- Chronic Myelogenous Leukemia with e13a3 (b2a3)Type of BCR-ABL Transcript Having a DNA Breakpointbetween ABL exons a2 and a3
- 作者:刘立根|发布时间:2013-06-21|浏览量:596次
chronic myelogenous leukemia with e13a3 (b2a3)
type of bcr-abl transcript having a dna breakpoint
between abl exons a2 and a3
li-gen liu,1,2 hideo tanaka,1* kinro ito,1 taiichi kyo,3 takuo ito,1 and akiro kimura1
1department of hematology & oncology, research institute for radiation biology and medicine, hiroshima university, hiroshima, japan上海市第五人民医院血液内科刘立根
2hematological department, shanghai fifth people’s hospital affiliated with fudan university, shanghai, china
3fourth department of internal medicine, hiroshima red cross & atomic bomb survivors hospital, hiroshima, japan
we describe a patient with chronic myelogenous leukemia (cml), in whom the dna
breakpoint in the bcr-abl fusion gene was determined to result in a rare e13a3 (b2a3)
transcript. the breakpoint in bcr was intron 13, which was 30 bp downstream from exon
13, and the breakpoint in abl was intron 2, and was 46 bp downstream from exon a2.
this case conforms to the mechanism of dna breakage occurring within abl intron 2,
but not at 50 to abl exon a2. with our review of this case and the literature, it seems that
cml with the bcr-a3 fusion product is associated with a low proportion of circulating
immature cells, mild or lack of splenomegaly, slow progressiveness, rather resistance to
ifn-a, and good response to imatinib mesylate. this is the first report of bcr-a3-type
cml in which the exact dna breakpoint was identified and located between exons a2 and
a3 of the abl gene. am. j. hematol. 74:268?272, 2003. ª 2003 wiley-liss, inc.
key words: chronic myelogenous leukemia; bcr; abl; dna breakpoint
introduction
the hallmark of chronic myelogenous leukemia
(cml) is the chimeric bcr-abl fusion gene, which
is usually formed as a result of the t(9;22) translocation
(philadelphia chromosome, ph). most bcr-abl fusion
transcripts are e13a2 (b2a2), e14a2 (b3a2), and less
commonly, e1a2, e19a2 [1]. cml with an atypical
hybrid transcript, in which bcr sequences are fused
to abl exon a3 rather than exon a2, is very rare, and its
characteristics are poorly understood [2?11]. until
now, the position of the dna breakpoint in the abl
gene with bcr-a3-type cml has been reported in only
three cases. abl exon a2 encodes 58 amino acids, the
last 17 of which formpart of a stretch of 50 amino acids
of src homology 3 (sh3) domain of the abl protein.
the sh3 is believed to negatively regulate the kinase
domain (sh1). thus, in theory, deletion of abl exon
a2 should result in increased tyrosine kinase activity
and, therefore, increased transforming activity. however,
recent data suggest that sh3 does not necessarily
contribute to aggressive phenotype. details of the clinical
phenotype of cml with this type of transcript are
unclear because of its very low incidence.
here, we report a case of cml with the e13a3 transcript
and the dna breakpoints in the bcr and abl
genes. this case was rather resistant to interferon a
(ifn-a) and showed rapid response to imatinib mesylate
(imatinib). this is the first report of the exact dna
breakpoint in the abl gene determined by nucleotide
sequencing in bcr-a3?type cml.
contract grant sponsor: tsuchiya foundation
*correspondence to: hideo tanaka, m.d., ph.d., department of
hematology & oncology, research institute for radiation biology
and medicine, hiroshima university, kasumi 1-2-3, minami-ku,
hiroshima 743-8553, japan. e-mail: dtanaka@hiroshima-u.ac.jp
received for publication 21 october 2002; accepted 15 july 2003
published online inwiley interscience (www.interscience.wiley.com).
doi: 10.1002/ajh.10429
american journal of hematology 74:268?272 (2003)
ª 2003 wiley-liss, inc.
materials and methods
rna and dna were extracted from the cells of a
bone marrow aspirate from a patient with cml before
treatment. informed and written consent was obtained
before bone marrow aspiration. reverse transcription
polymerase chain reaction (rt-pcr) for specific fusion
transcript of major bcr-abl was performed using
sets of primers for sequences within bcr exon 13 and
abl exon a3. the primer sequences were as follows
(the primer sequences were derived from genbank
accession numbers u07000 and u07563):
bcr1: 50-gcttctccctgacatccgtg-30
abl1: 50-ggcccatggtaccaggagtg-30
bcr2: 50-ggagctgcagatgctgaccaac-30
abl2: 50-gtttctccagactgttgactg-30
nested pcrwas performed with the set of primers of
first, bcr1 and abl1, and second, bcr2 and abl2.
genomicdna pcr was also performed with the same
sets of primers. the pcr product was cloned into a
vector using ta cloning kit (invitrogen corp, carlsbad,
ca) and followed by sequencing in abi prism 310
genetic analyzer (perkin elmer, foster city, ca).
case
a 49-year-old man was diagnosed with chronicphase
(cp) cml. he had mild splenomegaly at diagnosis
in august of 2000. peripheral blood analysis
results were as follows: white blood cell (wbc) count,
87 109/l; hemoglobin, 11.5 g/dl; platelet count,
331 109/l. thewbc differential count was as follows:
0.5% blasts; 3.5% promyelocytes; 20.5% myelocytes;
11% metamyelocytes; 16.5% band neutrophils; 29.5%
segmented neutrophils; 6.0% basophils; 4.5% eosinophils;
1.5% monocytes; 7.0% lymphocytes. the bone
marrow aspirate showed hypercellularity, and the karyotype
was 46, xy, t(9;22)(q34;q11) [20/20]. the positive
rate of bcr-abl fusion in peripheral blood was
97.2% as determined by fluorescence in situ hybridization
(fish). starting in september 2000, the patient
was treated with ifn-a and hydroxyurea. the peripheral
wbc count was 6.8 109/l in march 2001, but
the abnormal white blood cell differential (1% promyelocyte,
17% myelocyte, and 10% metamyelocyte) indicated
only a partial hematologic response (phr).
chromosome analysis in august 2001 showed that
ph-positive clone remained at 95%, indicating poor
response. fish analysis at november 8 2001 showed
that the positive rate of the bcr-abl fusion gene was
75.8%. thus, ifn-a with hydroxyurea did not induce
good response. then, from december 2001, imatinib
was administered. in april 2002, the karyotype was
normalized (46, xy [20/20]), and fish for bcr-abl
dropped to 0%, indicating complete cytogenetic response
(ccr).
rt-pcr was performed to detect bcr-abl transcript.
the product (fig. 1a, lane 2) showed one band
that was smaller than the common e13a2 (lanes 3) or
e14a2 (lane 4) transcripts. sequencing of the pcr
product revealed the e13a3-type bcr-abl transcript,
which was 174 bp shorter than that of the usual e13a2
type, meaning that the abl exon a2 transcript was
completely gone.
to determine the dna breakpoints of the bcrabl
fusion gene, genomic dna was amplified with
the preceding sets of primers. the pcr product showed
one band (lane 5) at the position of approximately 800
bp. sequencing of the product revealed that the dna
breakpoint of bcr was in intron 13, 30 bp downstream
from exon 13, and that the breakpoint of abl
was in intron 2 and was 46 bp downstream from exon
a2 (fig. 1b).
fig. 1. (a)agarose gel electrophoresis ofrt-pcrproduct of
bcr-abl mrna transcripts and dna pcr product of bcrabl
fusion gene fromgenomic dna. lane 1, 1-kb dna ladder
marker. lane 2, rt-pcr product of our patient’s bcr-abl
transcript. lane 3, rt-pcr product of e13a2 (b2a2) as a
positive control. lane 4, rt-pcr product of e14a2 (b3a2) as a
positive control. lane 5, dna pcr product of bcr-abl fusion
gene from our patient’s genomic dna. (b) sequencing result
of thepcrproduct fromgenomicdnaof our patient. thevertical
arrowindicates thednabreakpoint and bcr-abl junction
of the fusion gene. [color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
case report: cml lacking abl exon a2 269
discussion
to our knowledge, this is the first report of cml
with the bcr-a3 type, in which the exact dna breakpoint
of the abl gene was identified. we found that
the breakpoint of the abl gene was between exons a2
and a3. this is consistent with earlier reports of two
cases of ph-positive acute lymphocytic leukemia (all)
[12] and 1 case of cml with the e13a3 transcript [2].
however, in these cases, the breakpoints were determined
by southern blotting. dna breakpoint determined
by sequencing was reported only in one of those
two patients with all [12], in which the breakpoint
was in abl intron 2 just like our case. their breakpoint
was 22 bp downstream from exon a2, at a position
24 bp upstream from the breakpoint that we identified
in our case. in contrast, there has been one detailed report
and one short report of cml patients with the bcra3-
type, in which no dna rearrangement was detected
in abl intron 2 by southern blotting, suggesting that
deletion of exon a2 from the transcript was due to the
splicing mechanism in these cases [3,4]. thus, at least
two different mechanisms seem to be involved in the
formation of the bcr-a3 fusion transcript, one isdna
breakage within abl intron 2, and the other is dna
breakage at 50 to abl exon a2 and thereafter transcript
for exon a2 is spliced out.
the primary factors that determine preferential
breakage sites in the bcr and abl genes are presently
unknown. the low incidence of bcr-a3 fusion product
may be due to the relatively short length of abl intron
2 (0.6 kb) compared with the introns at the beginning of
the abl gene (>200 kb). the theoretically predicted
frequency of occurrence of a dna breakpoint between
abl exons a2 and a3 is 0.3%of bcr-abl?rearranged
patients, assuming that breakpoints in abl are randomly
distributed. this theoretical frequency may hold
true, because we found this case during the rt-pcr
study of 110cml cases. alu element is thought to be a
strong candidate involved in dna breakage and chimeric
gene rearrangement, because it is the most abundant
repeat sequence in both of these genes. however,
in our case, we could not find alu element at the breakpoints
of both bcr and abl genes. a lack of alu
elements was observed across the major and minor
breakpoint cluster regions of bcr and across a 25-kb
region with a high frequency of breakage in abl1 [13].
thus, the incidence and dna breakage of bcr-a3
type may not be determined only by the size of introns
or by alu element. thus, mechanisms of dna breakage
in bcr-abl gene seem more complicated.
a review of the literature revealed 13 cases of bcra3?
type cml including our case and 6 cases of bcra3?
type all. the clinical data of bcr-a3?type cml
are summarized in table i. there does not seem to be
any particular feature with regard to age or gender
distribution, wbc count, or platelet count. however,
there seemed to be an association with low proportion
of circulating immature cells and mild or no splenomegaly.
all but 3 of the 13 reported cases had a benign
prognosis. the longest lived patient has survived for at
least 126 months and was in cp at the last follow-up.
two patients did not require any treatment and
remained in the cp for at least 60 and 66 months.
this supports the notion that bcr-a3?type cml
patients may have a benign prognosis. in eight cases,
ifn-a has been administered. among the five cases in
which hematologic responses were assessed, our case
achieved phr, two cases achieved complete hematologic
response (chr), and two cases achieved remission
without precise statements from points of response
criteria. however, three of these latter four cases lost
responsiveness to ifn-a within 2 years. among the
four cases in which cytogenetic responses were assessed,
two showed partial cytogenetic response (pcr), one
showed no cytogenetic response (ncr), and our case
showed mcr, but the ph chromosome was 95% positive,
meaning close to ncr. imatinib was used only in
three cases including our case. all of them achieved
ccr. thus, imatinib treatment seems very promising
even for bcr-a3?type cml, in addition to common
bcr-a2?type cml.
a cml patient with bcr-a3 transcript is a nice
model in which to investigate the roles of sh3 domain
in bcr-abl protein in vivo. bcr-abl mutants lacking
sh3 resulted in reduced tissue invasiveness, and proliferation
of leukemic cells slowed down in vivo [14].
another study demonstrated that both sh3 and sh2
are required for stat5 activation by bcr-abl protein
[15]. in murine models of cml, e14a3-type of bcr-
abl/p210 induced cml-like myeloproliferative diseases
but showed small delay of the increase in peripheral
blood wbc counts and showed small, but consistent,
increase in survival compared with the common e14a2-
type [16]. these findings seem to agree with the generally
less progressive clinical histories of bcr-a3?type cml
patients.
in conclusion, cml with the bcr-a3 transcript
seems less progressive but rather resistant to ifn-a
and sensitive to imatinib as is seen in the common
bcr-a2 type. therefore, diagnosis of this type of
cml is important, because it may have a different
clinical expression and may affect treatment strategies.
acknowledgments
we thank ryoko yamaguchi, sachiko hidani, and
nanae nakaju for their excellent technical support.
270 case report: liu et al.
table i. clinical and laboratory characteristics of cml patients with bcr-abl transcripts lacking abl exon a2
case no. 1 2 3 4 5 6 7 8 9 10 11 12 13
gender male male female female male male na male female male female male male
age (y) 49 41 64 75 69 51 na 23 68 19 39 27 59
diagnostic phase cp cp cp cp cp cp cp cp cp cp cp cp cp
splenomegaly mild yes no no no no na no no na no na yes
karyotype ph ph ph ph t(4:9:22) ph ph ph ph ph ph/13q ph ph
bcr-abl transcript e13a3 e1a3/e1a2 e1a3 e1a3 e14a3 e14a3 e13a3 e14a3 e1a3/e13a3 e14a3 e14a3 na e13a3
hemoglobin level (g/dl) 11.5 na na 12.4 13.3 15.1 na 12.7 12.9 na na na (6.0 mmol/l)
wbc count (109/l) 87 189.5 53.2 18.5 18 19.9 na 95.8 38.4 42 9 na 254
wbc differentiation na na na
blast (%) 0.5 2 1 0 0 0 0 0 0 0
promyelocyte (%) 3.5 0 0 0 0 1 1 1 0 10
myelocyte (%) 20.5 0 0 0 0 0 0 14 3 42
metamyelocyte (%) 11 32a 22a 0 0 0 0 11 9 5
band +
segmented (%)
46 60 72 76 82 78 88 53 46 38
basophils (%) 6 0 0 1 4 1 1 6 24 0
eosinophils (%) 4.5 4 0 0 8 0 0 7 1 2
monocytes (%) 1.5 0 2 6 0 7 7 0 2 0
lymphocytes (%) 7 2 3 17 6 13 3 8 15 0
normoblasts (%) 3
platelet count (109/l) 331 184 156 257 527 566 na 485 629 381 na na 180
initial treatment ifn/hy ifn/hy ifn/hy no no!hy ifn na ifn/arac ifn ifn/hy no bu/ifn hy
ifn response
(hematologic)
pr cr na na remission!
progression
remission!
progression
cr!
progression
ifn response
(cytogenetic)
mr nr pr pr
second-line treatment imatinib imatinib imatinib bu/6mp autobmt hy bmt
imatinib response
(cytogenetic)
cr cr cr
duration of cp
before imatinib (mos)
16 56 62
duration of
follow-up* (mos)
24 56 62 66 36 126 96 60 20 34 60 24
clinical outcome cp cp cp cp cp cp cp cp bc cp bc ap
dna breakpoint of
abl gene
intron 2 upstream
of a2
upstream
of a2
intron 2
authors, published
year
present
case
al-ali,
2002
al-ali,
2002
roman,
2001
tribelli,
2000
tribelli,
2000
wilson,
2000
amabile,
1999
martinelli,
1999
pola´ k,
1998
iwata,
1994
pa´ ldi-
haris, 1994
van der
plas, 1991
*between the diagnosis and the last follow-up.
aall immature neutrophils included.
na, not applicable; cp, chronic phase; ap, accelerated phase; bc, blastic crisis; ph, t(9:22)(q34:q11); ifn, interferon; hy, hydroxyurea; bu, busulfan; 6-mp, 6-mercaptopurine: auto,
autologous; bmt, bone marrow transplantation; arac, cytarabine; cr, complete response; pr, partial response; mr, minor response; nr, no response (by criteria of talpaz).
case report: cml lacking abl exon a2 271
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8. tiribelli m, tonso a, ferrero d, et al. lack of sh3 domain does
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torres a. e1a3 as a unique, naturally occurring bcr-abl transcript
in an indolent case of chronic myeloid leukaemia. br j haematol
2001;114:635?637.
11. al-ali h-k, leiblein s, kovacs i, henning e, niederwieser d,
deininger mwn. cml with an e1a3 bcr-abl fusion: rare, benign,
and a potential diagnostic pitfall. blood 2002;100:1092?1093.
12. soekarman d, van denderen j, hoefsloot l, et al. a novel variant
of the bcr-abl fusion product in philadelphia chromosome-positive
acute lymphoblastic leukemia. leukemia 1990;4:397?403.
13. jeffs ar, wells e, morris cm nonrandom distribution of interspersed
repeat elements in the bcr and abl1 genes and its
relation to breakpoint cluster regions. genes chromosomes
cancer 2001;32:144?154.
14. skorski t, nieborowska-skorska m, wlodarski p, et al. the sh3
domain contributes to bcr/abl-dependent leukemogenesis in vivo:
role in adhesion, invasion, and homing. blood 1998;91:406?418.
15. nieborowska-skorska m, wasik ma, slupianek a, et al. signal
transducer and activator of transcription (stat) 5 activation by
bcr/abl is dependent on intact src homology (sh) 3 and sh2
domains of bcr/abl and is required for leukemogenesis. j exp
med 1999;189:1229?1242.
16. gross aw, zhang x, ren r. bcr-abl with an sh3 deletion retains
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activated by an sh3 deletion induces only lymphoid malignancy.
mol cell biol 1999;19:6918?6928.
272 case report: liu et al.
type of bcr-abl transcript having a dna breakpoint
between abl exons a2 and a3
li-gen liu,1,2 hideo tanaka,1* kinro ito,1 taiichi kyo,3 takuo ito,1 and akiro kimura1
1department of hematology & oncology, research institute for radiation biology and medicine, hiroshima university, hiroshima, japan上海市第五人民医院血液内科刘立根
2hematological department, shanghai fifth people’s hospital affiliated with fudan university, shanghai, china
3fourth department of internal medicine, hiroshima red cross & atomic bomb survivors hospital, hiroshima, japan
we describe a patient with chronic myelogenous leukemia (cml), in whom the dna
breakpoint in the bcr-abl fusion gene was determined to result in a rare e13a3 (b2a3)
transcript. the breakpoint in bcr was intron 13, which was 30 bp downstream from exon
13, and the breakpoint in abl was intron 2, and was 46 bp downstream from exon a2.
this case conforms to the mechanism of dna breakage occurring within abl intron 2,
but not at 50 to abl exon a2. with our review of this case and the literature, it seems that
cml with the bcr-a3 fusion product is associated with a low proportion of circulating
immature cells, mild or lack of splenomegaly, slow progressiveness, rather resistance to
ifn-a, and good response to imatinib mesylate. this is the first report of bcr-a3-type
cml in which the exact dna breakpoint was identified and located between exons a2 and
a3 of the abl gene. am. j. hematol. 74:268?272, 2003. ª 2003 wiley-liss, inc.
key words: chronic myelogenous leukemia; bcr; abl; dna breakpoint
introduction
the hallmark of chronic myelogenous leukemia
(cml) is the chimeric bcr-abl fusion gene, which
is usually formed as a result of the t(9;22) translocation
(philadelphia chromosome, ph). most bcr-abl fusion
transcripts are e13a2 (b2a2), e14a2 (b3a2), and less
commonly, e1a2, e19a2 [1]. cml with an atypical
hybrid transcript, in which bcr sequences are fused
to abl exon a3 rather than exon a2, is very rare, and its
characteristics are poorly understood [2?11]. until
now, the position of the dna breakpoint in the abl
gene with bcr-a3-type cml has been reported in only
three cases. abl exon a2 encodes 58 amino acids, the
last 17 of which formpart of a stretch of 50 amino acids
of src homology 3 (sh3) domain of the abl protein.
the sh3 is believed to negatively regulate the kinase
domain (sh1). thus, in theory, deletion of abl exon
a2 should result in increased tyrosine kinase activity
and, therefore, increased transforming activity. however,
recent data suggest that sh3 does not necessarily
contribute to aggressive phenotype. details of the clinical
phenotype of cml with this type of transcript are
unclear because of its very low incidence.
here, we report a case of cml with the e13a3 transcript
and the dna breakpoints in the bcr and abl
genes. this case was rather resistant to interferon a
(ifn-a) and showed rapid response to imatinib mesylate
(imatinib). this is the first report of the exact dna
breakpoint in the abl gene determined by nucleotide
sequencing in bcr-a3?type cml.
contract grant sponsor: tsuchiya foundation
*correspondence to: hideo tanaka, m.d., ph.d., department of
hematology & oncology, research institute for radiation biology
and medicine, hiroshima university, kasumi 1-2-3, minami-ku,
hiroshima 743-8553, japan. e-mail: dtanaka@hiroshima-u.ac.jp
received for publication 21 october 2002; accepted 15 july 2003
published online inwiley interscience (www.interscience.wiley.com).
doi: 10.1002/ajh.10429
american journal of hematology 74:268?272 (2003)
ª 2003 wiley-liss, inc.
materials and methods
rna and dna were extracted from the cells of a
bone marrow aspirate from a patient with cml before
treatment. informed and written consent was obtained
before bone marrow aspiration. reverse transcription
polymerase chain reaction (rt-pcr) for specific fusion
transcript of major bcr-abl was performed using
sets of primers for sequences within bcr exon 13 and
abl exon a3. the primer sequences were as follows
(the primer sequences were derived from genbank
accession numbers u07000 and u07563):
bcr1: 50-gcttctccctgacatccgtg-30
abl1: 50-ggcccatggtaccaggagtg-30
bcr2: 50-ggagctgcagatgctgaccaac-30
abl2: 50-gtttctccagactgttgactg-30
nested pcrwas performed with the set of primers of
first, bcr1 and abl1, and second, bcr2 and abl2.
genomicdna pcr was also performed with the same
sets of primers. the pcr product was cloned into a
vector using ta cloning kit (invitrogen corp, carlsbad,
ca) and followed by sequencing in abi prism 310
genetic analyzer (perkin elmer, foster city, ca).
case
a 49-year-old man was diagnosed with chronicphase
(cp) cml. he had mild splenomegaly at diagnosis
in august of 2000. peripheral blood analysis
results were as follows: white blood cell (wbc) count,
87 109/l; hemoglobin, 11.5 g/dl; platelet count,
331 109/l. thewbc differential count was as follows:
0.5% blasts; 3.5% promyelocytes; 20.5% myelocytes;
11% metamyelocytes; 16.5% band neutrophils; 29.5%
segmented neutrophils; 6.0% basophils; 4.5% eosinophils;
1.5% monocytes; 7.0% lymphocytes. the bone
marrow aspirate showed hypercellularity, and the karyotype
was 46, xy, t(9;22)(q34;q11) [20/20]. the positive
rate of bcr-abl fusion in peripheral blood was
97.2% as determined by fluorescence in situ hybridization
(fish). starting in september 2000, the patient
was treated with ifn-a and hydroxyurea. the peripheral
wbc count was 6.8 109/l in march 2001, but
the abnormal white blood cell differential (1% promyelocyte,
17% myelocyte, and 10% metamyelocyte) indicated
only a partial hematologic response (phr).
chromosome analysis in august 2001 showed that
ph-positive clone remained at 95%, indicating poor
response. fish analysis at november 8 2001 showed
that the positive rate of the bcr-abl fusion gene was
75.8%. thus, ifn-a with hydroxyurea did not induce
good response. then, from december 2001, imatinib
was administered. in april 2002, the karyotype was
normalized (46, xy [20/20]), and fish for bcr-abl
dropped to 0%, indicating complete cytogenetic response
(ccr).
rt-pcr was performed to detect bcr-abl transcript.
the product (fig. 1a, lane 2) showed one band
that was smaller than the common e13a2 (lanes 3) or
e14a2 (lane 4) transcripts. sequencing of the pcr
product revealed the e13a3-type bcr-abl transcript,
which was 174 bp shorter than that of the usual e13a2
type, meaning that the abl exon a2 transcript was
completely gone.
to determine the dna breakpoints of the bcrabl
fusion gene, genomic dna was amplified with
the preceding sets of primers. the pcr product showed
one band (lane 5) at the position of approximately 800
bp. sequencing of the product revealed that the dna
breakpoint of bcr was in intron 13, 30 bp downstream
from exon 13, and that the breakpoint of abl
was in intron 2 and was 46 bp downstream from exon
a2 (fig. 1b).
fig. 1. (a)agarose gel electrophoresis ofrt-pcrproduct of
bcr-abl mrna transcripts and dna pcr product of bcrabl
fusion gene fromgenomic dna. lane 1, 1-kb dna ladder
marker. lane 2, rt-pcr product of our patient’s bcr-abl
transcript. lane 3, rt-pcr product of e13a2 (b2a2) as a
positive control. lane 4, rt-pcr product of e14a2 (b3a2) as a
positive control. lane 5, dna pcr product of bcr-abl fusion
gene from our patient’s genomic dna. (b) sequencing result
of thepcrproduct fromgenomicdnaof our patient. thevertical
arrowindicates thednabreakpoint and bcr-abl junction
of the fusion gene. [color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
case report: cml lacking abl exon a2 269
discussion
to our knowledge, this is the first report of cml
with the bcr-a3 type, in which the exact dna breakpoint
of the abl gene was identified. we found that
the breakpoint of the abl gene was between exons a2
and a3. this is consistent with earlier reports of two
cases of ph-positive acute lymphocytic leukemia (all)
[12] and 1 case of cml with the e13a3 transcript [2].
however, in these cases, the breakpoints were determined
by southern blotting. dna breakpoint determined
by sequencing was reported only in one of those
two patients with all [12], in which the breakpoint
was in abl intron 2 just like our case. their breakpoint
was 22 bp downstream from exon a2, at a position
24 bp upstream from the breakpoint that we identified
in our case. in contrast, there has been one detailed report
and one short report of cml patients with the bcra3-
type, in which no dna rearrangement was detected
in abl intron 2 by southern blotting, suggesting that
deletion of exon a2 from the transcript was due to the
splicing mechanism in these cases [3,4]. thus, at least
two different mechanisms seem to be involved in the
formation of the bcr-a3 fusion transcript, one isdna
breakage within abl intron 2, and the other is dna
breakage at 50 to abl exon a2 and thereafter transcript
for exon a2 is spliced out.
the primary factors that determine preferential
breakage sites in the bcr and abl genes are presently
unknown. the low incidence of bcr-a3 fusion product
may be due to the relatively short length of abl intron
2 (0.6 kb) compared with the introns at the beginning of
the abl gene (>200 kb). the theoretically predicted
frequency of occurrence of a dna breakpoint between
abl exons a2 and a3 is 0.3%of bcr-abl?rearranged
patients, assuming that breakpoints in abl are randomly
distributed. this theoretical frequency may hold
true, because we found this case during the rt-pcr
study of 110cml cases. alu element is thought to be a
strong candidate involved in dna breakage and chimeric
gene rearrangement, because it is the most abundant
repeat sequence in both of these genes. however,
in our case, we could not find alu element at the breakpoints
of both bcr and abl genes. a lack of alu
elements was observed across the major and minor
breakpoint cluster regions of bcr and across a 25-kb
region with a high frequency of breakage in abl1 [13].
thus, the incidence and dna breakage of bcr-a3
type may not be determined only by the size of introns
or by alu element. thus, mechanisms of dna breakage
in bcr-abl gene seem more complicated.
a review of the literature revealed 13 cases of bcra3?
type cml including our case and 6 cases of bcra3?
type all. the clinical data of bcr-a3?type cml
are summarized in table i. there does not seem to be
any particular feature with regard to age or gender
distribution, wbc count, or platelet count. however,
there seemed to be an association with low proportion
of circulating immature cells and mild or no splenomegaly.
all but 3 of the 13 reported cases had a benign
prognosis. the longest lived patient has survived for at
least 126 months and was in cp at the last follow-up.
two patients did not require any treatment and
remained in the cp for at least 60 and 66 months.
this supports the notion that bcr-a3?type cml
patients may have a benign prognosis. in eight cases,
ifn-a has been administered. among the five cases in
which hematologic responses were assessed, our case
achieved phr, two cases achieved complete hematologic
response (chr), and two cases achieved remission
without precise statements from points of response
criteria. however, three of these latter four cases lost
responsiveness to ifn-a within 2 years. among the
four cases in which cytogenetic responses were assessed,
two showed partial cytogenetic response (pcr), one
showed no cytogenetic response (ncr), and our case
showed mcr, but the ph chromosome was 95% positive,
meaning close to ncr. imatinib was used only in
three cases including our case. all of them achieved
ccr. thus, imatinib treatment seems very promising
even for bcr-a3?type cml, in addition to common
bcr-a2?type cml.
a cml patient with bcr-a3 transcript is a nice
model in which to investigate the roles of sh3 domain
in bcr-abl protein in vivo. bcr-abl mutants lacking
sh3 resulted in reduced tissue invasiveness, and proliferation
of leukemic cells slowed down in vivo [14].
another study demonstrated that both sh3 and sh2
are required for stat5 activation by bcr-abl protein
[15]. in murine models of cml, e14a3-type of bcr-
abl/p210 induced cml-like myeloproliferative diseases
but showed small delay of the increase in peripheral
blood wbc counts and showed small, but consistent,
increase in survival compared with the common e14a2-
type [16]. these findings seem to agree with the generally
less progressive clinical histories of bcr-a3?type cml
patients.
in conclusion, cml with the bcr-a3 transcript
seems less progressive but rather resistant to ifn-a
and sensitive to imatinib as is seen in the common
bcr-a2 type. therefore, diagnosis of this type of
cml is important, because it may have a different
clinical expression and may affect treatment strategies.
acknowledgments
we thank ryoko yamaguchi, sachiko hidani, and
nanae nakaju for their excellent technical support.
270 case report: liu et al.
table i. clinical and laboratory characteristics of cml patients with bcr-abl transcripts lacking abl exon a2
case no. 1 2 3 4 5 6 7 8 9 10 11 12 13
gender male male female female male male na male female male female male male
age (y) 49 41 64 75 69 51 na 23 68 19 39 27 59
diagnostic phase cp cp cp cp cp cp cp cp cp cp cp cp cp
splenomegaly mild yes no no no no na no no na no na yes
karyotype ph ph ph ph t(4:9:22) ph ph ph ph ph ph/13q ph ph
bcr-abl transcript e13a3 e1a3/e1a2 e1a3 e1a3 e14a3 e14a3 e13a3 e14a3 e1a3/e13a3 e14a3 e14a3 na e13a3
hemoglobin level (g/dl) 11.5 na na 12.4 13.3 15.1 na 12.7 12.9 na na na (6.0 mmol/l)
wbc count (109/l) 87 189.5 53.2 18.5 18 19.9 na 95.8 38.4 42 9 na 254
wbc differentiation na na na
blast (%) 0.5 2 1 0 0 0 0 0 0 0
promyelocyte (%) 3.5 0 0 0 0 1 1 1 0 10
myelocyte (%) 20.5 0 0 0 0 0 0 14 3 42
metamyelocyte (%) 11 32a 22a 0 0 0 0 11 9 5
band +
segmented (%)
46 60 72 76 82 78 88 53 46 38
basophils (%) 6 0 0 1 4 1 1 6 24 0
eosinophils (%) 4.5 4 0 0 8 0 0 7 1 2
monocytes (%) 1.5 0 2 6 0 7 7 0 2 0
lymphocytes (%) 7 2 3 17 6 13 3 8 15 0
normoblasts (%) 3
platelet count (109/l) 331 184 156 257 527 566 na 485 629 381 na na 180
initial treatment ifn/hy ifn/hy ifn/hy no no!hy ifn na ifn/arac ifn ifn/hy no bu/ifn hy
ifn response
(hematologic)
pr cr na na remission!
progression
remission!
progression
cr!
progression
ifn response
(cytogenetic)
mr nr pr pr
second-line treatment imatinib imatinib imatinib bu/6mp autobmt hy bmt
imatinib response
(cytogenetic)
cr cr cr
duration of cp
before imatinib (mos)
16 56 62
duration of
follow-up* (mos)
24 56 62 66 36 126 96 60 20 34 60 24
clinical outcome cp cp cp cp cp cp cp cp bc cp bc ap
dna breakpoint of
abl gene
intron 2 upstream
of a2
upstream
of a2
intron 2
authors, published
year
present
case
al-ali,
2002
al-ali,
2002
roman,
2001
tribelli,
2000
tribelli,
2000
wilson,
2000
amabile,
1999
martinelli,
1999
pola´ k,
1998
iwata,
1994
pa´ ldi-
haris, 1994
van der
plas, 1991
*between the diagnosis and the last follow-up.
aall immature neutrophils included.
na, not applicable; cp, chronic phase; ap, accelerated phase; bc, blastic crisis; ph, t(9:22)(q34:q11); ifn, interferon; hy, hydroxyurea; bu, busulfan; 6-mp, 6-mercaptopurine: auto,
autologous; bmt, bone marrow transplantation; arac, cytarabine; cr, complete response; pr, partial response; mr, minor response; nr, no response (by criteria of talpaz).
case report: cml lacking abl exon a2 271
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