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A Possible Correlation Between the Type of bcr-abl Hybrid Messenger RNA and
Platelet Count in Philadelphia-Positive Chronic Myelogenous Leukemia
By Koiti Inokuchi, Tokiko Inoue, Arinobu Tojo, Makoto Futaki, Koichi Miyake, Takashi Yarnada, Yoshihiro Tanabe,
lchiro Ohki, Kazuo Dan, Keiya Ozawa, Shigetaka Asano, and Takeo Nornura
The Philadelphia (Phl) chromosome, in which the hybrid
bcr-ab/ gene is formed, is thought to be the initial event in
chronic myelogenous leukemia (CML). The position of the
breakpoint within the breakpoint cluster region (bcr) on Phl
chromosome and the splicing pattern determine the species
of the fused bcr-ab/ messenger RNA (mRNA). We tried to
detect the two types of fused mRNAs in 57 chronic-phase
cases of Phl-positive CML using the polymerase chain reaction procedure (RT-PCR). The bcr exon 2/ab/ exon 2 fused
mRNA (b2-a2) was detected in 17 patients, the bcr exon
Blab/ exon 2 fused mRNA (b3-a2) was detected in 34
patients, and both types of mRNA were detected in six
patients. The platelet counts of patients who expressed
b3-a2 mRNA or both types were significantly higher than
those of patients who expressed only b2-a2 (841.5 v
373.5 x 109/L: P < .015), although there was no significant
difference in the white blood cell counts or hemoglobin. This
finding suggests a possibility that the type of bcr-ab/ mRNA
may affect the thrombopoieticactivity in CML.
o 1991 by The American Society of Hematology.
A
The total RNA
RT-PCR for detection of bcr-ab1 fusion “A.
of mononuclear cells was extracted by the CsCl method.’’ RT-PCR
was performed using the methods described by Roth et all4and by
Kawasaki et al” with a slight modification.” Approximately 2 pg of
total RNA was used as the template with 10 U of avian myeloblastosis virus reverse-transcriptase (Boehringer-Mannheim, Indianapolis, IN) for 1 hour at 37°C in 50 mmol/L Tris-HCI, pH 8.3, 50
mmol/L KCl, 8 mmol/L MgCl,, 10 mmol/L dithiothreitol, dNTPs
at 500 pmol/L each in a total volume of 50 pL with 0.05 OD,, units
of oligo-primer (abl-RT 5’-AACGAAAAGGTTGGGGTC-3‘,
antisense strand). Ten microliters of the reverse transcriptase reaction mixture was adjusted to PCR conditions (10 mmol/L Tris-HCI
pH 8.3,50 mmol/L KCl, 15 mmol/L MgCI,, 2 mmol/L dithiothreitol, 200 pmol/L dNTPs) with 0.0125 OD,, units of one of two sets
of primers (the first set, A 5’-AGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAA3‘, coding strand, and B: 5’-TCACTGGGTCCAGCGAGAAGGTTMCCTTGGAGTT-3’,antisense
strand; the second set; C 5’-GGAGCTGCAGATGCTGACCAAC3‘, coding strand and D: 5’-TCAGACCCTGAGGCr‘CAAAGTC3’, antisense strand) and 2 U of Taq polymerase (Thermus
aquaticus DNA polymerase; Perkin Elmer Cetus, Nonvalk, CT) in
a 50 pL reaction volume (Fig 1).PCR of 40 cycles was performed,
consisting of 30 seconds at 94°C (denaturation), 30 seconds at 55°C
(annealing), and 1 minute at 75°C (extention). Finally, the PCR
products (20 pL) were phenol-extracted, ethanol-precipitated, and
electrophoresed through a 2% agarose gel. The PCR products
were visualized directly in ethidium bromide-stained gels, photographed, and transferred onto Gene Screen Plus membranes (New
England Nuclear, Boston, MA). Southern hybridization was performed using a 0.6HB probe corresponding to the bcr exon 2
(provided by Dr S. Hirosawa, Tokyo Medical and Dental University), or two kinds of oligonucleotides (E: 5’-GCTGAAGGGClT”-
SHORTENED chromosome 22, designated as the
Philadelphia (Phl) chromosome, is observed in the
leukemic cells of more than 90% of chronic myelogenous
leukemia (CML) patients.’ The Phl chromosome, which
can be considered to be the first karyotypic marker consistently present in a human neoplasm, is the result of a
reciprocal translocation between chromosomes 9 and 22,
t(9;22) (q34;qll).*This translocation moves the c-ab1protooncogene from chromosome 9 to chromosome 22.’ The
breakpoint of chromosome 22 has been shown to be
clustered within a limited region of about 5.8 kb, referred to
as the breakpoint cluster region ( b ~ r ) .Almost
~ , ~ all of the
patients have genomic breakpoints between exons b2 and
b3 or between exons b3 and b4 within bcr.‘ The two
chimeric genes may be distinguished by Southern blot
analysis. The first indication that the precise location of the
breakpoint on chromosome 22 might have some clinical
significance came from a study by Schaefer-Rego et al.’
Next, a study by Eisenberg et al’ showed that the mean
duration of the chronic phase for patients who had progressed to blast crisis was significantly shorter for patients
with a 3’-breakpoint site than for those with a 5’-breakpoint
site within the bcr. Many studies have reported on this
controversial
Even a small difference in the location of the breakpoint within the bcr gene may cause a
change in the amino acid sequence of the fused protein,
which in turn may affect the clinical features of CML
patients. Recently, we reported that CML patients with a 3’
breakpoint within the bcr gene have higher thrombopoietic
activity than those with a 5‘ breakpoint.“
Here, we tried to ascertain the relationship between
thrombopoiesis and the two potential types of fused bcr-ab1
messenger RNAs (mRNAs) using the polymerase chain
reaction (RT-PCR) in 57 CML patients. As expected, we
found that patients with fused b3-a2 mRNA had higher
platelet counts than those with fused b2-a2 mRNA.
MATERIALS AND METHODS
Patient population. Blood and bone marrow samples were
obtained from 57 Phl-positive CML patients in the chronic phase
of the disease. Most of the patients had not received therapy before
RNA analysis. The clinical data of each patient are that at
diagnosis.
Blood, Vo178, No 12 (December 15). 1991: pp3125-3127
From the Third Department of Internal Medicine, Nippon Medical
School; and the Department of Hematology-Oncology,the Institute of
Medical Science, the University of Tokyo, Tokyo, Japan.
Submitted August 26, 1991; accepted September 17, 1991.
Address reprint requests to Koiti Inokuchi, MD, PhD, The Third
Department of Intemal Medicine, Nippon Medical School, Sendagi
1-1-5,Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This aricle must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191 / 7812-0029$3.00/0
3125
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3126
INOKUCHI ET AL
I
1
b l b2 b3 a2
111
D B AT
Fig 1. Schematic representation of the bcr-ebl mRNA with position of oligomers numbered A, B, C, and D used in the PCR reaction,
and ab/-RT oligomer for making cDNA synthesis as described."" (0)
bcr-specific sequences; (0)
ab/-specific sequences.
analysis of 57 patients showed the presence of b3-a2 mRNA
in 34 cases and b2-a2 mRNA in 17 cases, and coexpression
of both junctions in six cases. The relationship between the
type of bcr-ab1 mRNA and platelet count is shown in Fig 3.
The platelet counts of patients expressing b3-a2 mRNA
were significantly higher than those of patients expressing
only b2-a2 mRNA (841.5 v 373.5 x lo9& P < .015). We
also examined the relationship with the white blood cell
count and hemoglobin, but no significant correlations were
found.
DISCUSSION
TGAACTCKY, specific for bcr exon31abl exon 2; and F 5'GCTGAAGGGCTTCTTCCTTATTGATG-3', specific for bcr
exon2Iabl exon 2)''
RESULTS
RNA samples from 57 CML patients were analyzed for
specific bcr-ab1 mRNA. The bcr-ab1 mRNA junctions were
amplified by the RT-PCR method using two pairs of
primers. The two junctions, bcr2-ab12 and bcr3-ab12, were
identified by their electrophoretic mobility and hybridization to bcr exon2 (0.6HB) probe or junction-specific oligoprobes. An example of the RT-PCR analysis in which a pair
of primers (primers A and B) was used is shown in Fig 2.
The RT-PCR products of RNA samples from K562 cells
and five CML patients were electrophoresed and subsequently visualized by ethidium bromide staining and hybridization to the 0.6HB probe. The 319-bp RT-PCR product is
the band specific for b3-a2 mRNA, while the 244-bp
RT-PCR product is specific for b2-a2 mRNA. The RT-PCR
Phl-positive CML is thought to be a clonal myeloproliferative disorder resulting from oncogenic transformation of a
bone marrow stem cell. In the chronic phase, the disease
phenotype is characterized by pronounced granulocytosis
and thrombocytosis in peripheral blood and increased
myeloid and megakaryocytecompartments in the hypercelMar bone marrow.
Recently, our understanding of the pathogenesis of CML
has been greatly advanced by the molecular studies. Phlpositive CML is characterized by the presence of a bcr
rearrangement. Attempts have been made to elucidate the
relationship between the breakpoint site and the duration
of survival or the other clinical features by Southern
analysis at the DNA level. Shtalrid et a1,9Jaubert et a1,Ioand
Morris et al" reported that there was a significant correla-
p<0.015
n
e
v)
g
l
2
3
4
5
6
e
bP
872
:&LA -
-
603
4
-
Y
~
234 194
-
A
w +b3-a2
b2-a2
+
0
Fig 2 Examples of Southern blot hybridization of RT-PCR products of the bcr-ebl mRNA in Phl-positive patients. Products from
RT-PCR analyses were size-fractionated by 2.0% agarose gel electrophoresis, transferred to a nylon membrane, and hybridizedto a 0.6HB
probe. K562 cells and patients 1, 4, and 6 have the b3a2 junction.
Patients 2 and 5 have the b2a2 junction only. Patient 3 produces both
junctional products.
b242 b3-a2 type
bcr-ab/ mRNA
Fig 3. Correlationof platelet count with the type of bcr-abl mRNA.
The platelet counts were obtained at diagnosis before the therapy.
The normal range is indicated by the hatched box. The bars indicate
the mean values. The significance of platelet counts in bothtypes was
tested with the Wlcoxon-2 sample test.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BCR-ABL
3127
MRNA AND PLATELET COUNT IN CML
tion between the site of the bcr breakpoint and the duration
of survival. CML patients have one of two bcr-ab1 mRNA
types or b ~ t h . ’ ~Application
,’~
of the RT-PCR method to
determine the type of bcr-ab1 mRNA in CML patients has
made it easier to identify the site of the bcr rearrangement.
The studies using the RT-PCR method are now prevailing
in this research area.””8 Recently, we found that CML
patients with a 3’ breakpoint site within the bcr gene have a
tendency to have higher thrombopoietic activity than those
with a 5’ breakpoint.’* Therefore, to confirm this preliminary observation, we extensively studied the relationship
between the type of bcr-ab1 mRNA and the patient’s
platelet count in 57 Phl-positive CML cases using the PCR
method. As expected, the platelet counts were higher in the
patients expressing b3-a2 mRNA.
A recent study reported similar finding in Phl-positive
essential thrombocythemia.” The b3-a2 type mRNA was
observed in five patients and the b2-a2 type mRNA was
observed in only one patient. In our present study, 16
patients who had platelet counts of more than 900 x 109/L
expressed only b3-a2 type mRNA, and almost all of the
patients with b2-a2 type mRNA had a platelet count of less
than 600 x 109/L. There were no apparent differences in
hematologic findings between the patients with b3-a2 type
mRNA and the CML patients with b2-a2 type mRNA,
except for the platelet count. Thus, we speculate that the
type of bcr-abl mRNA may affect the thrombopoietic
activity in CML, although we are currently unable to
explain why the platelet count correlates with the type of
bcr-ab1mRNA.
ACKNOWLEDGMENT
We thank Dr S. Hirosawa for the 0.6HB probe, the bloodresearch group of the Third Department of Internal Medicine of
the Nippon Medical School for supplying blood samples, and Dr S.
Inokuchi for help with the preparation of the manuscript.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1991 78: 3125-3127
A possible correlation between the type of bcr-abl hybrid messenger
RNA and platelet count in Philadelphia-positive chronic myelogenous
leukemia [see comments]
K Inokuchi, T Inoue, A Tojo, M Futaki, K Miyake, T Yamada, Y Tanabe, I Ohki, K Dan and K
Ozawa
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