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Consistent Amounts of Acute Leukemia-Associated P190BCR/ABL
Transcripts Are Expressed by Chronic Myelogenous Leukemia
Patients at Diagnosis
By Giuseppe Saglio, Fabrizio Pane, Enrico Gottardi, Ferdinand0 Frigeri, Maria Rosaria Buonaiuto, Angelo Guerrasio,
Daniela De Micheli, Adele Parziale, Maria Nella Fornaci, Giovanni Martinelli, and Francesco Salvatore
In chronic myelogenous leukemia (CML), the Philadelphia
in the formationof
(Ph) chromosome translocation results
BCR/ABL genes, normally transcribed in t w o types of hybrid transcripts with a b2a2 or b3a2 BCR/ABL junction,
which give origint o 210-kD fusion proteins(P210). A third
type of BCR/ABL (with ela2 type of junction) has been
identified in approximately 50% of the Ph-positive acute
lymphoblastic leukemia(Ph+ ALL) cases and resultsin the
production of a BCR/ABL protein of 190 kD (P190). The
presence of this transcript
has been associated almost exclusively with the presence of an acute leukemia phenotype. By contrast, here we describe that in addition t o
transcripts with the b2a2 and b3a2 types of junction corre-
sponding t o t h eP210 proteins, virtually all CMLs at diagnosis bear also BCR/ABL transcripts showing the ela2
type ofjunction, which correspondt o t h eacute leukemiaassociated P190 protein. With a quantitative polymerase
chain reactionassay we found that the amount of the ela2
mRNA present in CMLs in chronic phase, although in absolute amount much lower than that present in Ph+ ALLs,
represents in mostcases approximately 20% t o 30% of the
total BCR/ABL transcripts. Moreover, using a novel and
very sensitive Western blot technique, we detected relevant amounts of P190 protein in addition t o P210 from
peripheral cells of t w o of the patients.
0 1996 by The American Societyof Hematology.
THE
Cooperative Study Group on CML and sent to our institution for
OCCURRENCE OF the Philadelphia (Ph) chromosome translocationt(9;22) in human hematologic malig- molecular diagnosis and characterization. The series examined were
representative of the proportion between the three prognostic groups
nancies parallels the formation of BCWABL hybrid genes.’
classified according to the Sokal index present in the clinical study.‘
Whereas the breakpoints on chromosome 9 are always 5’ to
ABL exon 2, the breakpoints on chromosome 22 differ in
Qualitative Reverse-Transcriptase (RT)-PCR to Detect the
their position within the BCR gene, giving origin to hybrid
b2a2, b3a2, and ela2 Types of BCRLABL Junctions
transcriptswithdifferenttypes
of BCWABL junction. In
chronicmyelogenousleukemia(CML),thebreakpoints
on
Total RNA was obtained from the leukemic cells by the guanidium
thiocyanate and phenol-cloroform method. The methods used to amchromosome 22 are restricted in a central region of the BCR
plify in two different “nested” RT-PCR respectively, the b2a2, b3a2
gene called “mBCR” (majorbreakpointclusterregion),
types of BCWABL hybrid mRNAand the ela2 type have been
which contains 5 exons numered from 1 to 5.’ Two different
previously de~cribed.’,~
Briefly, 5 pg of total cellular RNA were
types of BCWABL junction may be present in CML.3 In the
dispensed
in
50
pL
appropriate
buffer containing 20 U RNAsin
first, “mBCR” exon 2 is joined to ABL exon 2 (b2a2 junc(Promega, Madison, WI), 1 mmol/L dNTPs (each of four), 100 pmoll
tion), whereas in the second, “mBCR” exon 3 is spliced to
L 3’ antisense ABL primer (see Table l), 200 U of MoMLV reverse
Ab1 exon 2 (b3a2 junction). The two chimeric mRNAs differ
transcriptase (BRL, Bethesda, MD). After al-hour incubation at
for the presence of the “mBCR” 3 sequences (75 bp), and
37°C two aliquots of 5 pL were diluted with 95 pL of a PCR
the corresponding P210 protein differs for 25 amino acids.
mixture (20 mmol/L each deoxynucleotide triphosphates (dNTPs),
A third type of BCWABL junction has been identified
50 mmol/L KCI, 10 mmol/L Tris HCI pH 8.3, 2.5 mmol/L MgClz,
almost exclusively in Ph-positive acute lymphoblastic leukebovine serum albumin (BSA) 2 mg/mL), and I .5 U of Taq polymermias (ALL)! In fact, whereas approximately 50% of Ph+
ase (Promega, Madison, WI), and two different PCR reactions with
ALL show the same molecular rearrangements found in
CML, in theremaining half, as a consequence of a breakpoint
falling within intron 1 of the BCR gene, only the first exon
From the Laboratorio di Medicina e Oncologia Molecolare, del
of the BCR gene is joined to ABL exon 2 (ela2 j~nction).~ Dipartimento di Scienze Biomediche e Oncologia, Umana dell‘UniThis results in the production of a BCWABL protein of 190
versita di Torino, Orbassano-Torino, Italy; CEINGE, Biotecnologie
kD in molecular weight (P190).5
Avanzate and Dipartimento di Biochimica e Biotecnologie Mediche,
Here we describe that,in addition to the b2a2 or b3a2 types Universitd “Federico 11” di Napoli; and Istituto di Ematologia
of junction corresponding to the P210 proteins, virtually all
“L. & A . Seragnoli”, Universita di Bologna, Bologna, Italy.
Submitted February 21, 1995; accepted September 6, 1995.
CMLs at diagnosis bear BCWABLtranscriptsshowingthe
Supported by grants from MURST,CNR (Progetto jinalizzato
ela2 type of junction. With a quantitative polymerase chain
FATMA Sp I , and Progettojnalizzato ACRO Sp 4, Rome), Regione
reaction (PCR) assaywe found that the amount
of ela2 mRNA
Campania, AIRC (Milan) and Agensud. A.D. is supported by an
represents in most cases approximately 20% to 30%
of the
AIDS fellowship of Minister0 della Sanita.
total BCWABL transcripts. Furthermore, we useda novel and
Address reprint requests to Giuseppe Saglio, MD, Laboratorio di
sensitive Western blot technique, optimizedin our laboratory,
Medicina e Oncologia Molecolare, Ospedale S. Luigi Gonzaga,
for the detectionof BCWABL proteins in chronic phase
Ch4L
10143 Orbassano-Torino, Italy.
blood cells, to confirm this finding at the protein level.
The publication costs of this article were defrayed in part by page
MATERIALS AND METHODS
Patients and Samples
We studied bone marrow samples from 25 consecutive Ph-positive
CML patients enrolled in a multicenter clinical study of the Italian
Blood, Vol 87,No 3 (February l), 1996:pp 1075-1080
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8703-0015$3.00/0
1075
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1076
SAGLIO
ET AL
Table 1. Sequences of the Oligonucleotides Used for RT-PCR Performed in the Study
Primers for the RT-PCR amplification the b2a2 and b3a2 BCR/ABLmRNA:
Step 1: 5'-TGTGAlTATAGCCTAAGACCCGGAG-3'
(3' ABL primer, antisense)
(5' BCR primer, sense)
5"CGGGAGCAGCAGAAGAAGTC-3'
Step 2: 5'-TCCACTGGCCACAAAATCATACAGT-3'
5"GTGAAACTCCAGACTGTCCACAGCA-3'
(3' ABL primer, antisense)
(5' BCR primer, sense)
Primers for the RT-PCR amplification the ela2 BCR/ABL m R N A
Step 1: 5'-TGTGAlTATAGCCTAAGACCCGGAG-3'
5"lTGTCGTGTCCGAGGCCACC-3'
Step 2: 5'-TCCACTGGCCACAAAATCATACAGT-3'
5"CAAGACCGGGCAGATCTGGCCC-3'
Primers used to in PCR amplification to incorporate the T7 promoter sequence into
(3' ABL
(5'BCR
(3' ABL
(5'BCR
primer, antisense)
primer, sense)
primer, antisense)
primer, sense)
b2a2 and b3a2 amplified products:
5"TGTGATTATAGCCTAAGACCCGGAG-3'
5'-TAATACGACTCACTATAGGGAGAGTGAAACTCCAGACTGTCCACAGC-3'
(3' ABL primer, antisense)
(5' BCR primer containing 7 promoter, sense)
Primers used to in PCR amplification to incorporate the T7 promoter sequence into
ela2 amplified products:
(3' ABL primer, antisense)
(5' BCR primer containing 7 promoter, sense)
5'-TGTGAlTATAGCCTAAGACCCGGAG-3'
5"TAATACGACTCACTATAGGGAGATTGTCGTGTCCGAGGCCACC-3'
Oligonucleotides used as probes (complementary to the junction sequences):
5"GCTGAAGGGClTCUCCTTATTGATG-3'
5"GCTGAAGGGCTmGAACTCTGClTA-3'
5'-GCTGAAGGGCTTCTGCGTCTCCAT-3'
50 pmol each of different amplimers were set up to amplify, respectively, in one the b2a2 and b3a2 types of junction and in the other
the ela2 junction. Allthe amplimers usedinthe
different PCR
reactions are listed in Table 1. Amplification reactions were performed on a temperature controller (Thermal Cycler, Perkin Elmer
C o p , Norwalk, CT) as follows: after an initial denaturation step at
95°C for 5 minutes, 40 cycles of denaturation, annealing, and extension were performed at 94°C for 30 seconds, 55°C for 30 seconds,
and 72°C for 30 seconds, respectively. Subsequently, 2 pL of the
first PCR reaction were used to set up a second round amplification
that was performed for 20 cycles as described above with amplimers
internal to the previous ones (see Table 1).
Next, 10 pL of each step of the different PCR reactions were run
on a 3% Nusieve, 1% Seakem agarose gel for ultraviolet (UV)
analysis and the type of BCWABL junction was established on the
basis of the molecular size of the amplified products obtained (b2a2
= 193 bp; b3a2 = 268 bp; ela2 = 190 bp) and of the primer pairs
used in the reaction (Fig 1A and B). The identity of the BCWABL
junction was confirmed by hybridization of the amplified products
with oligonucleotide probes corresponding to the specific BCWABL
junction (Table 1). Briefly, amplified mixtures were run on a 3%
Nusieve, 1% Seakem agarose gel electrophoresis and blotted on a
Gene Screen membrane (NEN-Du Pont, Milan, Italy). Prehybridizationand hybridization were performed in 6X SSPE (1X SSPE =
0.18 m o m NaCI, 10 mmol/L NaH2P0,, 1 mmol/L EDTA), 0.5%
sodium dodecyl sulfate (SDS), 5X Dehnard's solution and 100 pg/
mL of denatured herring sperm DNA. The probes used in thehybridization phase were previously labeled with y3'P-adenosine triphosphate (Amersham, Buckinghamshire, UK) by a T4 polynucleotide
kinase reaction. The hybridized membranes were washed with 2X
SSPE, 0. I % SDS for I O minutes at room temperature, and with 6X
SSPE, 1% SDS for I O minutes at denaturing temperature for each
probe. Moreover, to further prove the identity of the ela2 type of
BCWABL junction, we used a recently developed highly specific
colorimetric assay (c-TRAK t(9;22) Kit, Raggio-Italgene, PomeziaRome). Briefly, at the end of the PCRreaction, the mixture is diluted
with an equal volume of diluent, denatured, and allowed to hybridize
with two oligonucleotide probes corresponding, respectively, to the
(junction b2a2)
(junction b3a2)
(junction ela2)
ela2 BCWABL junction sequence fluorescein isothiocyandte
([FITCI-linked "capture" probe) andtoanABL
exon 2 internal
sequence (alkaline phosphatase [ALP]-linked "reporter" probe).
Separation of sequences bound by the FITC-linked capture probe is
performed using anti-FITC magnetic beads and visualized using
the appropriate substrate for ALP. Only when the ela2 BCWABL
-I
106
RNA mokculw
Fig 1. Ampliication efficiency (slope of the curves) of quantitative
PCR assay of el/a2 type of BCR/ABL hybrid mRNA and of the synthetic RNA used to plot the titrationcurve. Atotal of 500 and 250 ng
aliquots of total RNA extracted from a sample were reverse transcribed and amplified using the assay conditions described in Materials and Methods, and the radioactivity of amplified bands were plotted against the relative total amount of RNA. Two samples
containing 6 x 10' and 3 x lo6 molecules of synthetic RNA with the
same sequence as the ella2 type of BCRlABL hybrid mRNA were
also assayedin parallel with thesamples, and their radioactivity measured in pixel density units (PDUs) plotted against the starting number of molecules assayed. The similar slopes ofthe two curves demonstrate that amplification efficiency is roughly equal for sample and
standard RNA aliquots.
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1077
P190 IN CML
sequences are present, both “capture” and “reporter” probes can
bridge together, and absorbance values can be measured by a spectrophotometer.
In each series of RNA samples to be analyzed, a 5-pg aliquot of
yeast tRNA was analyzed as negative control to avoid false positive
cases due to carry-over contamination.
Quantitation of DifSerent Types of BCRLABL mRNAs
Quantitation of absolute amounts of BCWABL transcripts was
performed by using a recently devised noncompetitive PCR technique? Briefly, the technique consists of the reverse transcription of
two aliquots (500 and 250 ng, respectively) of total RNA extracted
from each sample, and the PCR amplification of the two cDNAs
obtained using a radiolabeled deoxynucleotide. The conditions of
the PCR reaction, ie, the total number of amplification cycles and the
reaction mixture, are standardized to ensure a constant amplification
efficiency throughout all the amplification cycles and to terminate
the reaction during the exponential phase of amplification. Under
these conditions there is a log-log linear relation between the number
of starting molecules of BCWABL mRNA contained in the sample
and the amount of PCR products estimated by the radioactivity of
the amplified band. The limiting number of amplification cycles
up to which amplification proceeds with a constant efficiency was
calculated, independently for the P210 and P190 hybrid BCR/ABL
transcripts, by amplifying scalar dilutions from 500 to 5 ng of total
RNA by RT-PCR using a decreasing number of cycles. After each
experiment, logarithmically transformed data were analyzed by linear regression, and the highest number of cycles that ensured a loglog linear relationship between sample RNA dilutions and amplified
products was used in the assays. The absolute amount of BCWABL
mRNA molecules was calculated by interpolating the amount of
PCR products with the titration curve obtained by amplifying, in
parallel with the samples, known amounts of a “synthetic” RNA
molecule with the same sequence as that of the type of BCWABL
mRNA to be quantitated. The synthetic RNA used as a standard was
synthetized in vitro using a two-step procedure. Total RNA from a
biological specimen containing the specific mRNA being studied
(P210 and P190 types of hybrid mRNAs) was reverse transcribed,
andthe cDNA was amplified byPCR using an upstream primer
containing the T7 phage promoter sequence’ (Table 1). The downstream primers were the same used for the first step amplifications
of the P210 and P190 BCR/ABL mRNAs. The modified upstream
primers were usedto incorporate the T7 promoter sequence into
amplified products at the 5’ ends. After a purification step, these T7
sequences containing double-strand amplified products were used
for in vitro transcription to produce synthetic RNA with the same
sequence as those to be quantitated.
The roughly twofold reduction of the number of molecules calculated in the 250-ng aliquot of total RNA analyzed as compared with
the sample containing 500 ng, represents a control of the constancy
of amplification efficiency in the samples being analyzed, and, hence,
of the accuracy of the assay (Fig 1).
The RT and PCR assay conditions for the b2/a2 and b3/a2 types
of BCWABL hybrid mRNAs were the following: the aliquots of
total RNA were incubated for 60 minutes at 37°C ina 20-pL reaction
mixture containing 20 mmol/L Tris HCI (pH 8.3), 5 mmol/L MgClz,
50 mmollL KCI, 0.5 mmoVL of each deoxyribonucleotide, 2.5 U of
RNAsin, 0.75 mmol/L of antisense ab1 primer (Table l), and 50 U of
MoMLV reverse transcriptase; the reaction was stopped by heating at
95°C for 10 minutes. The PCR amplification of cDNA obtained was
performed in a reaction mixture containing 20 mmol/L Tris HCl
(pH 8.3), 2.5 mmoVL MgClz, 50 mmol/L KC1, 0.1 of both sense
and antisense primers (Table l), 0.1 mmoVL of each deoxyribonucle-
otide, 2 mCi ofa3*PdCTP, and 2.5 U of Taq polymerase; the reaction
was performed using a total number of 18 cycles and thequantitative
frame, ie, the linearity range of the assay under these conditions was
between 1.2 X lo6 and 6 X lo3molecules. In the case of el/& type
of BCWABL transcript, the same RT and PCR reaction mixtures as
those used for the b2/a2 and b3/& types were used; however, given
the lower efficiency of this amplification reaction with respect to
that of the other types of BCWABL transcripts, a two-step “nested”
PCR was used; a total number of 18 + 20 cycles in the two steps
of amplification were chosen, and the quantitative frame of the assay
in these conditions was between 6 X l@ and 3 X l@ molecules.
Detection of BCRLABL Proteins in White Blood Cells
(WBC) From Peripheral Blood
Cell preparation. Fresh peripheral blood samples obtained from
two of the analyzed patients, containing more than 3 X lo7 WBC,
were processed to extract intact protein from myeloid cells according
to the method of Kuwao and Takahashi” with some modifications,
adopted to ensure a high-recovery of intact high molecular weight
proteins. Briefly, erythrocytes were removed after a 5-minute incubation in melting ice with 2 v01 of a pH 7.3 solution containing 155
mmoVL NbCI, 10 mmoVL KHC03, 0.1 mmol/L EDTA. WBC
pellets were washed twice with PBS solution containing 5 mmol/L
phenyl-methyl-sulfonyl fluoride (PMSF). Pellets were immediately
processed for protein extraction or alternatively stored at - 140°C.
Imrnunoblot procedures. WBC pellets were lysed by incubation
for 10 minutes in melting ice with a buffer containing 20 mmol/L
TRIS, 150 mmol/L NaCl, 5 mmol/L Na3V0.,, 2 mmol/L EDTA, 10
pg/mL aprotinin, 5 mmol/L benzamidine, and 5 mmol/L PMSF.
Samples were then centrifuged at 13,000, and supernatants were
supplemented with 2% sodium deoxycholic acid and 2% SDS. Supernatants were then concentrated to a final volume of 40 pL by
using Microcon 100 concentrators (Amicon Inc, Eeverly, MA) following the manufacturer’s directions. The protein solutions were
supplemented with volume of Laemli buffer (0.02 mmoVL Tris,
12.5% glycerol, 0.1% SDS, 5% 2-mercaptoethanol, at pH 6.8), and
analyzed by SDS polyacrylamide gel electrophoresis, at a gel concentration of 6.5%. After electrophoresis migration, the gel was
soaked for 30 minutes in a transfer buffer (0.05 m o m Tris, 0.380
mol/L glycine, 20% methanol and 0.1% SDS), and the proteins were
transferred on a 0.45 pm nitrocellulose membrane using a semidry
apparatus (Protean Mini-gel, BIO-RAD Laboratories, Hercules, CA)
for 40 minutes at 5.5 mA/cm2.
Block treatment of membranes was performed by overnight incubation in TST buffer (10 mmol/L TRIS pH 8.0, 0.15 mol/L NaCI,
0.05% Tween 20) containing 1% nonfat dry milk.
Detection of specific proteins blotted onto membranes was performed by a two-step procedure. First, the membrane was incubated
at room temperature with a 1:500 dilution of anti-ABL antibody
(Abl-Ab3, Oncogene Science Inc, Uniondale, NY) and then with a
1:1, o o O dilution of antimouse horseradish peroxidase (HRP)-labeled
secondary antibody (Amersham); in both cases, incubation time was
1 hour. Visualization of protein bands was then obtained using a
chemoluminescent detection method (ECL Western Blotting Detection System, Amersham) following the manufacturer’s directions.
RESULTS
Inall the CML cases at diagnosis,amplificationbands
corresponding in size to that expected for the b2a2 or b3a2
types of BCWABL fragments were seen even after a single
step (40 cycles) of the RT-PCR reaction (Fig 2A). This, of
course, is expected given the full expansion of the Ph-posi-
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SAGLIO ET AL
1078
1" step
1" step
2" step
2" step
Fig 2. (A) Picture of the gel examined under UV showing the results obtainedin six different cases of CML at diagnosis after the RT-PCR
devised t o amplify theBCRlABL transcripts with theb2a2 or b3a2 types of junction. Amplification fragments 401
of bp (b3a2 junction) or326
(b2a2 junction), respectively, are clearly visible after the first step(40 cycles) of the nested RT-PCR analysis. The results are confirmed by the
presence of fragments of 268 bp (b3a2 junction) and 193 bp (b2a2 junction), respectively, at the end of the second step (20 additional cycles)
with more internalprimers. After the second step, the bands of higher molecular weight, which are visible in addition to theexpected bands,
in
amount in the reaction
are the "half n e s t e d PCR products due to thecombination of the primersused in the first step (still present some
mixture) and those newly added during the second step. Case No. 5 shows some amount of b2a2 BCRlABL transcript in addition t o the
prevalent b3a2 type of transcript. (B) Picture of the gelexamined under UV showing the results obtained
in the same six cases of CML shown
of
No specific amplification bands are visible after
in (A) after RT-PCR devised t o amplify the BCRlABL transcripts with the ela2 type junction.
the first step(40 cycles) of RT-PCR, but amplification fragmentsof the same size (190 bp) are clearly visible after the second step (20 additional
cycles) with more internal primers. The specificity of the fragments has been confirmed by hybridization with the junction oligonucleotide
(see Table l),
and in one case, also by sequencing.
tive clone and the high amount of the BCWABL transcripts.
In our laboratory, however, to confirm the specificity of the
results. we checked the presence of amplification fragments
after 20 additional cycles of RT-PCR with primers internal
to the previous one (Fig 2A). By contrast, as shown in Fig
lB, in a parallel RT-PCR reaction designed to amplify the
ela2 type of BCWABL junction, no specific amplification
fragments were evident after 40 cycles, but became clearly
visible after 20 additional "nested"RT-PCR cycles (Fig
2B). This was constantly observed in all the cases tested.
The specificity of the ela2 fragment was confirmed by hybridization to an oligonucleotide probe complementary to
the specific sequence of the junction, by a highly specific
colorimetric assay and, in one case, also by direct sequencing
of the amplified fragment. Contamination was carefully excluded by running appropriate negative controls (see Materials and Methods).
To evaluate the relative amount of ela2 transcripts with
respect to the b2a2 and b3a2 transcripts, we used a recently
developed quantitative RT-PCR assay that detects attomoles
of specific mRNAs present in a given sample.' This procedure was specifically designed to monitor the amount of
minimal residual disease in CML patients after allogeneic
bone marrow transplantation and during treatment ofPhpositive ALL patients. To have the exact quantitation of the
relative amount of the two transcripts (P210 and P190 types
of BCWABL hybrid mRNA), we used the above-mentioned
procedure in seven cases of the 25 patients observed (see
Table 2). The amount of the b2a2 and b3a2 BCWABL transcripts is quite variable from case to case, ranging from 2.6
X 10' to IS X IO' molecules per pg of totalRNA,and it
seems apparently more elevated in cases with the b3a2 junctionthan in those withtheb2a2 junction. The number of
cases examined, however, is too low to establish a precise
correlation. The amount of the ela2 BCWABL transcripts
is also variable from case to case, ranging from 3.1 X I O 7
to 6.5 X IO4 molecules per pg of total RNA, but it shows a
rough correlation with the amounts of b2a2 or b3a2 transcripts expressed by the corresponding cases. Thus, the percentage of the eIa2 transcripts represents between 24% and
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1079
P190 IN CML
Table 2. Specific mRNA Levels (molecules per p g of total RNA) in
a Series of Samples From CML Patients
Sample
p210
mRNA
No.
1
2
3
4
5
6
7
Junction of
bcr/abl
b2/a2
b3/a2
b2/a2
b3/a2
b3/a2
b2/a2
b2/a2
p210 bcr/abl
mRNA
Molecules
2.6
1.3
3.3
5.0
1.5
4.9
7.8
x 10'
lo5
lo'
lo'
x lo5
x lo'
x
x
x
x 10'
p190 bcrlabl
mRNA
Molecules
1.3 x
1.8 x
3.1 x
2.1
29 x
6.5 x
1.624x
2.1
26x
lo'
lo'
lo3
10'
10'
10'
10'
p190 bcr/abl mRNA
Over Total bcrlabl
mRNA Molecules (96)
32
12
8
30
Each value is the mean between two replicates (500 and 250 ng of
total RNA). The mean ratio (expressed in percent) of the differences
between each pair of replicates and the corresponding mean values
was 16% for P210 transcripts and 19% for P190 transcripts.
32% of the total transcript in five of the seven cases, while
it is lower (8% and 12%) in the remaining two cases.
For two of the patients, a fresh peripheral blood sample,
containing at least 3 X IO' WBC, was available for analysis
of BCWABL hybrid proteins by Western blot analysis (Fig
3). In both cases the anti-ABL monoclonal antibody detected
three major bands: one with P145 mobility (as verified both
by comparison with proteins from WBC from a normal subject and from the size marker mobility curve), which corresponds to the normal ABL protein; and two bands whose
molecular weights, as calculated from the size marker mobility curve, were 185 and 210 kD corresponding tohybrid
BCWABL P190 and P210 proteins, respectively.
DISCUSSION
A relationship generally exists between the type of BCW
ABL rearrangement and the hematologic phenotype of Phpositive leukemias. The presence of the rearrangement that
gives origin to the P190 protein is almost constantly associated with an acute leukemia phenotype, mainly lymphoblastic."By contrast, although sporadic cases expressing only
P19OX." or bearing an alternative type of BCWABL junction" have been reported, CML patients generally show the
types of BCWABL rearrangement that give origin to P210.
C
1
2
Fig 3. Detection of both P210 and P190 in peripheral blood from
two chronicphase Ph+ CML patients (lanes 1 and 2) by Western
blotting. In lane C, as reference, isthe P145 ABL protein detected by
the same technique in peripheral blood from a normal subject. Each
lane was loaded with extract from 3 x lo7WBC, and the gel contained
molecular weight marker. Exposure time, 1 hour.
These observations would suggest a different biological activity of the different BCWABL proteins, and, as a consequence, a direct influence on the clinical and hematologic
phenotype. Indeed, several lines of in vivo and in vitro experimental evidence suggest that P190 is endowed with a higher
transforming activity than is P210.".'4
Here we describe that, in addition to the b2a2 or b3a2
types of junctions corresponding to the P210 proteins, virtually all CMLs at diagnosis bear also BCWABL transcripts
showing the ela2 type of junction, which corresponds to
the P190 protein. The simultaneous presence of BCWABL
transcripts able to codify for both P210 and P190 proteins
has been previously described to occur in sporadic cases of
Ph-positive acute leukemias"~" and of CML blastic crisis"
and ascribed to derive from a mechanism of alternative splicing of the BCWABL hybrid mRNA. This must also be the
mechanism responsible for the presence of ela2 transcripts
in chronic phase CML, a finding in accordance withthe
fact that the amount of the ela2 junction mRNA roughly
correlates with the total amount of BCWABL transcript present. However, the entity of the splicing must be variable in
different phases of the disease, because the presence of
mRNA with the ela2 junction is not constantly detectable
during blast crisis or in P~IO-ALLS,in which the total
amount of BCWABL transcripts is certainly higher than during chronic phase (data not shown).
The fact that the constant presence of ela2 BCWABL
transcripts in CML cases at diagnosis has never been previously detected is rather surprising. However, this is probably due to the fact that the PCR reaction devised to detect
the ela2 junction is not normally used in CML cases and
that, at least in our hands, this particular RT-PCR reaction
is less sensitive than that normally used to amplify the BCFU
ABL transcript with the b2a2 or b3a2 types of junction (see
Materials and Methods). Therefore, an exact measure of the
amounts of hybrid transcripts formed is essential to define
the relative ratio of the expression of different transcripts.
Moreover, many cases have probably been analyzed during
therapy, and our preliminary observations suggest that several types of treatment commonly usedin chronic phase
CML may affect the pattern of BCWABL expression.
With the quantitative RT-PCR assay described, we estimate that CML patients at diagnosis express about lo5molecules of P210 mRNA per microgram of total RNA, and that
the P190 mRNA is a significant proportion of the total BCFU
ABL hybrid transcripts (up to 25% to 30% in most cases).
Our results on the quantitation of P210 are consistent with
those of Cross et allxwho useda semicompetitive PCR assay,
and found 10' to lo6 molecules per microgram of total RNA
in four chronic phase CML patients.
The presence of the ela2 BCWABL transcript is likely to
result in a similar proportion of P190 protein, production.
Unfortunately, this cannot be easily assessed. In fact, it is
well known that, due to a low level of expression and to a
concomitant phosphotyrosine phosphatase and several proteolytic activities in the mature myeloid cells, the BCWABL
proteins are not easily detectable in CML patients during
the chronic phase of the disease." However, we optimized
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
SAGLIO ET AL
1080
a novel Western blot technique” to detect, with a high degree
of analytical sensitivity, intact high-molecular weight proteins from mature myeloid cells, and to show, usingthis
technique with an anti-ABL antibody for detection, the presence of significant amount of P190 BCWABL protein in two
patients.
At the moment, the biological significance of the P190
presence in CML patients at diagnosis is unclear. In qualitative terms, we have constantly found the presence of BCW
ABL transcript with the ela2 junction in all the CML patients
tested at diagnosis. However, in quantitative terms, we have
observed a wide degree of variability in its amount from
case to case, and this generally reflected an overall variability
in the total amount of BCWABL transcripts. Of course, a
higher number of cases need to be evaluated with the quantitive assay to establish an eventual correlation between the
total amount of BCWABL transcripts and the clinical and
hematologic data present at diagnosis. Preliminary results
seem to indicate that a correlation may exist. If confirmed,
these data will allow to establish an important prognostic
parameter in CML and willunderline the utility of a quantitative PCR analysis in the clinical management of CML patients.
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From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
1996 87: 1075-1080
Consistent amounts of acute leukemia-associated P190BCR/ABL
transcripts are expressed by chronic myelogenous leukemia patients
at diagnosis
G Saglio, F Pane, E Gottardi, F Frigeri, MR Buonaiuto, A Guerrasio, D de Micheli, A Parziale, MN
Fornaci, G Martinelli and F Salvatore
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