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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 574-588
By
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.
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.
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.
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.
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 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 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
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.
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).
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.
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.
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.
Submitted October 20, 1997;
accepted March 10, 1998.
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
5 mol/L) was added, and 17 hours
later the S-phase block was released with thymidine
(10
80°C in 10-µL aliquots.
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.
