MEDITERRANEAN JOURNAL OF HEMATOLOGY AND INFECTIOUS DISEASES

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Original Articles

A Study on the he Expression of BCR-ABL Transcript in n Mixed Phenotype Acute

Leukemia (MPAL) Cases Using the he Reverse Transcriptase Polymerase Reaction
Assay (RT-PCR) and its ts Correlation with
ith Hematological Remission Status Post
Initial Induction Therapy

Prateek Bhatia1, Jogeshwar Binota2, Neelam Varma3, Deepak Bansal4, Amita Trehan4, Ram Kumar Marwaha5,
Pankaj Malhotra5 and Subhash Varma6
1
Assistant Professor-Pediatrics, 2Senior Laboratory Technician, 3Professor and Head -Hematology, 4 Additional
Professor, 5Professor – Pediatric Hemato-oncology
Hemato and 5 Additional Professor, 6Professor & Head – Internal
Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh

Correspondence to: Prof. Neelam Varma,

Varma Professor & Head. Department of Hematology
Hematology, PGIMER, Chandigarh.
Phone: +91-01722755125. Email: [email protected]

Competing interests: The authors have declared that

th no competing interests exist.

Published: May 8, 2012

Received: February 18, 2011
Accepted: March 20, 2011
Mediterr J Hematol Infect Dis 2012, 4(1): e2012024,
e201 DOI 10.4084/MJHID.2012.024
This article is available from: http://www.mjhid.org/article/view/10111
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
cited

Abstract. Introduction: The MPAL comprise 2-5% 2 5% of all acute leukemia. The present WHO 2008
classification has separated two groups in MPAL based on t(9;22) positivity and MLL
rearrangement. Aims & Objectives: The aim of the present pilot study is to note the frequency of
BCR-ABL
ABL transcript in MPAL cases using the RT-PCR RT PCR assay and to correlate the status with
hematological remission post induction. Materials & Methods: A total of 10 MPAL cases classified
on Flow-cytometry
cytometry based on the current WHO 2008 criteria were enrolled. In all the cases Bone
marrow or peripheral blood sample in EDTA was processed for molecular studies and the RT RT-PCR
reaction carried out using primers specific to the t (9;22) and t(4;11) tra translocation. The post
induction check marrow slides were also reviewed. Results: Out of the total 10 MPAL cases, 7/10
(70%) were adult and 3/10 (30%) pediatric cases. A total of 4/10 (40%) cases showed positivity for
the t(9;22) transcript and none for t (4;11).
(4;11). Of the 4 positive cases, 3/10(30%) were adult cases and
1/10(10%) pediatric case. The BCR-ABL
BCR ABL transcript type in adult cases was b3a2 (p210) in 2/3
(66%) and e1a2 (p190) in 1/3 (33.3%) case. The single pediatric case was positive for b3a2
transcript. Discussion & Conclusion: All the 4 positive MPAL cases presented with high TLC and
low platelet count (p<0.05). The positive cases also showed hematological remission at post
induction check marrow (blasts<5%). This could partly be explained due to good rresponse to the
imatinib added to the treatment protocol.

Introduction. Acute leukemia with a mixed phenotype leukemias.. Immunophenotyping for B/T/Myeloid
is a rare disease and comprises 2–5%5% of all acute markers is necessary for detection of Mixed Phenotype

Mediterr J Hematol Infect Dis 2012; 4;; Open Journal System

acute leukemias. The WHO 2008 classification standardized in our laboratory in 2004 for detection of
identifies two subtypes of MPAL with characteristic p210 transcript in CML cases. A separate Multiplex
genetic lesion as separate entities:- i) MPAL with t RT-PCR was done for detection of p190 and MLL-
(9;22) and ii) MPAL with MLL rearrangements. The AF4 transcript as adapted from study by Pakakasama et
incidence of MPAL with t(9;22) and MLL al3. This Multiplex RT-PCR is routinely used in our
rearrangement in adults is 28-30% and 2-3% and in laboratory as a screening PCR for detection of common
children 3-5% and 10-15% respectively1. The aim of chimeric fusion transcripts in ALL cases. The PCR
the present study was to note the frequency of BCR- conditions for both the PCR are outlined below and the
ABL positivity in MPAL cases using the Reverse primer sequences and the product base size are
Transcriptase Polymerase Reaction Assay (RT-PCR) highlighted in the table 1. The BCR-ABL PCR for
and to correlate BCR-ABL positivity with status of p210 transcripts (b3a2 and b2a2) was carried out in
hematological remission at 1st check marrow. following conditions: Pre-Denaturation- 94c-4 mts –
one cycle; Denaturation-94c-1 mt, Annealing-63c-2 mt
Materials and Methods. The study was a prospective and Extension- 72c- 3 mt for 32 cycles. This was
study carried out over a period of one year from July followed by one cycle of final Extension at 72c-10 mt.
2010-June 2011. Inclusion criteria included: (i) All The Multiplex RT-PCR for p190 (e1a2) transcript and
cases of Acute leukemia coming for Bone marrow the MLL-AF4 transcript was carried out as follows:
examination to Department of Hematology, PGIMER, The PCR was carried out in a final volume of 25 ul
Chandigarh and diagnosed as Mixed Phenotype Acute with 1 ul cDNA, 1x PCR buffer, 200 uM dNTP, 1.5
Leukemia according to WHO 2008 criteria on Bone mM MgCl2, 120 nM of each primer pair and 1 unit of
marrow examination and Immunophenotyping. (ii) All Tag polymerase. After initial denaturation step at 940 C
MPAL Cases receiving treatment in the Institute and for 3 min, the 35 cycles of PCR condition including 940
having complete follow-up details of 1st check marrow. C for 45 s, 630 C for 1 min, and 720 C for 1.5 min were
Cases of Acute leukemia with aberrant B/T/Myeloid performed. The final extension for 7 min at 720 C was
marker expression on immunophenotyping, known set to ensure a complete extension of all PCR products.
cases of Chronic Myeloid leukemia (CML) in Blast The PCR products were then run on agarose gel and
crisis and cases with incomplete follow-up details/not stained with Ethidium Bromide and visualized under
receiving treatment in the institute were excluded from UV-Gel doc for b3a2 (385bp), b2a2 ( 310bp) and e1a2
the study. (521bp) bands. Appropriate positive and negative
controls were included in each run.
Methodology. RNA was extracted from 1-2ml Bone Review of Geimsa stained Bone marrow and
Marrow aspirate in EDTA or 3-5ml Peripheral blood peripheral blood slides at 1st check marrow (Day 14-
(if blasts > 20% and TLC> 50X109 /L) using the Pediatric cases & Day 28-Adult cases) for Remission
commercial kit (Qiagen Miniamp RNA Blood kit) status (Standard Remission criteria- Hb>10g/dl; TLC
according to manufacturer’s instructions. The RNA 4-12X 109/L; Platelet 150-450 x 109/L; Bone marrow
quality was checked in each case by absorbance at blasts<5%; No Auer Rod; No blasts in peripheral
260nm in a spectrophotometer and by running a 1% blood) was performed and relevant data analyzed.
formaldehyde gel for 18s and 28s RNA bands. This Cytogenetic samples were taken in all cases.
was followed by cDNA synthesis according to the
commercial kit protocol (Fermantas cDNA kit). The Ethical Justification. The blood sample used in the
quality of cDNA was checked using primers for β- study is withdrawn as a part of routine diagnostic
Actin housekeeping gene. Reverse Transcriptase work-up of the patient and no additional sample pricks
polymerase chain reaction was carried out using were performed. Prior informed consent was taken
primers specific for p210 (b3a2 and b2a2)2 adapted from all patients/guardians before withdrawl of sample.
from the study of Jones et al and the protocol was
Table 1. Primer sequences and band sizes for BCR-ABL and MLL-AF4 transcript
Product size
S. No. Transcript Primer sequence
(bp)
BCR-b2 5’-ACAGAATTCCGCTGACCATCAATAAG-3’
1. b3a2 (p210) 385
ABL-a2 5’-TGTTGACTGGCGTGATGTAGTTGCTTGG-3’
BCR-b2 5’-ACAGAATTCCGCTGACCATCAATAAG-3’
2. b2a2 (p210) 310
ABL-a2 5’-TGTTGACTGGCGTGATGTAGTTGCTTGG-3’
BCR-e1 5’-GAC TGC AGC TCC AAT GAG AAC-3’
3. e1a2 (p190) 521
ABL-a2 5’-GTT TGG GCT TCA CAC CAT TCC-3’
MLL 5’-CCG CCT CAG CCA CCT AC-3’
4. MLL-AF4 559
AF4 5’-TGT CAC TGA GCT GAA GGT CG-3’

Mediterr J Hematol Infect Dis 2012; 4: Open Journal System

Table 2. Clinical presentation and Hematological data in MPAL cases

WBC PLATELET
AGE Lymph Node
S.NO. SEX Hepatomegaly spleenomegaly COUNT (x COUNT
(YRS) Enlargement
109/L) (x 109/L)
CASE 1 3.5 M +* (Inguinal) + (Mild) + (Mild) 19.2 45

CASE 2 42 F - + (Mild) + (Mild) 202.4 30

CASE 3 26 F ++**(cervical) - - 3.1 243

CASE 4 7 M - - - 33.4 31

CASE 5 31 M - +(Mild) + (Mild) 51 25

CASE 6 15 M + (Cervical) - - 72 11

+++(cervical,
CASE 7 27 F axillary and - - 6.5 252
Inguinal)

CASE 8 28 M - - - 5.1 381

CASE 9 60 F - +(Mild) + (Moderate) 246.6 53

CASE 10 7/12 M - - - 97.4 118

Statistical Analysis. The X2 and the Fischer exact test Figure 1 and 2 show RT-PCR agarose gels for
were used to correlate the clinical and laboratory Housekeeping gene (β-actin) and BCR-ABL transcript
features in the two groups and a p value of ≤0.05 was positive MPAL cases. None of the MPAL cases
taken as significant. showed positivity for MLL-AF4 transcript.

Results. The total frequency of MPAL was 6% Discussion. In the present study all the MPAL cases
(10/170) of all acute leukemia cases that underwent positive for BCR-ABL transcript had high WBC count
routine diagnostic evaluation during the study period. and low platelet count (p<0.05). Many studies have
Out of the 10 MPAL cases, 7/10 (70%) were adult and also shown BCR-ABL positive MPAL’s presenting
3/10 (30%) were Pediatric cases. The age in adult cases with high WBC count4,5, but present study also shows
ranged from 26-60 years (Mean age 31.5 yrs) and M: F relation of BCR-ABL positivity with low platelet
ratio was 1.3:1. The age in Pediatric cases ranged from count. There were 4 cases of T/Myeloid MPAL,of
7 months – 7 years (Mean age 3.7 years) and all were which one showed BCR-ABL positivity (Figure 3).
Male children. The clinical and Hematological data of Mastutes et al6 in their study had also found BCR-ABL
the MPAL cases is outlined in table 2. positivity in 13% (2/15) cases with T/Myeloid
On Immunophenotyping, 60% (6/10) were phenotype.
B/Myeloid and 40% (4/10) were T/Myeloid. The The incidence of MPAL cases was 6% and the
incidence of BCR-ABL positivity in MPAL cases was BCR-ABL positivity in present study was 40% which
40% (4/10). All four MPAL cases positive for BCR- is quiet comparable to few other studies from the
ABL transcript presented with High TLC and Low subcontinent and west. Studies from Asian sub-
platelet count (p value ≤0.05). 3/4 (75%) BCR-ABL continent by Xu et al7, Lee et al8 and Mi et al9 found
positive MPAL’s had B/Myeloid phenotype while 1/4 incidence of MPAL as 4.6%, 2.1% and 3.4% and BCR-
(25%) had T/Myeloid phenotype. Molecular break- ABL positivity as 25.0%, 36.8% and 16.7%
point was p210 (b3a2 type) in 3/4 (75%) cases and respectively. Studies from west by Owaidah et al10,
p190 in 1/4 (25%) case. The Immunophenotyping, Carbonell et al11, Legrand et al12, Killick et al13 and
molecular and remission data in MPAL cases is Weir et al14 found the incidence of MPAL as 3.4%,
detailed in table 3. Cytogenetics revealed satisfactory 4.0%, 8.0%, 3.6% and 1.3% and BCR-ABL positivity
metaphases in 6/10 cases. Philadelphia positive as 9.1%, 30.8%, 35.0%, 38.1% and 18.8% respectively.
metaphases were noted in all four BCR-ABL positive Check marrow is performed at Day 14 in Pediatric
MPAL cases with a 100% correlation. However, no Acute leukemia cases and Day 28 in adult cases. The
additional cytogenetic abnormality could be identified Complete Hematological Remission rate (CHR) at first
in any of the cases. check marrow after induction therapy was 50% (5/10)
Mediterr J Hematol Infect Dis 2012; 4: Open Journal System
Table 3. Immunophenotype and Molecular
ar data and Hematological remission status in MPAL cases.

Post Induction check

MOLECULAR
IMMUNOPHENOTYPIC PROFILE marrow (Hematological
S.NO. PROFILE
Remission Status)

(B/MYELOID) (T/MYELOID) BCR-ABL

ABL (9;22)
CD 19 bright,10,20 & CD 13, 33, POSITIVE-p210
p210
CASE 1 In Remission
117, Anti-MPO (b3a2) Transcript

CD 2, 3, 4, 7, cCD3, CD 56
POSITIVE-p210
p210
CASE 2 (>90%) and CD 13, 117, Anti- In Remission
(b3a2) Transcript
MPO (> 30%)

cCD3, 2, 5, 7, 4, 8, TdT and Not in Remission (9%

CASE 3 -
CD13, 33, Anti-MPO blasts)

cCD3, 2, 5, 7, TdT, 4, 8, TCR dim

CASE 4 - No follow-up
and CD13, 33, Anti-MPO

CD 19 bright, 10 (>97% ) and

CASE 5 - In Remission
Anti-MPO (>70% Blasts)
CD 19 bright, CD 10 and CD 13, POSITIVE-
Not in Remission (60%
CASE 6 33, Anti-MPO,
MPO, 11b, 11c, 4dim, p190 (e1a2)
blasts)
34, 123, 45 Transcript
cCD3, 2, 5, 7, 4, 8, TdT and
CASE 7 - No follow-up
CD13, Anti-MPO
CD 19, 22, 79a, 34, DR, 123, 38,
CASE 8 Tdt and CD 13, 33, 117, Anti-
Anti - No follow-up
MPO
POSITIVE-
CD 19, 34, DR, 123, 38, Tdt, and
CASE 9 p210 (b3a2) In Remission
CD 13, 33, 117, Anti-MPO
Transcript
CD 19, 22, 79a (>40%) and CD 4,
14, 16, 33, DR, Anti-MPO
MPO (>
CASE 10 - In Remission
60% Blasts)- B/Monocytoid
Monocytoid-
Infantile Leukemia

TOTAL 10 6/10 (60%) 4/10 (40%) 4/10 (40%)

Figure 2. Agarose gel showing BCR BCR-ABL positivity in the four

Figure 1. Beta Actin band positivity as marker of internal cDNA MPAL cases. Lane 1: Case 1 (Positive; b3a2 transcript
transcript- 385bp).
quality in all 4 positive MPAL cases (Two per case) Lane 2: Case 6 (Positive; e1a2 transcript
transcript- 521bp). Lane 4: Ladder
pattern 100bp. Lane 5: Case 2 (Positive; b3a2 transcript
transcript- 385bp).
Lane 8: Case 10 (Positive; b3a2 transcript
transcript- 385bp).
in MPAL cases; 30% cases had no follow-up
follow details
while 20% were not in CHR. Of the four BCR-ABL
BCR
positive cases 75% (3/4) were in CHR and all these
Mediterr J Hematol Infect Dis 2012; 4:: Open Journal System
 in MPAL cases. The MPAL cases are treated with
ALL/AML standard induction protocol based on
predominant Blast immunophenotype. In addition
Imatinib is added to the treatment regimen if the
MPAL case is BCR-ABL positive on RT-PCR. All the
above four BCR-ABL positive MPAL cases received
Imatinib along with standard induction regimen drugs.
The good response in three cases could be attributed to
the possible favorable effect of Imatinib addition to the
standard induction regimen, which is quiet well
described in literature . However long term follow up
data in more number of cases is needed to further
substantiate the findings. None of our MPAL case was
positive for MLL-AF4 transcript. However we had not
looked for other MLL rearrangements in these cases.
Figure 3. Geimsa Stain- PBF (1000X); Two population of Blasts.
Arrow- Blast showing presence of Auer Rod like granules Conclusions. Immunophenotyping of all acute
leukemia cases is necessary with a complete panel of
were positive for b3a2 transcript type (p<0.05%). Only lineage specific markers to detect presence of Mixed
one BCR-ABL positive MPAL (p190 transcript) Phenotype Acute leukemias. RT-PCR for BCR-ABL is
showed 60% blasts in Bone marrow at 1st check also mandatory in all MPAL cases as the overall
marrow. However, the case number and positivity for prognosis in these patients may improve with addition
p190 transcript is low and the follow-up data is limited of Imatinib (Gleevac /STI571) therapy to the treatment
to derive any conclusive statement regarding regimen.
prognostic difference between the two transcript types

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