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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 574-588
Multiplex Reverse Transcription-Polymerase Chain
Reaction for Simultaneous Screening of 29 Translocations
and Chromosomal Aberrations in Acute Leukemia
By
Niels Pallisgaard,
Peter Hokland,
Dorthe C. Riishøj,
Bent Pedersen, and
Poul Jørgensen
From the Department of Molecular and Structural Biology, Aarhus
University, Aarhus, Denmark; the Department of Hematology and Medicine,
Aarhus University Hospital, Aarhus, Denmark; and the Department of
Cytogenetics, Danish Cancer Society, Aarhus, Denmark.
 |
ABSTRACT |
We have developed a multiplex reverse transcription-polymerase chain
reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid
leukemia (ALL) and acute myeloid leukemia (AML). Through the
construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and
reagents. We show the value of our set-up in a retrospective analysis
on cryopreserved material from 102 AML and 62 ALL patients. The
multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in
47% of the ALL cases with cytogenetic data, submicroscopic chromosomal
aberrations or masked translocations were shown that were not detected
in the cytogenetic analysis either for structural reasons or because of
an insufficient number of metaphases obtained. This multiplex RT-PCR
system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic
analysis in the up-front screening of acute leukemia patients and
should provide a rapid and efficient characterization of leukemia
cells, even in situations with sparse patient material.
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INTRODUCTION |
THE DIAGNOSIS OF acute leukemia is
multidisciplinary, with histology, immunology, and cytogenetics as the
most often used methodologies. Neither immunophenotyping nor histology
provides tools for prognosticating patients, whereas cytogenetic
evaluation has been shown to delineate patients with a defined
prognosis.1 The value of cytogenetics as a prognostic tool
in cancer is based on the existence of a number of balanced chromosomal
translocations. At present, more than 50 different consistently
occurring translocations have been described, many of which have been
found to be specific for particular subtypes of leukemia or lymphoma
(for a recent review, see Look2).
Molecular studies of these rearrangements have provided important
insights into the mechanisms of tumorigenesis. Thus, many genes
involved in translocations are transcription factors that appear to
have a direct role in hematopoiesis. Translocations may alter the
functions or activities of cellular proto-oncogenes located at or near
the breakpoint by at least two mechanisms, either (1) by juxtaposition
of a cellular proto-oncogene to the regulatory element of a
tissue-specific gene, eg, Ig or T-cell receptor genes in leukemia,
leading to inappropriate expression of the oncogene,3,4 or
(2) by creating fusion genes coding for chimeric proteins with
functional features different from the two parental proteins, eg,
t(1;19)(q23;p13) and t(8;21)(q22;q22).5,6
A translocational breakpoint gene may have several fusion partners, the
most promiscuous example being the MLL gene (also called
ALL1, HTRX1, and HRX) at chromosome band 11q23,
for which more than 40 different fusion partners together with an
internal duplication have been described.7-15 Thus,
depending on the fusion partner, the MLL gene can contribute to
the pathogenesis of lymphoid and myeloid malignancies. The
MLL/AF4 fusion gene, detected in t(4;11)(q21;q23)
translocations, is observed in acute lymphoid leukemia (ALL)
only,14,16-18 whereas the MLL/AF9 fusion gene
detected in t(9;11)(p22;q23) translocations is found primarily in acute myeloid leukemia (AML) patients.14,19 The dupMLL
has been described in both ALL and AML patients.15,20,21
Because cytogenetic analysis is time-consuming and yields sufficient
metaphases only in 60% to 80% of the bone marrow samples, efforts to
design polymerase chain reaction (PCR) reactions for translocations
have been ongoing for some time.22,23 The availability of
cDNA sequence information for an increasing number of fusion genes has
resulted in PCR protocols for individual translocations. PCR analysis
does not require much patient material, can be performed on resting
cells, and is very sensitive in detecting rare abnormal cells. Thus, it
should be of great potential benefit to bring the PCR methodology
up-front in the diagnosis of acute leukemia. However, considering the
great number of fusion genes and breakpoint variants presently
characterized, more than 50 separate PCR reactions are needed for the
screening of a patient with a standard procedure, which is at best
labor-intensive and material-demanding and probably not practically
feasible.
We describe here our efforts to establish a multiplex reverse
transcription-PCR (RT-PCR) analysis system that
facilitates the detection in 8 parallel PCR reactions of 29 translocations/chromosomal aberrations, including more than 80 mRNA
breakpoint or splice variants.
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MATERIALS AND METHODS |
Patient samples and cell lines.
For the combined purpose of optimizing the PCR primers and
obtaining unlimited amounts of material for positive controls for the
multiplex assay, we used cell lines as the RNA source. For the
t(1;19)(q23;p13), we used 697; for t(2;5)(p23;q35), Karpas-299; for
t(4;11)(q21;q23), RS4;11 and MV-4-11; for t(6;11)(q27;q23), ML-2; for
t(9;11)(p22;q23), Mono-Mac-6; for t(15;17)(q21;q22), NB4; for
t(17;19)(q22;p13), HAL-01; and for TALD, RPMI8402.
In addition, we used RNA from patients positive for dupMLL(11q23), inv(16)(p13q22), t(6;9)(p23;q34),
t(8;21)(q22;q22), t(9;22)(q34;q11), t(10;11)(p12;q23),
t(11;19)(q23;p13.3), and t(11;19)(q23;p13.1) that were identified
during the study.
Leukemic cell samples from patients admitted to the Departments of
Hematology and Pediatrics, Aarhus University Hospital (Aarhus, Denmark)
were subjected to Isopaque-Ficoll sedimentation and the mononuclear
cell suspensions used for routine immunophenotyping purposes. In cases
in which more than 5 × 106 cells were available,
these were cryopreserved in 10% fetal calf serum and 10% dimethyl
sulfoxide according to standard techniques. All cell
collection was performed according to protocols approved by the Local
Ethical Committee for the County of Aarhus. Likewise, the Biobase
containing cell material from the patients has been approved by the
Danish Data Protection Agency (Registertilsynet).
The cell lines Karpas-299, ML-2, Mono-Mac-6, NB-4, 697, JOSK-M, JOSK-I,
NALM-6, and RPMI8402 were obtained from DSM-Deutsche Sammlung von
Mikroor ganismen und Zellkulturen (Braunschweig, Germany). The cell
lines RS4;11 and MV-4-11 were obtained from the American Type Culture
Collection (Manassas, VA). The cell line HAL-01 was kindly provided by
Dr Kazuma Ohyashiki (Tokyo Medical College, Tokyo, Japan). All cell
lines were cultured in RPMI-1640 medium supplemented with 10% fetal
calf serum and antibiotics. The medium for the cell line Mono-Mac-6 was
supplemented with 9 µg/mL bovine insulin. An overview of the
characteristics of most of these lines can be found in Drexler et
al.24
Cytogenetic analysis.
Bone marrow cells were cultured at 37°C in an atmosphere of 5%
CO2 in air for approximately 24 hours. After 3 to 5 hours, methotrexate (10 5 mol/L) was added, and 17 hours
later the S-phase block was released with thymidine
(103 mol/L). Colchicine was added 10 minutes before
termination of the culture period.25 Cells were harvested
with conventional methods and slides were prepared. The slides were
aged by heating at 60°C for 17 hours before banding. Giemsa bands
were produced with Wright's stain as described
elsewhere.26
RNA preparation.
Cells were thawed out and washed twice in phosphate-buffered
saline. Total RNA was prepared either by the guanidinium
thiocyanate-phenol chloroform method27 or by using a RNeasy
Kit (Quiagen Gmbh, Hilden, Germany) according to the manufacturer's
recommendations. The RNA solution was subsequently treated with 0.1 U/µL RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) in 50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L MgCl2 at 37°C for 30 minutes. After DNase treatment, EDTA (pH 8.0) was added to a final
concentration of 10 mmol/L and the RNA solution was extracted once in
phenol/chloroform 1:1. Sodium acetate was added to a final
concentration of 200 mmol/L and RNA was precipitated with 1 vol of
isopropanol. RNA was pelleted in an Eppendorf centrifuge at
13,000 rpm for 30 minutes, washed with 80% ethanol, and resuspended in
25 µL diethylpyrocarbonate ddH2O. Five
microliters were withdrawn for quantification on a GeneQuant
RNA/DNA calculator (Pharmacia Biotech, Sollentuna, Sweden). Subsequently, RNA was diluted to 0.1 µg/µL in DEP ddH2O
and stored until use at  80°C in 10-µL aliquots.
The multiplex PCR setup.
We have designed a multiplex RT-PCR strategy to detect the transcripts
of chromosomal translocations/rearrangements found in leukemic
patients. Taking advantage of the fact that a number of the genes
involved in translocations can have different fusion partners, we
combined such genes in the assay, thus reducing the number of primers.
In 8 multiplex PCR reactions this assay tests for 29 translocations or
chromosomal rearrangements that may result in the generation of more
than 80 fusion gene variants because of heterogeneity of breakpoints
and/or alternative splicing. The translocations and the
resulting transcripts tested for in the multiplex PCR assay are shown
in Table 1. Table 1 also
includes a number MLL gene fusion variants (marked T), which in
theory may be expected to occur, but have to our knowledge, not yet
been described.
Internal positive control.
Because false-negative results are an inherent problem in RT-PCR assays
because of varying RNA quality and/or handling errors, we
included an internal positive control in which a 690-bp segment of the
ubiquitously expressed transcription factor E2A mRNA is amplified.28,29
Translocation specific cDNA primers.
The amount of patient RNA can be a limiting factor, and efficient cDNA
synthesis is therefore a critical step in RT-PCR. To increase the
sensitivity of cDNA synthesis and to reduce the background from
irrelevant RNA, we opted not to use random hexamer primers, but instead
designed a number of translocation-specific cDNA primers shown in
Table 2. The cDNA primers were 11 to 13 nucleotides (nt) long and located 10 to 100 nt downstream of the most
3 PCR primer. The melting temperature (Tm) of the cDNA primers
was approximately 40°C, which is sufficiently high to ensure
efficient cDNA priming and low enough to ensure that these primers
would not interfere with the subsequent PCR reaction that was performed
without purification of the cDNA.
Construction of primers.
All PCR oligonucleotide primers were designed with the primer analysis
software OLIGO version 5.0 (National Biosciences Inc, Plymouth, MN) and
published sequence data from the EMBL DNA database. Oligonucleotide
primers were purchased high-performance liquid chromatography-purified from DNA Technology (Science Park,
Aarhus, Denmark).
RT-PCR.
To achieve maximal sensitivity, a nested PCR protocol was used and, to
minimize the risk of contamination, filter-tips and four different
laboratory rooms with indigenous pipettes were used for (1) preparation
of stock solutions; (2) RNA preparation and cDNA synthesis/setup of
first PCR; (3) the first to second PCR transfer; and (4) gel
electrophoresis. One microgram of total RNA was incubated at 65°C
for 5 minutes with a mixture of translocation-specific cDNA primers
(2.5 pmol of each) and then reverse transcribed by incubation at
37°C for 45 minutes in a total volume of 25 µL containing 20 U
RNase inhibitor (Boehringer), 1 mmol/L of each dNTP, 10 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, and 400 U Moloney murine leukemia virus reverse
transcriptase (BRL, Bethesda, MD). After the incubation, the cDNA
reaction mixture was diluted with ddH2O to 50 µL. PCR
amplification was performed as 8 parallel nested (2-round) multiplex
reactions in a Perkin Elmer 9600 thermocycler (Roche Molecular Systems,
Branchburg, NJ). Five microliters of diluted cDNA reaction was added to
each of 8 20-µL multiplex mixtures that contained 11 mmol/L Tris-HCl, pH 8.3, 55 mmol/L KCl, 1.65 mmol/L MgCl2, 0.2 mmol/L of
each dNTP, a mixture of oligonucleotide primers (5 pmol of each
primer), and 1.5 U AmpliTaq-Gold polymerase (Perkin Elmer). The first
PCR consisted of an initial activation of the polymerase at 95°C
for 15 minutes, followed by 25 cycles of PCR amplification (annealing at 58°C for 30 seconds, elongation at 72°C for 1 minute, and
denaturation at 95°C for 30 seconds). After the first PCR, 1-µL
aliquots from each of the 8 PCR reactions were transferred to 8 24-µL
second-round multiplex mixtures that contained 10 mmol/L Tris-HCl, pH
8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L of each
dNTP, 5 to 12.5 pmol of each primer, and 1.5 U AmpliTaq-Gold
polymerase. The second PCR consisted of an initial activation of the
polymerase at 95°C for 15 minutes, followed by 20 cycles of PCR
amplification (annealing at 58°C for 30 seconds, elongation at
72°C for 1 minute, and denaturation at 95°C for 30 seconds),
and finally by 10 minutes of extension at 72°C. Fifteen
microliters of each PCR reaction was electrophoresed in a 1.5%
agarose gel for 60 minutes at 100 V and stained with ethidium bromide
as shown in Fig 1A. Negative controls
without DNA template were included for all PCR reaction mixtures.
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| Fig 1.
Multiplex RT-PCR and split-out analysis. (A) The two cell
lines MV-4-11 and Mono-Mac-6 and patient no. 243 were positive for a
translocation in multiplex reaction R5 (see Table 3). (B) To determine
the translocation, a split-out PCR analysis was performed using the
individual primer sets R5A, R5B, R5C, and R5D. M, DNA molecular weight
marker VI (Boehringer).
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Split-out PCR and DNA sequence analysis.
Because each multiplex reaction identifies a number of translocations,
many of which may be found in several variants, the number of possible
translocation-positive PCR fragments is large (Table 1). To determine
and verify a fusion gene in a positive multiplex reaction, we therefore
performed split-out analysis using individual primer sets, as outlined
in Fig 1B and detailed in
Table 3. The split-out was
performed using the same reaction conditions as for the multiplex PCR,
except that only 1 U/reaction of AmpliTaq-Gold polymerase was used.
Split-out of samples with a limiting amount of RNA was performed with 1 µL from the first round multiplex PCR as template and the
second-round individual PCR primer sets. These analyses were performed
with and without the internal positive control primers. Negative
controls without DNA template were included for all PCR reaction
mixtures. The presence of translocations were confirmed by
determination of the sequence of the translocation specific DNA
fragment. The DNA sequencing was performed using agarose gel-purified
PCR fragments as a template and a Taq DyeDeoxy Terminator Sequencing
kit (Perkin Elmer). The product was analyzed using an automated 373A
DNA sequencer (Applied Biosystems, Foster City, CA).
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RESULTS |
Optimization of multiplex RT-PCR conditions.
The aim of the multiplex RT-PCR procedure is to detect and define
translocation-specific mRNA related to leukemia. To set up the
procedure, we first defined primers with a binding sequence near
putative breakpoints of the corresponding mRNA sequence. Primers were
designed to allow identical conditions in all PCR reactions. For cDNA
synthesis, we elected to use primers specific for the recombined mRNA
rather than random or poly-dT primers. Use of specific primers improved
sensitivity 25- to 125-fold relative to random hexamer primers. When
cell lines or patient material were available with known translocations
that lead to recombination-specific mRNA, we tested the constructed
primer pairs in PCR reactions before and after combining the different
primer sets into the multiplex PCR reaction. If not working properly,
the primers were redesigned. Finally, the sensitivity of the multiplex
PCR assay was evaluated by limiting dilution experiments in which
fivefold dilutions of RNA from translocation-positive cell lines were
mixed with RNA from the NALM-6 cell line. Several series of multiplex RT-PCR experiments consistently showed that the translocation positive
band could be readily detected in the 3,000- or 15,000-fold dilutions,
indicating that the multiplex assay, at least for the cell lines
tested, may detect 1 malignant cell out of 5,000 normal cells.
Translocations detected in AML and ALL patients by the multiplex PCR
analysis.
To verify the value of the multiplex PCR system, we reproduced
cytogenetic findings by multiplex PCR with material from cell lines and
patients with known translocations. To evaluate the multiplex RT-PCR as
a potential diagnostic tool used in up-front leukemia diagnosis, we
next applied the multiplex PCR assay in a retrospective analysis of RNA
purified from cryopreserved mononuclear blood or bone marrow samples of
102 AML and 62 pediatric ALL patients. RNA quality was evaluated by
inspection of the 8 internal positive control bands in the multiplex
PCR. In 8 of 102 of the AML and in 6 of 62 of the ALL cases, the
internal positive control band had a weak and scattered appearance or
was absent. This could be ascribed to insufficient amount or quality of
RNA due to low cell number or cell lysis.
As seen in Tables 4 and
5, the multiplex PCR analysis detected a
fusion gene in 45 of 102 of the AML and in 28 of 62 of the ALL cases.
The frequencies and distributions between the two patient groups of the
16 different chromosomal aberrations resulting in a fusion gene that
could be detected in the multiplex PCR are compared with cytogenetic
data in Table 6. Examples of various aberrations detected by the multiplex RT-PCR are shown in
Fig 2. The value of the described multiplex
PCR analysis is demonstrated by the finding of 3 previously undescribed
fusion gene variants: (1) a duplication of the MLL gene in
which exon 5 was fused to exon 2 (patient no. 44), (2) a new breakpoint
of the AF10 gene in a t(10;11)(p12;q23) (patient no.
22),30 and (3) a new breakpoint in the TLS gene in
a t(16;21)(p11;q22) (patient no. 93).
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| Fig 2.
Examples on chromosomal aberrations found by the
multiplex RT-PCR. The loading order and DNA molecular weight marker are
as in Fig 1. The band specific for the translocation is indicated by an
arrowhead beside the band. The dots indicate activation of HOX11 (lane
4) or EVI1 (lane 7). Because HOX11 and EVI1 represent activation of
native gene products and not of chimerical products, these are not
further discussed in this presentation. *Patient no. 335 was a chronic
myeloid leukemia case not included in this study.
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In this material, we did not identify patients positive for the
following translocations: TAL1D, t(X;11)(q13;q23),
t(1;11)(q21;q23), t(1;11)(p32;q23), t(3;5)(q25.1;q34), t(3;21)(q26;q22), t(5;12)(q33;p13), t(5;17)(q35;q22), ?t(9;9), t(9;12)(q34;p13), t(11;17)(q23;q21), and t(17;19)(q22;p13). These translocations should be detected by the PCR primers used and their
absence in our material can be ascribed to the low frequency of these
chromosomal aberrations. Thus, the t(X;11)(q13;q23), t(5;17)(q35;q22),
?t(9;9), and t(9;12)(q34;p13) have, to our knowledge, been described
only in single cases, and only a few cases of the t(1;11)(p32;q23),
t(1;11)(q21;q23), t(3;5)(q25.1;q34), t(5;12)(q33;p13), t(11;17)(q23;q21), and t(17;19)(q22;p13) have been reported. The t(3;21) has been described primarily in therapy-related AML, and only
recently in 1 de novo AML patient.31 The
TAL1D, with the fusion gene SIL/TAL1, has
been found in 25% of T-ALL cases.32 Because only 6 T-ALL
patients were included in this study, the absence of patients with the
SIL/TAL1 fusion-gene may be ascribed to statistical variation.
However, we cannot exclude that one or more of the PCR primers for
these translocations may be incompatible with the multiplex PCR
conditions, with the exception of the t(17;19)(q22;p13) and
TAL1D, for which we have positive controls. This
issue awaits the availability of positive patients, cell lines, or in
vitro synthesized control RNA.
Comparison between the multiplex PCR and the cytogenetic analysis.
Cytogenetic data were available for 66 AML cases. In 62 of the cases,
metaphase cells were obtained, but cytogenetic aberrations were
detected in only 28 cases. The corresponding numbers for ALL were in
total 45 cases, 34 with available metaphase cells and 17 with
chromosomal aberrations. When the cytogenetic analysis showed one of
the translocations included in the multiplex PCR, the corresponding
fusion gene mRNA was detected, except in 1 case in which the multiplex
PCR could not be performed because of insufficient RNA. The chromosomal
rearrangements dupMLL, TALD,
t(12;21)(p13;q22), and t(6;11)(q27;q23) cannot be detected (or are
easily overlooked) by classical cytogenetic analysis. In the multiplex
PCR analysis, 14 of 102 AML and 15 of 62 ALL cases were positive for
this group of aberrations. A new finding in this work is the high
frequency of dupMLL in ALL. Thus, in 6 AML and in 2 ALL cases
with an adequate cytogenetic analysis (10 or more metaphases obtained
at presentation), a fusion gene was detected by PCR that was not
detected by the cytogenetic analysis. Masked translocations, which are
not apparent in cytogenetic analysis but detectable by Southern
blotting or RT-PCR, have previously been observed.33,34 In
contrast, numerical aberrations, which cannot be detected by PCR, were
found by cytogenetic analysis in 17 of 66 of the AML and in 11 of 45 of
the ALL cases. The combined results from multiplex PCR and
cytogenetic analysis are presented in Table 6. Taken together, the
two methods uncovered chromosomal aberrations in 42 of 66 of the AML
and in 27 of 45 of the ALL cases and supplemented each other
in detecting chromosomal aberrations.
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DISCUSSION |
During the last decade, the multidisciplinary diagnosis of acute
leukemia has been expanded by the description of an ever-increasing number of balanced translocations that are amenable to detection by
classical karyotype analysis and by PCR analysis when sequence information is available for the designing of PCR primers.
Multiplex PCR has been used previously for characterization of
individual or small groups of translocations found in leukemic cells.22,23 We have scaled up this method of analysis to
cover most of the published translocations and describe here our
experience with a multiplex PCR procedure on cryopreserved material
from more than 160 patients diagnosed and treated at Aarhus University Hospital. We emphasize that the material is not necessarily
representative as an unselected material, because cryopreservation of
cells in the Biobase at the Laboratory of Immunohematology, Aarhus
University Hospital, was performed only if the sample contained at
least 5 million cells after immunophenotyping. Consequently, patients with scarce cell material at diagnosis were not included. Moreover, in
8 of 102 of the AML and 6 of 62 of the ALL cases, too small amounts of
RNA to warrant PCR analysis were obtained from cryopreserved cells
because of cell lysis. In fresh material, the fraction of patients with
insufficient RNA should therefore, in theory, be lower, a supposition
that is substantiated by our recent results from applying the method
prospectively (Hoklandet al, unpublished data). Our cell
material was cryopreserved over a period of 14 years; therefore, we
caution against a close comparison between cytogenetics and PCR
results. Banding techniques have improved considerably over the years,
and the percentage of translocation positive cases has, in our hands,
increased over time. Also, cytogenetic analysis on patients dating back
to the 1980s was performed on only 10 metaphase cells per sample.
Given these limitations, we believe that the data presented here
clearly prove the usefulness of the multiplex PCR concept. First, the
assay can be performed (including the split-out phase) within 2 to 3 days and is amenable to the analysis of up to 10 samples simultaneously
by 1 person. Second, the reaction clearly increases the number of
translocation-positive patients relative to cytogenetic analysis,
especially in cases of material with sufficient numbers of high-quality
metaphase cells. This is exemplified by the demonstration of 22 of 66 AML and 21 of 45 ALL patients in whom additional chromosomal
rearrangements were found by the multiplex PCR. Importantly, these
translocations were not restricted to submicroscopical chromosomal
aberrations, which are difficult, if not impossible, to detect by
cytogenetic analysis. Rather, we found a wide range of translocations,
including both those that are frequent [eg, t(8;21) and t(15;17)] and
those that are infrequent [eg, t(11;19)]. Of equal importance, the
additional translocations were found in both AML and ALL patients.
The multiplex PCR detects the expression, on the RNA level, of fusion
genes generated by chromosomal rearrangements. It does not detect
rearrangements in which native oncogenes are deregulated, as described
in, eg, t(8;14), t(11;14), and t(1;14). Such rearrangements may be
detected by PCR on DNA level. Similarly, Ig and T-cell receptor gene
rearrangements35 cannot be detected by the proposed multiplex system. Although not generally considered to be of
independent prognostic significance, these aberrations can be very
important in the detection of minimal residual disease. Finally, the
relative contribution of this reaction and fluorescent in situ
hybridization (FISH) techniques36 cannot presently be
directly evaluated, but (as with karyotypic analysis) we would expect
these methodologies to be complementary.
For at least two reasons, the PCR methodology cannot fully replace
karyotypic analysis. First, numerical aberrations and abnormalities other than balanced translocations cannot be detected. Second, unknown
balanced translocations are obviously not detected. Cytogenetic analysis will thus form an important platform for molecular
characterization of new genetic aberrations.
Because of its versatility and sensitivity, we believe that this novel
multiplex PCR procedure holds promises as a screening tool for the
initial diagnostic phase of acute leukemia. In addition, moving the PCR
methodology up-front will allow its use for remission evaluation, when
bone marrow material is often scarce. Here, the very high sensitivity
of the PCR reaction may yield information in cases in which the
translocation found at diagnosis would also be detected at remission.
Our retrospective data (patients no. 70, 90, 108, 259, and 265) and the
preliminary clinical experience clearly support the notion given
above, because we have positive multiplex reaction in several
marrow preparations at remission for the corresponding translocation
identified originally by cytogenetic analysis at diagnosis.
It might be argued that the multiplex concept is weakened by the
decrease in sensitivity relative to single PCR reactions, particularly
when the multiplex reaction is used for detection of minimal residual
diseases (eg, in Ph+ patients). In acute leukemia at
diagnosis, the cell source used for RNA preparations is usually greater
than 90% leukemic blasts. However, as demonstrated in this report, the
sensitivity of our reactions is comparable to single-pair reactions
probably because of the use of two (nested) primer sets. Moreover, the
split-out primer sets will have a sensitivity comparable to the
individual multiplex reactions.
We believe that the multiplex PCR assay is clinically useful as an
efficient and fast procedure for the detection of genetic changes in
acute leukemia and that it complements the cytogenetic analysis in a
fruitful manner. Clearly, this novel approach for addressing the
multitude of genetic changes in acute leukemia is open for addition of
new primer sets as information of novel translocations accumulates.
However, in our hands, this reaction has already yielded new
information in the retrospective setting as well as in the initial
diagnosis. Finally, our approach can be extended to the detection of
minimal residual leukemia, because the split-out reaction has a
sensitivity equivalent to that of single PCR assays. Thus, the
multiplex approach would be of significance not only at diagnosis, but
also for subsequent clinical decision-making.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted March 10, 1998.
Supported by Research Grants from the Danish Cancer Society and the
Karen Elise Jensen Foundation.
Address reprint requests to Poul Jørgensen, PhD,
Institute of Molecular and Structural Biology, Aarhus University, C.F.
Møllers Allé Bld. 130, DK-8000 Aarhus C, Denmark; e-mail:
pj{at}mbio.aau.dk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Niels Clausen (Aarhus University Hospital) for
supplying the pediatric ALL samples and Dr Kazuma Ohyashiki (Tokyo
Medical Collage) for the HAL-01 cell line. We are indebted to Dr K. Paludan for critical reading of the manuscript.
| |
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Abstract
Introduction
Abstract