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BCR-ABL Protein Expression in Peripheral Blood Cells of Chronic
Myelogenous Leukemia Patients Undergoing Therapy
By Jie Qiang Guo, Jin Ying Lian, Yong Ming Xian, Ming-Sheng Lee, Albert B. Deisseroth, Sanford A. Stass,
Richard E. Champlin, Moshe Talpaz, Jean Y.J. Wang, and Ralph B. Arlinghaus
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with thePhiladelphia chromosome
(Phl) in more than 95% of these patients. The Ph' and the
resulting BCR-ABL fused genes are markers for this type of
leukemia. In CML, the product of the fused BCR-ABL gene
is typically a protein of approximately 2,000 amino acids
termed P210 BCR-ABL. We have developed an assay for the
BCR-ABL protein involving Western blotting of circulating
white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph'-positive leukemia
patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before
and after various treatments. In parallel to changes in percentages of Ph'-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein
expression in blood cells decreased or increased aspatients
entered remission or underwent relapse. Of interest, six Ph'negative CML patients were BCR-ABL protein-positive. All
except one had a rearrangement in the major breakpoint
cluster region and that patientexpressed P185 BCR-ABLand
not P210. Our results indicate that the BCR-ABL Western
blotting assayhasclinical applications for both diagnosis
and prospective evaluation of Ph'-positive and Ph'-negative
CML patients.
0 1994 by The American Society of Hematology.
C
of CML." Using this assay, we have also been able to detect
the P185 form of BCR-ABL protein in white blood cells
(WBC) from a patient in chronic phase of CML." Cells
from this patient lacked P210 BCR-ABL and lacked a DNA
rearrangement in the major breakpoint cluster region (BCR)
of the BCR gene. Furthermore, RNA extracted from this
patient contained only transcripts with the BCR exon 1 fused
to ABL exon 2.
We now report results of analyses of CML patients undergoing treatment, either chemotherapy or bone marrow transplantation. Of importance, six patients lacking a detectable
Phl at diagnosis were found to express BCR-ABL proteins
in their peripheral WBC.
HRONIC MYELOGENOUS leukemia (CML) is characterized most frequently by its association with an
abnormal chromosome 22, known as the Philadelphia chromosome (Ph').'.' It is estimated that at least 95% of CML
cases possess the Ph'.3 In CML this abnormal chromosome
fuses a central portion of the BCR gene to the second exon
of the ABL gene?5 The protein product produced by this
fusion is termed P210 BCR-ABL. In one form the protein
contains 927 amino acids encoded by the 5' portion of the
BCR gene fused to 1096 amino acids encoded by the ABL
gene. A second form fuses 902 amino acids of BCR fused
to the same ABL sequences. These two forms of BCR-ABL
protein together with alternate splicing patterns result from
the fusion of one of two small central exons termed either
b2 or b3 to the second ABL exon termed a2. RNA analyses
indicate that CML patients can have either form or both,
known as b2a2 or b3a2 junctions.6
Another form of the BCR-ABL protein is detected typically in Ph'-positive acute lymphocytic leukemia
This form fuses the relatively long first exon of BCR (426
amino acids) to ABL exon 2 (aQ9 This protein termed P190
or P185 BCR-ABL has a higher specific protein kinase activity than P210 BCR-ABL." P185 BCR-ABL also has more
neoplastic transformation activity than P210 as measured in
cell culture systems." Consistent with its increased potency,
P185 BCR-ABL expression is typically butnot always"
detected in Phl-positive ALL, which is a subgroup of ALL
with a poor p r o g n o s i ~ . ~ . ~ ~ " ~
One important goal in CML is to develop methods to
monitor the level of BCR-ABL proteins and their activity
during treatment. A first attempt to do this was accomplished
several years ago"
by
using an
in vitro kinase
to
measure the autophosphorylation activity of the activated
ABL tyrosine protein kinase present in these BCR-ABL proteins. Unfortunately, although useful for detecting active
BCR-ABL tyrosine kinase activity in terminal stage patients
(blast crisis), this assay is not useful in monitoring patients
in the early stage of this disease known as the chronic or
benign stage.15 Lysis of mature blood cells harboring the
BCR-ABL protein causes rapid destruction of this and other
proteins. A Western blotting assay was developed that allows
detection of BCR-ABL protein at early as well as late stages
Blood, Vol 83, No 12 (June 15), 1994: pp 3629-3637
MATERIALS AND METHODS
Patient samples. Western blotting assays for all patient samples
were performed in a BCR-ABL protein screening laboratory within
the Department of Molecular Pathology at The University of Texas
M.D.Anderson Cancer Center in Houston. Blood samples were
obtained from patients as part of a progradproject study sponsored
by The National Cancer Institute (NCI). All patients signed an appropriate informed consent form. Peripheral blood samples yielding at
least 2 X lo7 WBC were cryopreserved. Ph' percentages were obtained by cytogenetic analysis of bone marrow samples obtained on
the same day as the blood sample thus allowing comparison between
our results and Ph' chromosome percentage. Cytogenetic analyses
From the Department of Molecular Pathology, Divisionof Laboratory Medicine, and the Departments of Hematology, and Clinical
Investigation, Houston, T X ; and The Department of Biology, The
University of California at San Diego, La Jolla.
Submitted July 29, 1993; accepted February 10, 1994.
Supported by Grants No. CA49369 and CA16672 from the National Institutes of Health.
Address reprint requests to Ralph B. Arlinghaus, PhD, The University of Texas, M.D., Anderson Cancer Center, 1515 Holcombe
Blvd, Houston, TX 77030.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8312-0015$3.00/0
3629
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GUO ET AL
3630
were performed by the Division of Laboratory Medicine. CML and
ALL were diagnosed according to standard criteria. Normal WBC
were donated by volunteers from our laboratory.
Cell lines. The K562I9 (positive control), HL-60,2"and KG-12'
cell lines (negative controls) were grown inRPM1medium containing 10% fetal bovine serum. K562 cells expressing P210 BCRABL were derived from a CML patient in blast crisis." KG-l cells
were derived from an acute myelogenous leukemia patient lacking
BCR-ABL proteins.
8E9 antibody. The SE9 antibody was originally isolated by Richardson etIt
is a mouse monoclonal antibody directed toward
the SH2 region of the mouse gag-ABL protein.ln
Processing of patient cells. Peripheral bloodwas collected in
heparanized tubes atroom temperature. Bloodwas treated with a
mixture of protease inhibitors immediately after receiving the specimen.".'* Red blood cells were removed by two cycles of treatment
with NH4CI.I' Of interest, heparanized blood received 4 days after
being drawn from the patient gave informative results from both
Ph'-positive and negative leukemia patients, but a 1 to 2 day time
period is preferred to reduce protein degradation. After removal of
red blood cells, intact WBC were treated with diisopropyl fluorophosphate (DIFP), a very potent protease inhibitor as described."
The manufacturer's precautions were strictly adhered to when handling DIFP. WBC were stored immediately at -72°C.
Western blotting procedure. The BCR-ABL Western blotting
test was performed as described,"." butmodified to increase the
sensitivity. The more sensitive test involved two fundamental
changes. First the enhanced chemiluminescent (ECL) detection system was usedin place ofthe ["'I] protein A detection system.
Second, the backgrounds were dramatically reduced by substituting
powdered milk for bovine serum albumin (BSA) in our blocking
solutions. The detailed procedure is as follows: Western blotting
was performed withan anti-ABL (8E9) monoclonal antibody as
described previously. Briefly, frozen WBC were lysed in boiling
sodium dodecyl sulfate (SDS) sample buffer for 5 to 7 minutes, and
the lysate was clarified by centrifugation. Aliquots of the extracts
corresponding to IO' cells were applied to each gel lane. Samples
were electrophoresed through 6.5% polyacrylamide gels, the gels
were electroblotted at 4°C overnight and transferred to lmmobilon
P filters (Millipore, Bedford, MA).
The ECL Western blotting detection system was used to probe
for BCR-ABL protein according to themanufacturer's protocol (Amersham, Arlington Heights, IL). Filters were preblocked by washing
with 10% non-fatmilk (NFM) in Tris-buffered saline-Tween 20
(TBS-T) buffer (20 mmol/L Tris base, 137 mmol/L NaCI, 0.0038
N HCI) for 2 hours and then incubated with 1:15,OOO to 20,000
dilution of SE9 in5% NFM TBS-T buffer overnight at room temperature. The filters were then incubated with a 1:3,000 to3,500 dilution
of horseradish peroxidase (HRP)-labeled sheep anti-mouse IgG
(Amersham, Cat. No. NA 9310) for 2 hours. The filters were mixed
with ECL reagents and exposed to X-ray film for I to 10 minutes.
Where indicated, the intensities of P210 BCR-ABL, the lower molecular weight BCR-ABL proteins, and P145 ABL within appropriate
autoradiograms were scanned with a soft laser scanning densitometer
(Zeineh Biomed Instruments, Inc. Fullerton, CA).
Southern blotting and polymerase chain reaction (PCR) assays.
Sample DNA was extracted, digested with restriction enzymes, separated on agarose gels, blotted on to nylon filters, and probed with a
universal BCR probe (UBCR) (Transprobe-l); Oncogene Sciences,
Manhasset, NY) and a 3' probe (human BCR DNA Probe-l, Oncogene Sciences), as described." PCR analyses was performed as described."
RESULTS
Increased sensitivity of the ECL detection system. After
a number of unsuccessful attempts to improve the sensitivity
A
1 2 3 4 5 6 7 8 9 101112
P2 10-
-200
P145-
0 " ' " " " "
0 100 200 300 400 500 800 700 800 900 1000
K582 C E U NUMBER
C
0-35
c
50.051
/
ow.
0
m
'
10
I
20
I
30
40
50
80
K582 CELL NUMBER (x10 -3 )
Fig 1. ECL Western blotting procedure for detection ofBCR-ABL
proteins. (A) Various mixtures of cell extracts from K562 cells and
KG-l cells were analyzed.Each lane was loaded with cell extract
from 10' cells. The amount of K562 + KG-l cells used in each lane is
as follows: lane 1, 1 x 10'K562 cells; lane 2, 5 x lo5 K562 cells + 5
x lo5 KG-l cells; lane 3,2.5 x lo5 K562 cells + 7.5 x 10'KG-l cells;
lane 4, 1.2 x lo5; lane 5, 6 x lo'; lane 6, 3 x lo'; lane 7, 1.5 x lo';
lane 8, 7 x 10'; lane 9, 3.5 x 10'; lane 10, 1.7 x 10'; lane 11, 8.5 x
10': lane 12, 1 x 10' KG-l cells. The samples were analyzed on 6.5%
gel, molecular weight marker were included. Exposuretime: 8 minutes. BCR-ABL/ABL protein ratios were plotted versus the number
of K562 cells ranging from 6 x 10' to 1 x 10' cells in a mixture with
KG-l cells, totaling 1 x 10' cells (B) and 0 to 6 x 10'K562 cells IC).
of the test by various methods, we found that theECL system
could improve the sensitivity more than 100-fold. A typical
example is shown in Fig 1A. In this experiment, increasing
concentrations of K562 cells were added to KG-I cells so
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BCR-ABL PROTEIN EXPRESSION INCML
3631
PATIENTS
1 2 3 4 5 6 7 8 9101112
P210P145-
-200
2
"
W -95
Fig 2. Sensitivity of the ECL Western blotting procedure for BCR-ABL protein with extracts from two
chronic phase CML patients. Mixtures
of cell extracts
of 100% Ph'-positive chronic phase CML samples (patients J.S. and 0,s.)mixed with normalWBCs. Each
lane was loaded with extract from
lo' cells. Amount
of CML cell + normal WBC in each lane is as follows:
lane 1, 1 x lo' CML cells; lane 2, 5 x 10' CML cells
+ 5 x 10' normal cells; lane 3, 2.5 x 10' CML cells +
7.5 x 10' normal cells; lane 4, 1.25 x 10' CML cells;
lane 5,6 x lo'; lane 6,3 x lo'; lane 7,1.5 x lo5;lane
8, 7 x lo'; lane 9, 3.7 x lo'; lane 10, 1.8
x lo'; lane
11,9 x 10'; lane 12,l x 10' normal WBCs. The samples were analyzed on 6.5% gel, molecular weight
markers were included. Exposure time: 12 minutes.
1
2
3 4 5 6 7 8 9 1 0 1 1 12
1
P210P145-
that in all cases the total number of cells was kept constant
at IO'. The results show that approximately 2,000 to 3.000
K562 cells gave a detectable BCR-ABL signal in a mixture
of 10" KG-l cells (ie, 0.3%) (Fig IA, lanes 9 and 10). About
3.000 K562 cells could also be detected in a mixture of 10
million KG-l cells (results not shown). A plot of the BCRABL protein response versus the number of K562 cells demonstrated that at either low or high concentrations. the relationship in this more sensitive assay was linear (Figs I B and
C) and very significant:Fig IB, r = 0.99. P < 0.01: and
Fig IC. r = 0.98. P < 0.01. In these experiments to account
for possible differences in cell number and blotting technique
(ie, band intensity). the ratios of BCR-ABL to ABL proteins
were plotted versus the K562 cell number.
Similar results were obtained with mixtures of WBC from
CMLchronic phasepatients that were 100% Ph'-positive
when mixed with normal WBC (Fig 2). These results clearly
showed that with cells from one patient (J.S.).as few as I .8
X IO4 scored positive for BCR-ABL protein expression (Fig
2, lane IO). In other studies, there was a positive correlation
between the ratio of BCR-ABL to ABL proteins in the peripheral blood sampleand the percentage of Phi-positive
cells in the bone marrow (manuscript in prep).
BCR-ABL protein e.rpression in inclividrtrrlpccticwtsrrrnples. Using the more sensitive assay. Western blot analysis
has been performed on more than 250 blood samples from
Ph'-positive CML patients at various stages of treatment.
many of which had cytogenetic analyses performed on bone
marrow cells on the same day. Typically, the relative intensity of the BCR-ABL protein bands compared with the ABL
protein band increased or decreased as the Ph' percentage
in the marrow increased or decreased. As an example. we
show Western blotting data on peripheral WBC together with
cytogenetic results of nine such patients a s they underwent
therapy. Figure 3A shows blots of a patient (patient M.J.)
who was treated with interferon and homoharringtonine simultaneously. PCR analyses indicated that M.J. had a W a 2
BCR-ABL junction. After a brief cytogenetic remission in
May of 1992. the patient relapsed because the therapy was
stopped due to severe anemia. Thepatient's peripheral blood
cells showed amarkedincrease
in the BCR-ABLprotein,
seen a s early as November of 1992. Patient J.J. (b3/a2) underwent 24 months of homoharringtonine/interferon treatment. Cytogenetic analyseson marrow indicated that diploid
cells were predominant in this patient (Nov 1993). The blood
sample analyses indicate a low level of BCR-ABL protein
throughout the course of treatment. consistent with the cytogenetic results (Fig 3A).
Blood samples from twoother patientswereanalyzed
during the course of their chemotherapy. Figure 3B shows
Western blot resultsfrompatientJ.R.
(b3/a2)who was
treated with interferon alone. The patient entered into remission a s of January of 1992. as indicated by cytogenetic studies where the bone marrow contained 40% Ph'-positive cells
(6 of 15 cells analyzed). BCR-ABL protein expression decreased dramatically at that time and remained undetectable
o n two other occasions in 1993. Figure 3B also shows the
results of patient S.F.. who was resistant to homo-harringtonine treatment but after changing to interferon. responded
well based on BCR-ABL protein analysis in March of 1993.
At that time the bone marrow showed 30% of the cells to
be Ph'-positive. In September of 1993. the patient lacked
detectable BCR-ABL in peripheral blood cells and contained
one Ph'-positive metaphase in 90 analyzed.
Figure 4 shows BCR-ABL Western blotting results of five
patients undergoing bone marrow transplantation
receiving
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GUO ET AL
3632
A
P.MJ
U
P.JJ
P210-
-200
P 145-
I"-"
8/8/97 9/5/91 1/3/92 4/24/926/28/9310/5/9212/10/924/1/93 4/29/93 6/1/936/30/93((/30/90
ph' 96%
NO
10%
12%
ND
10%
ND
10%
ND
ND
ND
5%
(7/12/91)
(1131192)
(10/30/92)
(11/2/93)
B
P.JR
P210-
-
-200
-
r
v
U
Ph'
112219l
100%
7124191
90%
1!30/92
40%
5/21/93
9/16/93
0%
0%
(12/ 13/90)
P.SF
p210P145-
FE
8/7/92
Ph'
100%
-
-3"m
1018192
1111Ql92
ND
-200
3/4/93
9/24/93
50%
30%
5%
Fig 3. Changes in BCR-ABL protein expression in
peripheral blood cellsfollowing
chemotherapy. BCRABL Western blotting analyses and cytogenetic tests
were performed on blood and bone marrow samples, respectively, from fourCML patients atvarious
stages of treatment with chemotherapy. Western
blot analyses were performed ongels in which positive and negative control cell lines were analyzed at
withthe
along
same time
molecular weight markers.
Dates of BCR-ABLWestern blotting are shown at the
bottom of gel lane; results (Ph'%) and dates of bone
marrow cytogenetic tests also are shown
bot- at the
tom of eachlane, if performed. (AI Patient M.J. initially showed partial
cytogenetic remission (5/26/92)
and thenrelapsed as is evident in the11/12/92sample. Patient J.J. shows decreasing Ph' percentage
from 96%t o 10% after undergoing about 20 months
HHT/IFN therapy. (B) Patient JR. was treated with
interferon. Patient SF.wasinitiallytreatedwith
Homo-harringtonine
laterand
with interferon.
OTEIN
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IN CML PATIENTS
BCR-ABL
A
P.TS
rALL0
3633
BMT(10/91)
B
C
P.KH
P210-3
--
P.AH
r ALLO BMT(2/26/93)
,-
4
-200
P210-
W
Pl45-1
P1452/1/93
2/16/93
4/19/93
9/1/93
0%
0%
(114/93)
(1/4/93)
(5128193)
m95%95%
P.DS
ALLO BMT(10/90)
ALLO BMT(4/93)
_.
P.SH
3
ALLO W(10190)
-200
200
~
pH
3/15/90
8/28/91
100%
3116192
M)
0%
4/8/83
94%
6/7/93)
Fig 4. Changes in BCR-ABL protein expression in peripheral blood followingallogeneic bone marrow transplantation. Bloodsamples and
bone marrow were taken for
BCR-ABL protein analyses and Ph' measurements at various pointsbefore and after transplant. (A) Patient T.S.
cytogenetics indicate this patientt o be
received an allotransplant in October of 1991. BCR-ABL Western blotting patterns and bone marrow
in complete remission through May of1993. (B)Patient K.H. underwent an allotransplant in February of 1993. Of interest, although marrow
cytogenetics failedt o detect Ph', low levels of BCR-ABL protein were expressed in this patient. Patient D.S. underwent an allotransplant in
April, 1993. (C) Patient A.H. underwent an allotransplant in October of 1990 and had a relapse some time before March of 1992. Patient S.H.
underwent an allotransplant in October of 1990 and relapsed before April of 1993. The 3/15/90 assay of patient S.H. was done by the ['=l1
procedure.'' All analyses were performed ona gel with positive and negative control cell
lines and molecular weight markers. Dates of BCRABL Western blot analyses are shown at the bottom ofeach gel lane; dates of cytogenetic bone marrow analyses are shown at the bottom
of each lane.
cells from a healthy donor. Patient T.S. (b2la2) (Fig 4A) had
high levels of BCR-ABL proteins in his blood cells before
the transplant. After transplant, patient T.S. containqd no
detectable BCR-ABL protein as analyzed on six occasions
through May of 1993, but was consistently b2la2 positive
after transplant except on May 15, 1992 and May 28, 1993,
which were negative by PCR. There was an excellent correlation with cytogenetic studies, as the patient was 80% Ph'positive before transplant (16 of 20 cells positive for the
Ph'), but lacked the Ph' after transplant. Patient K.H. (b2/
a 2 ) (Fig 4B), although lacking detectable BCR-ABL protein
and containing only diploid cells in the marrow shortly after
receiving an allotransplant, was weakly positive for BCRABL protein in peripheral blood cells at about 7 months
posttransplant. Of importance, cytogenetic analysis failed to
detect a Ph'-positive cell in the bone marrow of this patient
analyzed on the same day. Of interest, patient K.H. had the
b2la2 junction on June 30,1993.Patient D.S. (b2/a2) received an allogeneic bonemarrow transplant in April of
1993. Before that, patient D.S. was strongly BCR-ABL pro-
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GUO ET AL
3634
4
a
0
a9
J
a
p210-
?$
--
"
B3
7
7
a
- . . ".
a
-200
-95
Fig 5. BCR-ABL protein analyses ofperipheral blood cells from CML, ALL, and non-CML disease patients. Peripheral blood cells from patients
were harvested as usual and processed for Western blotting. P.7, ALL patient expressing P210 BCR-ABL; P.8, two samples of the same Ph'positive CML patient from peripheral blood (P) or bone marrow (B), respectively; P.9, a Ph'-positive ALL patient expressing P185 BCR-ABL;
P.10,chronic lymphomatic leukemia (CLL) patient; P.ll, lymphoma patient; P.12, Down's syndrome; P.13, vasospastic angina. All rasulto
obtained with the ECL assay. Every assay contained K562 cells and KG-l cells as positive and negative controls, respectively.
tein positive and 100% Ph'-positive (Fig 4B). After the transplant, the patient lacked detectable BCR-ABL proteinin
blood cells and no detectable Phl in marrow cells. Patient
A.H. (b2/a2) (Fig 4C) underwent an allotransplant in October
of 1990. Western blot analyses on three occasions in 1991
did not detect BCR-ABL protein expression in circulating
blood cells, although the patient was b21a2 positive on September 13, 1991. After 17 months, this patient relapsed as
indicated in the Western blot of March of 1992. This was
confirmed by PCR as the patient was b21a2 positive. Subsequent analyses through June of 1993 showed a high level
of BCR-ABL protein in blood cells and 100% Ph'-positive
marrow cells in samples analyzed on the same day. Patient
S.H. (b2la2) (Fig 4C) showed a relapse following an allotransplant some 30 months earlier. This patient had the b2/
a2 junction before and after transplant. BCR-ABL proteins
were present in high amounts in his peripheral blood cells
at that point and bone marrow cytogenetic analyses showed
18 of 19 cells to be Phl-positive. In summary, all nine patients showed a strong correlation between cytogenetic findings within marrow cells and the level BCR-ABL protein
expression in peripheral blood cells. The use of P145 ABL
as an internal control clearly showed a diminution of the
intensity of the BCR-ABL bands compared with the P145
ABL band as the percentage of Ph'-positive cells decreased.
Moreover, as patients underwent cytogenetic relapse, the
intensity of BCR-ABL bands showed a corresponding increase relative to the P145 ABL band.
Our results show that we can detect the BCR-ABL protein
both in peripheral blood samples and bone marrow samples
(Fig 5 , patient 8, P & B). Also, our Western blot assay not
only detected P210 BCR-ABL in Ph'-positive CML patients,
but also detected the BCR-ABL protein in Ph'-positive ALL,
either P210 BCR-ABL (patient no. 7) or P185/P190 BCRABL (patient no. 9).
Using the more sensitive assay, we also analyzed clinical
samples from patients with various other medical problems
(Fig 5 ) . Analysis of a large number of blood samples (more
than 95 at this point) have not yet detected a false positive,
as BCR-ABL proteins were not detected in these samples.
Similarly, in more than 231 CML patients (357 blood samples) that were 100% Ph'-positive, we have not had a false
negative (manuscript in preparation).
BCR-ABL protein detection in Ph'-negative patients.
We also analyzed blood samples from six patients that were
found to lack a detectable Ph' at diagnosis of disease (originally Ph'-negative) (Fig 6A-C). Western blotting results detected P210 BCR-ABL in five of these patients. Patients J.N.
(b2/a2), T.D. (b3/a2), G.S., and J.L. (b2/a2) were BCR-ABL
protein-positive in morethan one blood sample taken on
different dates (Fig 6A and B). Of interest, blood cells from
patient J.M.lacked P210 BCR-ABL expression but contained P185 BCR-ABL (Fig 6C). To verify the presence of
a rearranged BCR gene, Southern blotting using a bcr probe
was performed on these six patients. The results indicated
that five of these BCR-ABL protein-positive patients had a
rearrangement in the major bcr site and one patient (J.M.)
lacked detectable major bcr rearrangement (germ line) (Table l). The finding that patient J.M. expressed P185 and not
P210 BCR-ABL is consistent with the lack of rearrangement
in the major bcr region. Presumably, this patient has a break
in the first intron of the bcr gene. A seventh patient (P.H.)
also diagnosed as Ph'-negative, bcr-positive, was not informative because her blood cells gave only a faint P145 ABL
signal (not shown). These findings emphasize the medical
utility of the BCR-ABL Western blotting test in Phl associated leukemias. Our results indicate that it is more reliable
for diagnosing these leukemias than either classical cytogenetics or the widely used bcr Southern blotting assay.
DISCUSSION
The results presented here indicate that the level of BCRABLprotein expression in peripheral WBCmimicsthe
changes in percentages of Ph'-positive cells in bone marrow
cell populations. An improved assay was developed that can
detect BCR-ABL protein expression in mixed populations
of normal and leukemic cells. BCR-ABL expression can be
detected in mixtures of BCR-ABL negative cells containing
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BCR-ABL PROTEIN EXPRESSION IN CML PATIENTS
3635
A
P.TD
P.JN
.L
2/22/93
7120193
P.GS
I
*
12/7/90 12/28/90
2119/93
81 13/93
B
P.JL
C
P.JB
Fig 6. Detection of BCR-ABL
protein in peripheral blood cells
from Ph'-negative CML patients.
(A) Patient J.N., patient T.D., and
patient G.S.; (B) patient J.L.; (C)
patient J.B. and patient J.M.;
samples were analyzed by BCRABL Western blotting.Patient
J.M. was assayed by the ['2511
procedure." As in Figs 3 and 4,
appropriate positiveand negative controls as well as molecular weight markers were run on
the same gel in each case.
P.JM
K562
as fewas 2,000 to 3,000 K562 (Fig 1A). Moreover, the
intensity of the P210 BCR-ABL band is proportional to the
number of BCR-ABL cells in the mixture even at very low
levels (Fig 1B and C). In studies carried out with 100% Ph'positive chronic phase CML patients, BCR-ABL protein expression can be detected in mixtures of WBC that contain as
fewas 0.2% to 0.4% BCR-ABLexpressingleukemiccells
(approximately 20,000 to 40,000 leukemic cells in a mixture
of 10 million cells) (Fig 2). Thus, in addition to its medical
utility for diagnosis, the BCR-ABL protein test performed on
peripheral blood provides a convenient way to monitor CML
patients as they enter remission or as they undergo relapse.
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3636
GUO ET AL
Table 1. Ph’-Negative CML Patients Express BCR-ABL
Patient
J.N.
T.D.
G.S.
J.L.
J.B.
J.M.
BCR-ABL Protein
bcr Analyses
+
+
i
+
+
i
+
i
+
+
t*
-
* No P210 BCR-ABL, but cells express P185 BCR-ABL. These results
were obtained by [‘2511proteinA Western blotting assay.”
However, it is clear that even with the improved BCRABL test, mixtures of lo7 circulating WBC containing significantly less than lo4 cells leukemic cells will score negative. Moreover, in CML patients who are in remission and
have less than 20% Phl-positive bone marrow cells, BCRABL protein detection in circulating blood cells may in some
cases fall below the level of detection. Of interest, patients
that lack detectable Ph’-positive cells in bone marrow cytogenetic assays may in some cases score positive for BCRABL protein expression in blood cells (Fig 4B, patient K.H.).
Thus, cytogenetic bone marrow analyses and BCR-ABL protein expression in circulating blood cells maynot always
agree in CML patients with less than 20% Ph’-positive bone
marrow cells (manuscript in preparation). Nevertheless, the
assay should be quite useful to physicians, as reduction of
leukemic cells by more than two logs can be discerned by
assay of peripheral WBC from the patient. Although further
studies are needed to determine the percentage of false-positives, our experience with more than 350 patients with various types of leukemias, lymphomas, and other diseases
shows a perfect correlation between diagnosis of CML and
BCR-ABL protein expression (manuscript in preparation).
Studies of nine CML patients at various stages of treatment andremission andor relapse showed a clear correlation
between the patients cytogenetic analyses for the Ph’ in marrow cells and BCR-ABL protein levels in circulating WBC.
Examples of these types of analyses are shown in Figs 3
and 4. The BCR-ABL Western blot test was also shown to
detect both forms of BCR-ABL proteins, either P210 or P185
BCR-ABL, in Phl-positive ALL patients (Fig 5). A study is
underway to establish firmly the test’s usefulness for diagnosing Ph’-positive ALL (H. Kantarjian, J.Q. Guo, and Arlinghaus, in prep).
Also of importance, analysis of blood samples from six
patients with a history of being Phl-negative revealed that
they were BCR-ABL protein-positive despite being Ph’-negative (Table 1, Fig 6). One of these patients lacking the Phl
was also bcr negative, whereas the other five were bcr positive. Of interest, this patient expressed P185 BCR-ABL and
not P210 BCR-ABL, suggesting the presence of a BCR exon
1: ab1 2 junction. A similar phenotype has been previously
reported by us in a chronic phase CML patient.” Further
studies are in progress on cells from this Ph’-negative patient
to identify the type of junction.
Considering the 100%correlation with the presence of the
Phl in newly diagnosed CML patients and the lack of fdsepositives in patients with other diseases (manuscript in prep-
aration), the BCR-ABL Western blotting assay has value as
a relatively easy means of assessing whether or not patients
with leukocytosis andor myeloproliferative syndrome have
Ph’-positive leukemia.
The sensitivity of the BCR-ABL Western blotting assay
does not compare with that of the polymerase chain reaction
(PCR) methodology, as indicated by comparing PCR results
with BCR-ABL Western blotting data on patients T.S., A.H.,
and S.H. (Fig 4). However, the detection of the BCR-ABL
gene product in peripheral blood cells has importance based
on two considerations. First, it indicates that the patient’s
cells are expressing the BCR-ABL gene product (which may
or may not be kinase active). Second, detection of leukemic
cells in the peripheral blood indicates that the leukemic clone
is actively dividing. Further, studies are needed to determine
whether low levels of detectable BCR-ABL protein in circulating blood cells has clinical prognostic significance in patients who are in remission.
ACKNOWLEDGMENT
RBA is the recipient of the Hubert L. Stringer Chair in Cancer
Research. The ABL monoclonal antibody was either produced in
tissue culture or by production of ascites in mice maintained in
facilities approved by the American Association for Accreditation
of Laboratory Animal Care, and in accordance with current United
States Department of Agriculture, Department of Health and Human
Services, and National Institutes of Health Regulations and Standards. We thank Leslie Calvert for obtaining patient samples and
providing patient information, Dr Hagop Kantarjian for helpful comments, and Tammy Trlicek for assistance in manuscript preparation.
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1994 83: 3629-3637
BCR-ABL protein expression in peripheral blood cells of chronic
myelogenous leukemia patients undergoing therapy
JQ Guo, JY Lian, YM Xian, MS Lee, AB Deisseroth, SA Stass, RE Champlin, M Talpaz, JY Wang
and RB Arlinghaus
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