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From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1991 77: 238-242
Hybridization protection assay: a rapid, sensitive, and specific method
for detection of Philadelphia chromosome-positive leukemias
K Dhingra, M Talpaz, MG Riggs, PS Eastman, T Zipf, S Ku and R Kurzrock
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RAPID COMMUNICATION
Hybridization Protection Assay: A Rapid, Sensitive, and Specific Method for
Detection of Philadelphia Chromosome-Positive Leukemias
By Kapil Dhingra, Moshe Talpaz, Michael G. Riggs, P. Scott Eastman, Theodore Zipf, Stella Ku, and Razelle Kurzrock
The Philadelphia (Ph’) chromosome is present in greater than
90% of patients with chronic myelogenous leukemia (CML)
and in 2% t o 20% of those with acute leukemias, for which it
is an important prognostic marker too. The chimeric BCRABL mRNAs resulting from the translocation encode either a
210-Kd or a 190-Kd protein. The techniques used t o detect
Ph’ chromosome include karyotyping, Southern analysis t o
demonstrate bcr rearrangement, and polymerase chain reaction t o amplify the BCR-ABL transcripts. However, the routine performance of these methods by clinical laboratories is
cumbersome, time consuming, and exposes laboratory personnel t o radioisotopes. We describe here the clinical application of a new method, the hybridization protection assay
(HPA), which uses chemiluminescent acridinium-esterlabeled probes in conjunction with PCR for detection of the
amplified BCR-ABL sequences. The method is sensitive,
specific, and can reliably distinguish between the transcripts
In contrast t o the 2 days or
for P190”R-ABLand P21OBCRpgL.
longer required for conventional hybridization, HPA analysis
can be completed in less than 30 minutes. We have successfully used this method t o analyze 60 leukemia samples (34
from Ph’-negative acute leukemias; 6 from Ph’-positive acute
leukemias; and 20 from CML) with complete correlation (of
BCR-ABL positivity or negativity) with the results of karyotype or Southern Blot analysis of genomic DNA for bcr
rearrangement. Therefore, the HPA, in conjunction with PCR,
appears t o provide a rapid and reliable test for the diagnosis
of Ph‘-positivity.
0 1991by The American Society of Hematology.
THE
70% of their Ph’-negative counterparts are alive at 5 years.
Similarly, in adults with this disorder, the median remission
duration is less than 1 year. Therefore, alternative treatment strategies have been advocated for patients with
Phl-positive acute leukemias.I6Hence, it is desirable to have
a method that allows rapid and easy diagnosis of Ph’
translocation, both in acute leukemia and CML.
Amplification of the chimeric BCR-ABL transcripts by
PCR has been used to diagnose Ph’ positivity, to detect
minimal residual disease in patients with CML, and to study
the presence and significance of alternative splicing of
BCR-ABL mRNA.I7 However, the technique, as currently
used in most laboratories, is time consuming and requires
radioisotopes. We describe here the clinical application of a
hybridization technique involving acridinium-ester-labeled
oligonucleotide probes that is sensitive, specific, and does
not require the use of radioisotopes. This technique, in
conjunction with PCR, allows same-day detection of BCRABL positivity from small amounts of peripheral blood.
PHILADELPHIA (Ph’) translocation [t(9;22) (q34;
qll)], a hallmark of chronic myelogenous leukemia
(CML)’-*results from a break within a 5.8-kb region (the
breakpoint cluster region or bcr) in the central part of the
BCR gene on chromosome 2Z3and leads to juxtapositioning
of theABL oncogene (chromosome 9) with the BCR gene:,’
The resulting hybrid BCR-ABL mRNA codes for a tumorspecific 210-Kd protein6 with enhanced tyrosine kinase
enzymatic a~tivity.~,’
At least two different species of this
hybrid mRNA exist depending on whether exon 2 or exon 3
of bcr is spliced to exon 2 of the ABL gene, thereby giving
rise to two closely related 210-Kd proteins? In contrast, in
about half the cases of Ph’-positive acute leukemias, the Ph’
chromosome, although cytogenetically indistinguishable
from that in patients with CML, results from a breakpoint
in the first intron of the BCR gene (proximal to the bcr)‘0,1’2’2
and leads to the production of a truncated BCR-ABL
transcript encoding a 190-Kd protein.”’14 The Ph’ chromosome is discernable in approximately 20% of adults with
acute lymphoblastic leukemia (ALL),” 2% of adults with
acute myeloblastic leukemia (AML), and 5% of children
with ALL.’6 It is an important prognostic marker for both
adult and pediatric ALL. Less than 30% of children with
Phl-positive ALL are long-term survivors,’6whereas 60% to
From the Department of Clinical Immunology and Biological
Therapy, and the Department of Pediatrics, The University of Texas
M.D. Anderson Cancer Center, Houston; and Gen-Probe, Inc, San
Diego, CA.
Submitted September IO, 1990; accepted October 29, 1990.
Address reprint requests to Kapil Dhingra, MD, Department of
Clinical Immunology and Biological Therapy, Box 41, The University
of T m s M.D. Anderson Cancer Center, 1515 Holcombe Blvd,
Houston, TX 77030.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971l91/7702-0021$3.00/0
238
MATERIALS AND METHODS
Patients. Blood samples were collected from patients with
diagnosis of CML (chronic phase or blast crisis), or acute leukemia
(both Ph’-positive and Ph’-negative). Informed consent was obtained from all patients before venesection.
Sample preparation and amplification. Total cellular RNA was
extracted by previously described methods.” RNA from K562 cells,
an erythroid blast crisis cell line, and RNA from patient W, a
Ph’-positive CML patient, were used as positive controls for
mRNA containing bcr exon 3-ABL exon III9 and bcr exon 2-ABL
exon I1 junctions: respectively. The ALL-1 cell line (kindly
provided by Dr G. Rovera, Wistar Institute) was used as a positive
control for the transcript encoding P19@CR-ABL.”
HL-60 (a promyelocytic leukemia cell line) and normal human endometrial RNA
were used as negative controls. One microgram of total RNA from
cell lines or Xothof the total amount of RNA from 50 to 200 million
white blood cells (WBCs) from patient samples was used for
amplification reactions. The amplification method and the primers
have been previously described.” Amplification was performed for
40 cycles.
Blood, Vol77, No 2 (January 15). 1991: pp 238-242
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
BCR-ABL HYBRIDIZATION PROTECTIONASSAY
Because contamination has proven to be a significant problem in
some laboratories using this exquisitely sensitive technique, we
took the following precautions to ensure the accuracy of our
results: (1) the thermal cycler was kept in a separate laboratory,
away from the room where cell collection, RNA processing, and
cDNA synthesis was performed; (2) no amplified samples were
allowed to be brought back into the room where RNA processing
was performed; (3) at least one negative control was run for each
experiment; and (4) samples on each patient were run on at least
two different occasions.
Hybridizationprotection assay (HPA). Acridinium-ester-labeled
oligonucleotides complementary to the BCR-ABL junction sequences were synthesized by Gen-Probe, Inc (San Diego, CA). The
basic methodology for preparation of these probes has been
described previously?' The chemical labeling of the DNA probes
with acridinium-ester was achieved by reacting alkylamine linkerarms, which were introduced during DNA synthesis, and an
N-hydroxysuccinimide ester of a methyl acridinium phenyl ester.
The bcr2-ABLII probe was a 28mer with 22 bases in bcr exon 2 and
the bcr3-ABLII probe was a 25mer with 11 bases in bcr exon 3.
These probes were used to detect transcripts encoding P2108CR.ABL.
The BCRI-ABLII probe was a 26mer with 17 bases in BCRI and
was used to detect transcripts coding for P190BCR-ABL.
For hybridization, 10 pL of the amplified product was diluted to 50 pL in a 12 X
75-mm polypropylene tube. The sample was heated to 95°C for 3
minutes to denature the DNA and quick chilled on ice. Fifty
microliters of the hybridization solution containing 0.05 pm of the
probe was added to this (the 2X hybridization solution is a 0.1
mol/L lithium succinate buffer, pH 4.7, containing 20% lithium
lauryl sulfate, 1.2 m o m lithium chloride, 20 mmol/LEDTA, and 20
mmol/L of [ethylenebis(oxyethylenitrilo)]tetraacetic acid [EGTA]).
The sample was lightly vortexed, incubated at 60°C for 10 minutes,
and allowed to cool at room temperature for 1minute. Differential
hydrolysis of the bound versus free probe was performed by the
addition of 300 p L of hydrolysis buffer (0.6 m o m sodium tetraborate buffer, containing 10 mL of Triton X-100 surfactant [Sigma, St
Louis, MO] per liter, at pH 8.5) and incubating at 60°C for 6
minutes. After the sample had cooled at room temperature for a
few minutes, chemiluminescence was measured in a Leader I
luminometer (Gen-Probe, Inc) using an automated reagentinjection method involving two detection reagents. Injection of 200
pL of Detection Reagent I (0.1% H,O,, vol/vol; 1 mmol/L nitric
acid) was followed, after a 1-second delay, by injection of 200 FL of
Detection Reagent I1 (1 mol/L NaOH). The resulting chemiluminescence was integrated for 2 seconds and the reading expressed in
relative light units (RLUs). All these steps were performed in a
single 12 x 75-mm polypropylene tube.
Southem hybridization. To verify the results obtained by HPA,
conventional Southern blotting and hybridization of all amplified
samples was performed. Ten microliters of the amplified product
was run on 3% Nusieve/l% Seakem (FMC, Rockland, ME)
composite gels, transferred ovemight to Genescreen Plus membrane (New England, Nuclear, Boston, MA), and baked at 80°C for
2 hours. Oligonucleotide probes complementary to the junctional
BCR-ABL sequence^'^ were 5' end-labeled with 32Pand hybridization performed overnight using hybridization buffer with (for
bcr3-ABLII and BCRI-ABLII) or without (for bcr2-ABLII probe)
formamide. The membranes were washed as recommended by the
manufacturer and exposed to Kodak XAR film (Eastman Kodak
Co, Rochester, NY) for 3 to 48 hours. The bcr3-ABLII and
bcr2-ABLII probes detect 200-bp and 125-bp long amplification
products, respectively, while the BCRI-ABLII probe detects a
307-bp product.
239
RESULTS
To determine the time of differential hydrolysis that will
maximize the chemiluminescence from hybridized probe
while reducing the signal from unhybridized probe to a
minimum, hybridization reactions were performed in the
presence or absence of the target. The hybridization mixtures were then incubated with the differential hydrolysis
buffer at 60°C for various lengths of time and the residual
chemiluminescence measured. The results of such an analysis for the bcr3-ABLII probe are presented in Fig 1. The
half-life of the hybridized probe was approximately 9
minutes while that of the free probe was 19 seconds. Thus,
the TIn of the hybridized probe compared with the free
probe results in a differential hydrolysis ratio of 28. Similarly, the bcr2-ABLII and the BCRI-ABLII probes both
demonstrated differential hydrolysis ratios of 30. Accordingly, a 6-minute differential hydrolysis step was chosen.
In our initial experiments we attempted to establish the
sensitivity and specificity of the amplification process and
detection of amplified products by HPA. These results are
shown in Fig. 2. Starting with 1 pg of total RNA from the
K562 or the ALL-1 cells, serial 10-fold dilutions were
performed with normal human endometrial RNA. After 40
cycles of amplification, the BCR-ABL message could be
detected at a dilution of 1:106 and 1:104 with K562 and
ALL-1 RNA, respectively. Because one-tenth of the final
amplification product was used for analysis, this represents
the equivalent of total RNA from "th of a cell and one
cell, respectively (at 10 pgkell). The sensitivity of detection
of chimeric transcripts with HPA was identical to that on
Southern blots and there was no cross-reactivity between
the probes detecting P190BcR-ABL
and P210BCR-ABL.
Subsequently, we used HPA to analyze samples from
patients with CML and various types of acute leukemias.
These included 20 cases of childhood ALL (19,Ph'-
0
h
dp
b
4
0
10
20
30
40
Time (minutes)
Fig 1. Kinetic analysis of ester hydrolysis rates of hybridized and
free acridinium-ester-labeled bcr3-ABLII probe. The chemiluminescent measurements were plotted as log percent initial chemiluminescence with time.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
DHINGRA ET AL
240
Fig 2. Comparison of the sensitivity of radiolabeled probes
and HPAfordetecting PCR-amplified products. Lane 1, K562 RNA;
lanes 2 through 8, Serial 10-fold
dilutions of K562 RNA; lane 9,
ALL-1 RNA; lanes 10 through 14,
serial 10-fold dilutions of ALL-1
RNA; lane 15, HL-60 (negative
control) RNA. HPA counts on the
samples are shown below each
lane. bcrB-ABL II and BCRI-ABLII
probes were used t o detect amplified products from K562 and
ALL-1, respectively.
negative; 1, Ph'-positive), 20 cases of CML (18, Phipositive; 2 Phl-negative but bcr-rearrangement positive)
and 20 adult acute leukemias (15, Phl-negative; 5, Ph'Represenpositive-3 for P190wR'"''i.and 2 for P210""~"'"~).
tative results from these are shown in Fig 3. In every case,
the resultsof HPA were consistent with those obtained with
karyotypes and Southern blots. Interestingly, two acute
leukemia patients who were positive by HPA were not
known to be Ph'-positive because of inadequate metaphases
on karyotyping at thc time of presentation. Subsequent
cytogenetic evaluation at thc time of leukemic relapse in
both these patients showed the presence of the Ph' chromosome. In addition, two CML patients who had a diploid
karyotype but were positive by HPA were also found to
have bcr rearrangement on Southern blots. All samples
from Ph'-positive leukemias had HPA counts greater than
100,000 RLUs on HPA and could be clearly distinguished
from samples from Phi-negative leukemias, which were
usually less than 1,OOO RLUs. We have also used this
technique successfully to dctcct minimal residual disease in
individuals who are in complete cytogenetic remission
following a-interferon therapy or allogeneic bone marrow
transplantation (data not shown).
DISCUSSION
Fig 3. Comparison of the sensitivity of radiolabeled probes and
HPA in detecting PCR-amplfied p r o d u m from clinical specimens.
Lane 1, Ph'-positive pediatric ALL; lanes 2 through 4; Ph'-negative
pediatric ALL; lanes 5 and 6, Ph'-positive adult acute leukemia; lanes
7 and 8. Phhegative adult acute leukemia; lanes 9 and 10, chronicphase CML; lanes 11 and 12, blast-crisis CML; lane 13, K562; lane 14,
ALL-1; lane 15, HL-60 (negative control). (A) BCRI-ABLII probe; ( 6 )
bcr-ABLII probe HPA counts on the specimens are shown below each
lane.
Rapid advances in our understanding of the molecular
basis of human neoplasia have led to an increasing emphasis on the potential clinical applications of these discovcries. While the use of polymerase chain reaction (PCR) has
increased the sensitivity of detection of the Ph' chromosome, confirmation of the amplified product has required
Southern blotting, hybridization with radiolabeled probes,
extensive washing, and, finally, autoradiography. As an
alternative, HPA was investigated for the dctection of
PCR-amplified product. HPA is an entirely homogeneous
format that not only is nonradioactive, but also requires no
physical separation of free versus hybridized probe. In the
presence of the differential hydrolysis buffer, the rate of
hydrolysis of free probe is much faster than that of the
hybridized probe. As a consequence, separation Of hybridized probe from free probe on a solid Support iS UnnCCeSsary. Furthermore, elevated backgrounds traditionally associated with physical separation techniques are not a problcm
with HPA.
Acridiniumesters have high chcmilumin~scentquantum
yield and rapid chemiluminescent reaction kinetics. Alkyl-
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
241
BCR-ABL HYBRIDIZATION PROTECTIONASSAY
amine linker-arms can be attached to synthetic DNA
probes during DNA synthesis. The alkylamines are then
used as labeling sites for acridinium esters. Such acridiniumester-labeled probes are fully compatible with solution
hybridization methods. Solution hybridizations, as used by
the HPA format, allow for faster kinetics relative to
hybridizations performed on solid supports. Furthermore,
with the ability to hydrolyze all free probe before detection
with HPA, the amount of input probe can be adjusted to
further drive the reaction kinetics. The amount of probe
used and the time of hybridization for each of the probes
was based on COTlndeterminations (data not shown).
Using the HPA format described in this report, the
sensitivity of detection of chimeric BCR-ABL transcripts is
equal to that with radiolabeled probes while the background signal from unhybridized probe is in the 0.002% to
0.005% range, thus allowing a clear distinction between the
positive and negative samples. The probes used allow a
differentiation between the transcripts encoding P190BcR-ABL
and P210BCR-mL.
Because this method is used in conjunction
with PCR, it requires a much smaller sample than Southern
blotting and, unlike cytogenetics, can be performed on
peripheral blood. Furthermore, it can be successfully applied in instances where karyotyping is unsuccessful because of a lack of adequate metaphases. The rapidity of
HPA allows the nonradioactive detection of the Phl translocation on the same day that the sample is obtained and,
therefore, can help the physician make a more accurate
assessment of prognosis before initiating therapy. Finally,
our dilution experiments indicate one leukemic cell in a
population of a million or more normal cells will give a
positive result by this method, indicating that it may be used
to detect minimal residual disease. It should be noted that
the counts obtained by HPA may not increase linearly with
the increase in the quantity of the starting template or the
final amplification product. This result may be because of
(1) nonlinearity inherent in the PCR system (due to varying
efficiency of amplification depending on the amount of
starting template); (2) the inability of the photomultiplier
tube in the luminometer to discriminate individual chemiluminescent events above a certain signal-input level, especially signals greater than 100,000 RLUs; and (3) the
competition between the opposite strand of the PCRamplified product and the probe for the probe target region
during hybridization.
Besides its use for the detection of Phl-positive leukemias, HPA has potential for other clinical applications, too.
Because of its ability to provide high stringency between
closely related target sequences, the technique may be used
to detect point mutations in cancer-related genes. Indeed,
it has already been shown that one- or two-base mismatches
in DNA from closely related bacteria such as meningococcus and gonococcus can be detected by HPA.” Preliminary
results suggest that it can also be exploited to discern point
mutations in RAS protooncogenes.*I Finally, an increasing
number of cytogenetic defects are being discovered in
various tumors and, recently, other translocations encoding
novel proteins have been cloned?2 This method should be
easily applicable to the diagnosis and follow-up of these
neoplasms.
ACKNOWLEDGMENT
We thank Kristie Lykstad for excellent technical assistance and
Mehrdad Majlessi for synthesis and purification of the acridiniumester-labeled probes.
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