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./ 0"c/0. : Acute Promyelocytic Leukaemia is the third
commonest AML subtypes among Malaysian. Reciprocal
translocation between chromosome 17 and 15 is the hallmark for
APML. The treatment of APML was with all-transretinoic acid and
more recently with arsenic trioxide (As2O3) has increased long-
lasting complete remissions (CRs). Three different ÷

isoforms have been described; S-form, L-form and V-form. Our aim
was to determine the clinical implication of these three different
isoforms of PML/RARĮ in APML patients. /0": Nineteen
patients diagnosed as APML were identified. Clinical and
laboratory data was retrieved from record office and haematology
laboratory respectively. Total RNA was extracted from nineteen
bone marrow samples within 24 hours of collection using standard
methods as described in manufacturers¶ protocol for QIAamp®
RNA Blood Mini Kits (Qiagen GmbH, Hilden, Germany). Multiplex
RT-PCR was performed on all patients diagnosed as APML.
,/, : ÷
 fusion transcript was detected in all them.
Of these patients, 57.9% (11patients) exhibited V-form, 36.8% (7
patients) S-form, and 5.2% L-form. Total white blood cell count was
higher in S-form compared to V-form (P < 0.05). Four years
survival was 100% and 42.8% for V-forms and S-forms
respectively. (p<0.005). c0.c,0. : Patients with ÷


V- forms survived better that S-form. Molecular detection of
isoforms is indicated in all patients diagnosed as APML. Further
study is in progress to identify the underlying mechanism

$

 form was found to be higher than S-form in population such as the
Acute promyelocytic leukemia (APML) is a form of acute leukemia
representing 10 ± 15% of all acute myeloid leukemia (AML) cases.
[1]. Translocation (15;17)(q22;q11.2) is a balanced reciprocal
RT-PCR step above was used as template. The PCR mixture
contain 2ȝl of PCR product and 1U of Qiagen HotStarTaqŒ
polymerase (Qiagen GmbH, Hilden, Germany) with final
concentration of 1´ Qiagen Buffer, 100ȝM dNTP and 500nM of
each PML-INT and RAR-INT primers. PCR was performed in
Mastercycler Gradient at 94oC for 20 seconds, 55oC for 20
seconds and 72oC for 30 seconds followed by a 5 minute final
extension at 72oC. PCR amplification was preceded by 1 cycle of
enzyme activation or preheating at 95oC for 15 minutes. Positive
control was performed for every run and no template control (NTC)
for the detection of PCR contamination.
Each PCR product was run on 1.7% agarose gel prepared stained
with ethidium bromide. Sample amplified with the PML INT and
RAR INT primers which show bands sized either 714 base pair
(bp), 714-312bp (often several bands due to alternative splicing) or
214bp in the presence of 366bp PCR product of ABL housekeeping
gene were reported as positive for L-form, V-form and S-form
respectively.
 
RNA was extracted from nineteen Malay patients who was newly "


diagnosed as APML. PML/RARĮ fusion transcript was detected in
all them. Of these patients, 63.1% (12 patients) exhibited L/V- Studies have shown that incidence of various fusion transcripts
form,and 36.8% (7 patients) S-form (Figure 1) varies significantly among different populations. Incidence of L-
form, S-form and V-form were 50-55%, 27-49% and 8-20%
Those exhibited S-form were older with male predominant respectively as reported in literatures from America [4]. The L/V-
compared to L/V-form. Blood counts were almost similar except for
Latinos as reported by Dan Douer et al., 2003 [5] which was similar
platelet counts which were lower in S-form (Table 1). Coagulation
to our study. We noted that there was no significant difference in
profiles were normal but D-dimer was present in all of them.
Hb, and TWBC count at diagnosis between the isoforms.
chromosomal translocation that fuse the PML gene on / *c

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 1 Although there was also no significant difference in platelet counts
chromosome 15 with the RARĮ gene on chromosome 17. The 2   %



 between the two forms but S-form presented with a lower count.
resulting PML/RARĮ chimeric gene is expressed and subsequently Fibrinogen, APTT and PT were normal in all cases. We also
PML/RARĮ fusion protein inhibits the granulocytic differentiation PML/RAR alpha isoforms observed elevated D-Dimer level in both forms denoting occult
and promoting survival of hematopoietic progenitor cells. DIC. We therefore recommend screening of D-Dimer level in all
Short-form L/V-form
cases of APML as part of the management protocol. Debate exists
Three types of PML/RARĮ isoforms have been identified. Type A or n=7 (SD) n=12
over the clinical relevance of molecular heterogeneity of APML
the short form (S-form) arises when the breakpoint occurs within Age yrs 38.4 (15.9) 28.6(9.9)
intron 3 of PML (breakpoint cluster region 3, Bcr-3) and type B or Sex (M:F) 5:2 4:8
the long form (L-form) occurs within intron 6 (Bcr-1) whereas the Studies on the prognostic significance of PML/RAR isoforms have
Hb g/L 8.2 (1.8) 8.3 (2.9) reported contradictory results. In our study, four years survival was
third type B variant or variable form (V-form) occurs within exon 6
(Bcr-2). Breakpoint of RARĮ is invariably in intron 2. [2] Some TWBC x 109/L 2.1 (1.0) 2.5 (2.0) 100% and 42.8% for L/V-forms and S-forms respectively. Kaplan
literatures have described correlation between PML/RARĮ Meier analysis was performed and patients with L/V-form have
Platelet x 109/L 24.7 (22.4) 43.6 (52.6)
isoforms and several patient characteristics and outcomes but with significantly better survival than the S-form. (p<0.005).
discrepant results.
Median survival for S-form was 19.9 months while all patients with
In the study done by Gonzalez et al., 2001, there was no difference L/V- form has survived to date. A larger cohort of patients is
was observed between the three isoforms in complete remission required to consider the presence of the S-form as a high-risk
(CR) rate. However 3-year disease-free survival was lower for V feature and to design the future risk-adapted treatment strategies
form than L- and S-form (62% vs. 94% and 89%). Both V-form and for APML.
S form were associated with some negative prognostic features at
time of diagnosis. [3] Thus the aim of this study was to to
determine the clinical implication of these three different isoforms c
 


of PML/RARĮ in APML patients.
Patients with ÷
 V- forms survived better that S-form.
Identification of molecular isoform is important in the management
 

 $  %
$ of patients with APML. Further study is in progress to identify the
underlying mechanism
Nineteen Malay patients diagnosed with APML from September
2004 to October 2008. Informed and written consent were taken
from them and the study has been approved by ethical committee. 



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were designed similar to the ones used in the BIOMED-1 ‡Lane 2: Patient showed PML/RARĮ fusion gene exhibit the
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