloid Leukemia,” Genetics and Molecular

Research, Vol. 4, No. 4, 30 December 2005, pp. 803-811.

[25] D. Verma, H. M. Kantarjian, D. Jones, R. Luthra, G.

Borthakur, S. Verstovsek, M. B. Rios and J. Cortes,

“Chronic Myeloid Leukemia (CML) with P190 BCR-ABL:

Analysis of Characteristics, Outcomes, and Prognostic Sig-

nificance,” Blood, Vol. 114, No. 11, 10 September 2009,

pp. 2232-2235.

doi:10.1182/blood-2009-02-204693

[26] P. Sharma, L. Kumar, S. Mohanty and V. Kochupillai,

“Response to Imatinib Mesylate in Chronic Myeloid

Leukemia Patients with Variant BCR-ABL fusion Tran-

scripts,” Ann Hematology, Vol. 89, No. 3, March 2010,

pp. 241-247. doi:10.1007/s00277-009-0822-7

[27] Z. Iqbal, M. Iqbal and T. Akhter, “Frequency of

BCR-ABL Fusion Oncogene in Pakistani Childhood

Acute Lymphoid Leukemia (ALL) Patients Reflects Eth-

nic Differences in Molecular Genetics of ALL,” Journal

of Pediatric Hematology/Oncology, Vol. 29, No. 8, Au-

gust 2007, p. 585.

doi:10.1097/MPH.0b013e3180f61bcf

[28] F. X. Gruber, T. Lundán, R. Goll, A. Silye, I. Mikkola, O.

P. Rekvig, S. Knuutila, K. Remes, T. Gedde-Dahl, K.

Porkka and H. Hjorth-Hansen, “BCR-ABL Isoforms As-

sociated with Intrinsic or Acquired Resistance to Imatinib:

More Heterogeneous than just ABL Kinase Domain Point

Mutations?” Medical Oncology, 8 January 2011.

[29] Z. Iqbal, M. Iqbal, M. Akhtar, M. I. Naqvi, A. H. Tahir, T.

J. Gill, et al., “Presence of Prior-to-Treatment BCR-ABL

Mutations In CD34 + CD38-Stem Cells of Newly Diag-

nosed Chronic Phase CML Patients and Their Correlation

with Imatinib Resistance: Implications of Cancer Pharma-

cogenomics and Pre-Therapeutic Genetic Testing in Per-

sonalized Treatment of BCR-ABL+Leukemia Blood,” Vol.

116, No. 21, November 2010, p. 2278.

[30] C. M. Lucas, R. J. Harris, A. Giannoudis, A. Davies, K.

Knight, S. J. Watmough, L. Wang, R. E. Clark, “Chronic

Myeloid Leukemia Patients with the E13a2 BCR-ABL

Fusion Transcript have Inferior Responses to Imatinib

Compared to Patients with the E14a2 Transcript,”

Haematologica, Vol. 94, No. 10, October 2009, pp.

1362-1367. doi:10.3324/haematol.2009.009134