| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
At diagnosis, the bone marrow of ALL patients contains 1012 malignant cells. Complete remission is conventionally defined as <5% leukemic blasts according to morphological assessment, hence implying the presence of a significant amount of residual leukemic cells in individual patients. The term MRD indicates the presence of leukemic cells below the detection level of conventional methods, i.e., <10-2 leukemic cells. Several methodologies are available for MRD analyses in ALL patients, namely multiparameter immunophenotyping and PCR analysis using tumor-specific chromosomal translocations or clone-specific immunoglobulin and TCR rearrangements as markers (2) . The latter technique is based on the fact that B- and T-cell leukemias exhibit a distinct immunoglobulin and TCR gene rearrangement at the V(D)J junctional region that can be used as a specific marker for that particular leukemia clone. PCR analysis based on patient-specific junctional regions enables the detection of 1 leukemic cell in up to 106 normal cells (2) . Recently, two large prospective clinical trials using respective PCR targets demonstrated unequivocally the clinical relevance of MRD status during the first 3 months of treatment in children with ALL (3 , 4) .
To date, mainly two PCR strategies using immunoglobulin or TCR targets have been applied in MRD studies. In one approach, the respective junctional region of a immunoglobulin or TCR rearrangement is amplified and hybridized with a clone-specific probe in dot-blot or liquid hybridization analysis (2) . In the ASO-PCR technique, an ASO corresponding to the clone-specific junctional region is designed and used as a 5' or 3' primer in combination with a consensus immunoglobulin/TCR primer to amplify the target DNA (2 , 5, 6, 7, 8, 9) . The MRD status of a remission sample is determined semiquantitatively by comparing the amount of the respective PCR product (ASO-PCR) or hybridization signal (dot blot and liquid hybridization) with the PCR products of target DNA (leukemic cells) serially diluted in normal BC DNA of healthy individuals. ASO-PCR has several advantages because it is simple, rapid, less laborious, and nonradioactive. However, this technique has some limitations if applied in prospective MRD analysis to guide treatment stratification of individual patients. For example, in a standard PCR the amount of the respective PCR product reaches a plateau, thus preventing its reliable quantification in a serial dilution analysis. Therefore, multiple PCRs are required to determine the optimal number of cycles that allow a correlation between the PCR products in a serial dilution experiment with the amount of leukemia DNA in a remission sample. When the junctional region shows limited variability, i.e., a small N-nucleotide region, a second problem occasionally occurs, namely, the amplification of target DNA from normal lymphocyte DNA. Here again, several ASO-PCR reactions with varying cycle numbers are necessary to determine the optimal number of cycles that allows maximum sensitivity without false-positive signals from normal BC DNA.
Recently, new technologies have become available that allow "real-time and on-line" monitoring of PCR products by measuring fluorescence in every cycle with a built-in microvolume fluorimeter, i.e., the TaqMan (10, 11, 12, 13) and LightCycler technology (14, 15, 16, 17) . These techniques allow the measurement of PCR kinetics and reliable quantification of residual disease. Moreover, the LightCycler also allows the determination of melting curves, thus enabling the detection of mutations and the assignment of different PCR products (15 , 17) . Basically, both instruments can be used to explore two different techniques for the specific detection of amplification products: (a) hybridization and detection of fluorescence-labeled sequence-specific hybridization probes (oligonucleotides); and (b) detection of PCR products by the DNA intercalating dye SYBR Green I, as applied in this study. Both methods are quantitative over a large dynamic detection range, thereby avoiding the laborious testing of PCR conditions with varying cycle numbers. Moreover, post-PCR processing (DNA gel electrophoresis) can be omitted, saving time and money. Here we describe ASO-PCR protocols for the most frequent types of immunoglobulin/TCR rearrangements to quantify MRD in ALL patients by LightCycler technology and SYBR Green I staining. We compared the sensitivity and practicability of the LightCycler method for use on a routine basis with the conventional ASO-PCR technique on a block thermocycler followed by quantification by DNA gel electrophoresis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
DNA/HindIII-digested size marker
(Amersham Pharmacia Biotech, Freiburg, Germany) to verify the
concentration and size of each DNA sample.
Characterization of Rearranged Immunoglobulin/TCR Genes.
Rearrangements of the immunoglobulin/TCR genes were identified by
duplex or single PCR strategies. For the analysis of IGH
recombinations, we used mainly a "framework" 1 consensus primer
(FR1c) and a JH consensus primer
(JHcons; Ref. 20
). Alternatively,
each of the following primers, VH1/7 (5'-TCT GGG
GCT GAG GTG AAG AA-3'), VH3 (5'-GGG GGT CCC TGA
GAC TCT C-3'), and VH4 (5'-GCC CAG GAC TGG TGA
AGC-3'), was used in combination with a JH
consensus primer, JH21, (5'-ACC TGA GGA GAC GGT
GAC C-3'; kindly provided by Prof. J. J. M. van Dongen, Erasmus
University, Rotterdam, the Netherlands). One hundred ng of genomic DNA
were amplified in 70 µl of PCR reaction mixture containing 1x PCR
buffer, 1 unit of Taq polymerase (Life Technologies, Karlsruhe,
Germany), 1.5 mM MgCl2, and
40 pmol of each primer. The reaction was heated initially for 4 min at
94°C, followed by amplification for 30 s at 94°C, 45 s at
60°C, and 45 s at 72°C, with a final elongation step of 10 min
at 72°C on a block thermocycler (Primus 96; MWG AG Biotech,
Ebersberg, Germany).
IGK-Kde rearrangements were characterized by duplex PCR as described elsewhere (21) .
For screening of 12 different TCRD and TCRG
rearrangements, six duplex PCR reactions were performed (Table 1)
. The sequences of the PCR primers have been reported previously
(23)
. The reaction mixture (70 µl) and PCR conditions,
except for duplex PCR target 4 (Table 1)
, were basically identical to
the method described for the detection of IGK-Kde
rearrangements (21)
. The amount of each primer in the
70-µl reaction mixtures is shown in Table 1
. For duplex PCR target 4
(Table 1)
, we basically used the same PCR conditions as described for
the detection of IGH gene rearrangements except that 2.5
mM MgCl2 was used. Ten µl
of each PCR product were resolved on a 2% agarose gel (FMC). Twenty
µl of the PCR reaction were further analyzed by a heteroduplex
analysis (23)
. Forty µl of the PCR reaction that showed
monoclonality by heteroduplex analysis were electrophoresed on a 2%
agarose gel (FMC) and purified by a QiaexII kit (Qiagen, Hilden,
Germany). The junctional regions were characterized either by direct
sequencing or by cloning of the PCR fragment into a plasmid vector
(TOPO TA cloning kit; Invitrogen, Groningen, the Netherlands).
As sequencing primers, we used the inner primers for each rearrangement
(22)
. For sequence analysis of IGH
rearrangements, the PCR fragment was reamplified with a FR1c or one of
the VH-specific primers and an M13-tailed
JH21M primer (5'-TCA CTA TAG GGT GTA AAA CGA CGG
CCA GTG GAC CTG AGG AGA CGG TGA CC-3'), followed by direct sequencing
using an M13F primer (5'-TGT AAA ACG ACG GCC AGT-3'). Sequencing
reactions were carried out using a Cy5 Thermo Sequenase Dye Termination
Kit (Amersham Pharmacia Biotech) and analyzed on an autosequencer
(ALFexpress II; Amersham Pharmacia Biotech).
|
. For all but
IGH rearrangements, DNA was amplified using a touch down
program (21)
. Briefly, the mixture was heated initially at
94°C for 4 min. Amplification was initialized at an annealing
temperature of 66°C with a decrement of 0.5°C for every cycle for
45 s, using a thermal cycler (Primus 96; MWG AG Biotech).
Denaturation was performed at 94°C for 30 s and extension at
72°C for 45 s. After an annealing temperature of 52°C was
reached in the 28th cycle, 12 additional cycles were performed at
52°C. The PCR program for the first-round amplification of
IGH rearrangements was the same as that of the screening
step. In every first-round PCR, H2O used for the
preparation of the PCR reaction was simultaneously amplified as a
negative control. For the analysis of bone marrow follow-up samples
(CR) during the course of therapy, we used 1 µg of DNA for
amplification. Ten µl of the PCR products were electrophoresed on an
agarose gel (FMC). For the following ASO-PCR step, 10 µl of the first
PCR product were diluted in 990 µl of 5 mM
Tris-HCl, 0.5 mM EDTA (pH 8.0).
ASO-PCR (Second-Round) on a Block Thermocycler.
ASO primers corresponding to each junctional region were designed
according to the criteria published previously (22)
. All
ASO and PCR primers for this method were obtained from BIG Biotech
(Freiburg, Germany). For IGH analysis, the ASO was derived
from the third complementarity-determining region and was always
used as a reverse primer in combination with an inner forward primer
(Fig. 1
). For the second-round PCR of IGH rearrangements, we
designed primers according to the recently published
VH sequences (Ref. 24
and Table 2
). For the analyses of the IGK, TCRG, and
TCRD gene rearrangements, ASO primers were used as either
forward or reverse primers (Fig. 1
). The sequences of most of the inner
primers have been reported previously (22)
. Some inner
primers have been newly designed: K9.5 (for Kde), 5'-GTT TAC AGA CAG
GTC CTC AG-3'; V
1-33 (for V1), 5'-CCA GGG TTC TGA TGA ACA GAA
TGC-3'; V2-33 (for V2), 5'-TCA ACT GGT ACA GGA AGA CC-3'; 11.5
(for D3), 5'-AAG CTG CTT GCT GTG TTT GTC-3'; and G9.5 (for
J
1.3/2.3), 5'-CTA TGT TCT CTT TTA GTA TGA GC-3'.
|
|
and 3)
. Using a pause function of the thermocycler, 20 µl of the PCR
reaction were taken from the PCR reaction five cycles before the end of
the program. A final elongation step of 7 min at 72°C was performed.
|
ASO-PCR (Second-Round) on the LightCycler.
A LightCycler DNA Master SYBR Green I kit (Roche Molecular
Biochemicals) was used to perform ASO-PCR on the LightCycler for each
rearrangement. The kit provides PCR-grade H2O, 25
mM MgCl2, and 10x LightCycler DNA
Master SYBR Green I containing 10x PCR buffer, a deoxynucleotide
triphosphate mixture (with dUTP instead of dTTP), 10 mM
MgCl2, SYBR Green I dye, and Taq DNA polymerase.
One µl of the diluted PCR products from the first-round PCR reaction
was amplified in duplicate in a 20-µl reaction containing 2 µl of
10x LightCycler DNA Master SYBR Green I, 1.6 µl of 25 mM
MgCl2 (final concentration 3 mM;
Roche Molecular Biochemicals), and 10 pmol of each primer. Two negative
controls (H2O1 and H2O2)
were included in every PCR. The running protocol was programmed on the
LightCycler software, version 1.2 or 3.39. The program consisted of the
following three steps. The first step was an initial denaturation where
the reaction was incubated for 2 min at 95°C. In the second step, DNA
was amplified for 35 cycles of 1 s at 95°C, 10 s at
50–65°C, and 10 s at 72°C. Finally, the temperature was
raised gradually (0.2°C/s) from the annealing temperature to 95°C
for the melting curve analysis. If the sensitivity did not reach a
level of 10-4 leukemic cells, an
additional 10 cycles were performed before completion of the
amplification program (version 3.39).
Prevention of Cross-Contamination.
The PCR mixtures were prepared in a closed room separated from any PCR
product (pre-PCR room). After the PCR mixture was prepared, the
pipettes and stands that were only used in the pre-PCR room
were placed under UV light for 15 min. All other steps of the analyses
were performed in another post-PCR room.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To assess the sensitivity and practicability of ASO-PCR by the
LightCycler technology, we investigated 35 junctional regions of
immunoglobulin/TCR genes from these 25 ALL patients (Tables 2
and 3)
.
They comprised 11 IGH (1
VH1-JH, 8
VH3-JH, and 2
VH4-JH), 6 IGK
(1 V
I-Kde, 2 VII-Kde, 1 VIII-Kde, 1 VIV-Kde, and 1
RSS-Kde), 6 TCRD (2 V2-D3, 1 D2-D3, 1
D2-J1, 1 V1-D-J1, and 1 V2-D-J1), and 12
TCRG (7 VI-J1.3/2.3, 1 VII-J1.3/2.3, 2
VIII-J1.3/2.3, 1 VI-J2.1, and 1 VIV-J1.1)
rearrangements. The mean number of nucleotides inserted at the
junctional regions was 19.3 (range, 12–30) for IGH loci,
5.8 (range, 4–8) for IGK, 10.0 (range, 5–23) for
TCRD, and 6.7 (range, 2–10) for TCRG, and the
mean number of nucleotide deletions was 10.0 (range, 0–32) for
IGH, 17.0 (range, 9–42) for IGK, 22.0 (range,
0–67) for TCRD, and 14.0 (range, 2–62) for
TCRG. The mean size of the PCR product was 174 bp (range,
106–239 bp).
ASO-PCR on a Block Thermocycler.
To compare ASO-PCR data obtained by the LightCycler technology with
conventional ASO-PCR data, we analyzed 35 ASO-PCRs on a conventional
block thermocycler (Tables 2
and 3)
. The conventional ASO-PCR revealed
a detection limit of 10-3 in 2 cases (6%),
10-4 in 16 (46%), 10-5
in 15 (43%), and 10-6 in 2 (6%). The mean
number of cycles optimal for MRD analysis of IGH
rearrangements (end point) was 28, whereas that for
IGK/TCRD/TCRG was 23. The mean number
of ASO-PCR experiments necessary for the determination of the optimal
end point was 3.7.
ASO-PCR on the LightCycler.
Representative results of ASO-PCR on the LightCycler and a comparison
with conventional PCR data are depicted in Figs. 2
3
4
5
. Fig. 2
illustrates typical ASO-PCR results of two different
junctional regions of the TCRD and TCRG genes
obtained on the LightCycler. Fig. 2B
shows the amplification
profiles. The fluorescence (F1, fluorescence channel 1 for SYBR Green
I) is depicted on the Y axis, whereas the X axis
shows the number of PCR cycles. As expected, more PCR cycles are
required to amplify target DNA in a sample containing less template
DNA. For example, it takes 
15 PCR cycles before fluorescence can be
detected in the highest dilution sample of patient 32, which contains
the lowest amount of template DNA (Fig. 2B
,
1E-6), whereas fluorescence at a lower dilution containing a
high amount of template DNA (Fig. 2B
, 1E-1) can
already be measured after 5 PCR cycles. On the other hand, samples
containing a high amount of template DNA reach their plateau after
fewer cycles than those containing a smaller amount of template DNA.
When one of the PCR reactions of an MRD experiment has reached its
plateau, the linearity of such a reaction is lost, and in case of a
conventional ASO-PCR reaction, additional PCR reactions will have to be
performed. Because the LightCycler measures the amount of generated PCR
product after every cycle, such steps will not be required, thus saving
time and manpower. After 18 (patient 32) or 24 (patient 17) cycles,
target DNA is also amplified from control BC DNA.
|
|
|
|
).
For each sample with a known template concentration, the PCR cycle at
which the amplification enters the log linear region is determined
(starting point of the amplification curve; see Fig. 2B
).
The cycle number at which an adjustable baseline (crossing line) and an
amplification curve cross each other is defined as the crossing point
(Fig. 2B
). By plotting the crossing points of the serial
dilutions against the relative number of target molecules in the serial
dilutions, a calibration curve is obtained (Fig. 2C
). By
varying the baseline of the amplification curve, we determined the
calibration curve with optimal linearity, which was used for
quantification of the bone marrow follow-up samples.
Fig. 2A
shows the negative derivatives of melting curve
characteristics of two different junctional regions of the
TCRD and TCRG genes at the end of the PCR
reaction (35 cycles), respectively. The two peaks (83°C) represent
the Tm, i.e., the temperature at which
50% of the DNA PCR product melted. The melting curve shows only one
peak, indicating that only one target-specific PCR product was
amplified.
During the analysis of various samples with ASO-PCR by the LightCycler
technology, we observed three different types of melting curves (Figs. 3
4
5
). Fig. 3B
shows for patient 121C a type of melting curve
similar to the one discussed previously for Fig. 2
after a standard PCR
protocol of 35 cycles. There is only one melting peak, the
H2O1 control is negative, and BC shows the same
melting characteristics in all dilution steps (only
10-1 is depicted; melting curve profile type A
in Tables 2
and 3
). The corresponding conventional ASO-PCR reaction is
shown in Fig. 3A
. The sensitivity after 25 cycles was
10-4. Evaluation of MRD levels by visual
inspection and densitometric analysis indicated
10-2 residual blasts for remission sample 1
(CR1; 5 weeks after initial diagnosis), 10-4 for
CR2 (13 weeks), and a negative result (below
10-4) for CR3 (21 weeks). The amplification
profiles are illustrated in Fig. 3C
. The crossing points of
the templates 10-5, 10-6,
and BC are close together, indicating that the detection limit is
10-4, identical to that determined by
conventional ASO-PCR. Examination of the crossing points for the three
remission samples (CR1–3) revealed the same MRD levels as discussed
for Fig. 3A
and (Table 4)
.
|
depicts an example of the second type of melting profile
(melting curve profile type B; Tables 2
and 3
). The melting curves of
the ASO-PCR on the LightCycler after 35 cycles clearly show two
different peaks, suggesting the presence of two different DNA products.
DNA gel electrophoresis of this PCR material demonstrated (not shown)
the presence of small amplification products (primer-dimer) in the
10-5, 10-6, BC, and
H2O1 samples, demonstrating the melting curve
characteristics on the LightCycler. Gel electrophoresis of PCR material
after 30 cycles (Fig. 4A
) does not show respective
primer-dimers. These data indicate that the additional amplification
product in the three samples does not represent the amplification of
the target DNA (junctional region) and must therefore be omitted. The
amplification profiles are depicted in Fig. 4C
. The MRD
level of this patient (patient 27) after termination of treatment (CR1)
was quantified as 2.8 x 10-2.
Fig. 4A
shows the results of a conventional ASO-PCR.
Quantification of the serial dilutions is almost impossible, making it
very difficult to determine the MRD level (between
10-1 and 10-3) of the
remission sample (CR1).
Fig. 5B
(patient 127C) shows an example of the third type of
melting profile after 35 cycles (melting curve profile type C; Tables 2
and 3
). The melting curves show peaks at three different melting
temperatures (Tm); H2O1 at
82°C, serial dilutions at 87°C, and BC at 89°C. The
origins of the first two peaks have already been discussed. The
explanation for the third peak can be derived from the conventional
ASO-PCR results shown in Fig. 5A
. After 25 cycles, an
amplification product is observed (not visible after 20 cycles) that
differs in size from the amplified target sequence and represents a PCR
artifact that should be omitted in the amplification profile and
further quantification analyses.
Comparison of Sensitivities between Conventional and LightCycler
ASO-PCR.
For comparison of the conventional ASO-PCR on a block thermocycler and
ASO-PCR by LightCycler technology, the same target sequences were
amplified using identical primers. The sensitivities of the two methods
are comparable (Tables 2
and 3)
. In eight ASO-PCR reactions, the
sensitivity of the LightCycler ASO-PCR was superior to that of the
conventional ASO-PCR. The sensitivity of the LightCycler ASO-PCR ranged
from 10-4 to 10-6: 15
rearrangements with 10-4 (43%), 14 with
10-5 (40%), and 6 with
10-6 (17%).
MRD Assessment of Remission Samples.
Twenty-seven bone marrow follow-up samples from 15 ALL patients were
monitored by conventional ASO-PCR followed by gel electrophoresis and
by ASO-PCR with LightCycler technology (Table 4)
. This MRD analysis has
been included in the present report (a) to demonstrate the
accurate determination of MRD levels in various CR samples from several
ALL patients by LightCycler technology and (b) to compare
both methods. It was not our intention to draw clinical conclusions
from the limited number of patients and respective bone marrow
follow-up samples. In most of the cases, MRD levels as determined by
both methods match fairly well. However, in some patients
(e.g., patient 27; Fig. 4
) it was almost impossible to
determine the precise MRD level by conventional ASO-PCR because serial
dilutions reached an early plateau level, indicating an advantage of
the LightCycler approach. The MRD data from patient 7 are likewise
notable. The bone marrow follow-up sample at 33 weeks scored negative
for the IGK marker (sensitivity,
10-4) and the IGH rearrangement
(sensitivity, 10-5) by conventional ASO-PCR,
whereas the IGH analysis by LightCycler technology
(sensitivity, 10-6) showed MRD of 1.8
x 10-6. In fact, this patient relapsed
after the termination of treatment.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
.
Its major drawback, clonal evolution, can be compensated by application
of at least two different markers. Despite the small number of
N-nucleotides in the junctional region of IGK-Kde
rearrangements, the sensitivity of this marker in LightCycler analysis
is comparable to that of dot-blot analysis (21
, 22)
. Even
in case 47, exhibiting an insertion of only 2 bp at the V9-J1.3
junctional region, a sensitivity of 10-4 was
obtained. In line with our previous notion (4)
, case 7
clearly demonstrates the relevance of monitoring MRD at the most
sensitive level that can be achieved (Table 4)
. Compared with dot-blot analysis and conventional ASO-PCR, LightCycler technology is less laborious, nonradioactive, and rapid. Steps such as spotting of serial dilutions, radioactive labeling of oligonucleotides, hybridization, washing of filters, autoradiography, DNA gel electrophoresis, photography, quantification, and repetition of the ASO-PCR to determine the optimal number of cycles (conventional ASO-PCR) can be omitted. The MRD data are available immediately after a run on the LightCycler, and no post-PCR processing steps are required. Moreover, ASO-PCR analysis on a LightCycler allows real-time and on-line monitoring, i.e., fluorescence (generated PCR product) is measured after each cycle, enabling reliable quantification of the remission status.
A minor disadvantage of the LightCycler is its small reaction volume
(20 µl), which limits the initial amount of DNA (200 ng) that can
be analyzed and hence its sensitivity. For this reason we used a nested
PCR approach and started with a first round of PCR in a normal block
thermocycler with 1 µg of target DNA, followed by a second round of
PCR in the LightCycler. Because each clone-specific oligonucleotide
(ASO) is characterized by a distinct melting temperature and behaves
differently in a PCR reaction, it is also not possible to design
standard ASO-PCR protocols. An additional, albeit infrequent problem is
the amplification of non-target sequences, as depicted in Fig. 5
(melting curve type C). This problem increases with the number of
cycles performed (Fig. 5A
; compare gel electrophoreses data
at 20 and 25 cycles, respectively). At high cycle numbers, such as the
35 cycles usually used for LightCycler ASO-PCR analysis, target and
non-target PCR products may reach plateau levels, thereby limiting the
reliable quantification of the data. However, melting curve analysis at
the end of the PCR run easily allows the detection of this problem, and
reduction of the number of PCR cycles can circumvent this shortcoming.
Albeit less likely, a non-target PCR product with an identical
Tm as a specific target PCR product will become
invisible in a melting curve analysis. In this case, BC and lower
dilutions will show high background levels and display melting curve
profile type A (Fig. 3
). As a consequence, the sensitivity of the
LightCycler ASO-PCR will be low.
Recently, a real-time quantitative PCR strategy for the detection of MRD in ALL using junctional regions based on TaqMan technology was published (13) . Its sensitivity was compared with that of two conventional methods, i.e., dot blot and liquid hybridization of amplified immunoglobulin/TCR gene rearrangements using clone-specific radioactive probes. The TaqMan technology allowed the reproducible and quantitative detection of MRD. The sensitivity was comparable to that of the dot-blot method, but was less sensitive than liquid hybridization. The sensitivity of the LightCycler ASO-PCR method described here appears higher. However, one should take into account that only a small number of junctional regions (n = 10) were investigated in the previous study (13) . The costs of the relatively expensive fluorochrome-labeled TaqMan probes should likewise be considered.
It has been reported that TaqMan reverse transcription-PCR and
fluorescent probe design can be used in the LightCycler system
(16)
. Whether the application of TaqMan or LightCycler
hybridization probes (HybProbe) can further improve the sensitivity of
MRD analysis is still under investigation. The design and testing of
two optimal hybridization probes, each with 25 nucleotides covering
the junctional region, might be a disadvantage for the HybProbe
technique of the LightCycler. Hybridization characteristics and
primer-dimer formation of the four oligonucleotides in the reaction
mixture might lead to a loss of sensitivity. One should also consider
that hybridization probes add only one single label to a PCR product,
whereas multiple SYBR Green I labels intercalate in a PCR fragment,
suggesting that SYBR Green I represents a more sensitive approach.
A major and inevitable problem of MRD analysis is posed by the presence of normal B or T lymphocytes with immunoglobulin or TCR rearrangements similar to those characterizing the leukemic target cells. The application of hybridization probes cannot solve this problem. One of the advantages of HybProbes is that primer-dimers will not result in false-positive signals. Although primer-dimer formation does not occur very frequently, the application of HybProbes could in this case lead to an improvement of sensitivity. However, primer-dimer formation usually occurs in more highly diluted DNA samples with negligible amounts of MRD product and is therefore virtually of no significance. However, in PCR reactions using hybridization probes, primer-dimer formation may occur, although not visibly, and still interfere with the PCR reaction. In the case of SYBR Green I ASO-PCR, primer-dimers will be detected and provide the opportunity to design a new set of primers.
Because the purchase of fluorescence-labeled oligonucleotides is rather expensive, we decided to use the more cost-effective variant, i.e., the SYBR Green I detection method. Taking into account (a) the rather high sensitivity of our LightCycler ASO-PCR method, (b) the fact that the sensitivity threshold theoretically is maximally 10-5 or 10-6, and (c) the sometimes very limited variability of the junctional regions, we conclude that the optimal sensitivity has almost been reached. The amount of DNA that can be tested in a reaction vial determines the threshold level. Testing higher amounts of DNA in one reaction vial or raising the number of reaction vials analyzed per patient appears to be the only way to further increase the threshold level. Our study demonstrates that "real-time" ASO-PCR by LightCycler technology is a rapid, reliable, sensitive, and cost-effective approach for the routine monitoring of MRD in ALL patients.
|
FOOTNOTES |
|---|
1 This work was supported by grants from the
Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft (to
C. R. B.). M. N. is a recipient of a fellowship from the Alexander
von Humboldt-Stiftung. 
2 To whom requests for reprints should be
addressed, at Institute of Human Genetics, University of Heidelberg, Im
Neuenheimer Feld 328, D-69120, Heidelberg, Germany. Phone
49-6221-565152; Fax: 49-6221-565155; E-mail: Cr_bartram{at}med.uni-heidelberg.de
3 The abbreviations used are: ALL, acute
lymphoblastic leukemia; MRD, minimal residual disease; TCR, T-cell
receptor; ASO, allele-specific oligonucleotide; BC, buffy coat;
IGH, immunoglobulin heavy chain gene; Kde, -deleting
element; IGK, immunoglobulin gene;
TCRD, TCR- gene; TCRG, TCR- gene;
CR, complete remission; RSS, recombination signal sequence.
Received 10/ 1/99. Accepted 4/ 7/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(IGK) gene rearrangements in acute lymphoblastic leukemia. Leukemia, 14: 218-219, 2000.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. A. Morley, S. Latham, M. J. Brisco, P. J. Sykes, R. Sutton, E. Hughes, V. Wilczek, B. Budgen, K. van Zanten, B. J. Kuss, et al. Sensitive and Specific Measurement of Minimal Residual Disease in Acute Lymphoblastic Leukemia J. Mol. Diagn., May 1, 2009; 11(3): 201 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Steinbach, A. Schramm, A. Eggert, M. Onda, K. Dawczynski, A. Rump, I. Pastan, S. Wittig, N. Pfaffendorf, A. Voigt, et al. Identification of a Set of Seven Genes for the Monitoring of Minimal Residual Disease in Pediatric Acute Myeloid Leukemia Clin. Cancer Res., April 15, 2006; 12(8): 2434 - 2441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bruggemann, T. Raff, T. Flohr, N. Gokbuget, M. Nakao, J. Droese, S. Luschen, C. Pott, M. Ritgen, U. Scheuring, et al. Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia Blood, February 1, 2006; 107(3): 1116 - 1123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S L Kong, M Salto-Tellez, A P K Leong, Y H Chan, and E S C Koay Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications J. Clin. Pathol., August 1, 2005; 58(8): 839 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Y. Yang, D. Xu, J. Du, H. Kamino, J. Rakeman, and H. Ratech Rapid Detection of Clonal T-Cell Receptor-{beta} Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis J. Mol. Diagn., February 1, 2005; 7(1): 81 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Wallace, M. I. Block, W. Gillanders, J. Ravenel, B. J. Hoffman, C. E. Reed, M. Fraig, D. Cole, and M. Mitas Accurate Molecular Detection of Non-small Cell Lung Cancer Metastases in Mediastinal Lymph Nodes Sampled by Endoscopic Ultrasound-Guided Needle Aspiration Chest, February 1, 2005; 127(2): 430 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Wallace, M. Block, B. J. Hoffman, R. H. Hawes, G. Silvestri, C. E. Reed, M. Mitas, J. Ravenel, M. Fraig, S. Miller, et al. Detection of Telomerase Expression in Mediastinal Lymph Nodes of Patients with Lung Cancer Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1670 - 1675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Astier, R. Xu, M. Svoboda, E. Hinds, O. Munoz, R. de Beaumont, C. D. Crean, T. Gabig, and A. S. Freedman Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival Blood, February 1, 2003; 101(3): 1118 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xu, J. Du, C. Schultz, A. Ali, and H. Ratech Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System J. Mol. Diagn., November 1, 2002; 4(4): 216 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Husson, E. G. Carideo, D. Neuberg, J. Schultze, O. Munoz, P. W. Marks, J. W. Donovan, A. C. Chillemi, P. O'Connell, and A. S. Freedman Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays Blood, January 1, 2002; 99(1): 282 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Watzinger, E. Horth, and T. Lion Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site Nucleic Acids Res., June 1, 2001; 29(11): e52 - e52. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Seeger, K.-A. Kreuzer, U. Lass, T. Taube, D. Buchwald, C. Eckert, G. Körner, C.-A Schmidt, and G. Henze Molecular Quantification of Response to Therapy and Remission Status in TEL-AML1-Positive Childhood ALL by Real-Time Reverse Transcription Polymerase Chain Reaction Cancer Res., March 1, 2001; 61(6): 2517 - 2522. [Abstract] [Full Text]
|
|
|
|
|
|
M. Germeshausen, M. Ballmaier, H. Schulze, K. Welte, T. Flohr, K. Beiske, I. Storm-Mathisen, and T. G. Abrahamsen Granulocyte colony-stimulating factor receptor mutations in a patient with acute lymphoblastic leukemia secondary to severe congenital neutropenia Blood, February 1, 2001; 97(3): 829 - 830. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
