Note: Descriptions are shown in the official language in which they were submitted.
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Title: Detection of minimal residual disease in lymphoid
malignancies.
This invention relates to the field of cancer
diagnosis, more specifically to the monitoring of disease
development during and after treatment.
Cytostatic or cytotoxic treatment induces remission
in the majority of patients with lymphoid malignancies.
Nevertheless many of these patients relapse. Apparently
the current cytostatic or cytotoxic treatment protocols
are not capable of killing all malignant cells in these
relapsing patients, although they reached so-called
complete remission according to cytomorphoiogical
criteria. Since the detection limit of cytomorphological
techniques is not lower than 1-5o malignant cells, it is
obvious that such techniques can only provide superficial
information about the effectiveness of treatment.
Techniques with a higher sensitivity to detect "minimal
residual disease" or minimal disease (MRD) are needed to
obtain better insight in the reduction of tumor mass
during induction treatment and further eradication of the
malignant cells during maintenance treatme::t (Figure 1).
Techniques for detection cf "minimal residual disease"
(MRD) at levels of 10-3 to 10-5 (one malignar.~ cell between
103 to 105 normal cells) during follow-up c~ children with
acute lymphoblastic leukemia (ALL) can provide insight
into the effectiveness of cytostatic treatment. However
it is not yet clear whether and how MRD information can
be applied to the clinical decision process, e.g. for
stratification of treatmer.~ protocols. We monitored 240
childhood ALL patients who were treated according to
national protocols of the International BFT" Study Group.
Sixty patients relapsed and the patients i:-: continuous
complete remission (CCR) had a median ever. free follow-
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up of 48 months. Bone marrow (BM) samples were collected
at up to 9 time points during and after treatment: at the
end of induction treatment, before consolidation
treatment, before re-induction treatment, before
maintenance treatment, three samples during maintenance
treatment, at the end of treatment, and one year after
cessation of treatment. Classical polymerise chain
reaction (PCR) analysis of patient-specific
immunoglobulin and T cell receptor gene rearrangements
and TAL1 deletions were used as targets for semi-
quantitative estimation of MRD levels: >-10-2, 10-3, and _<
10-4 together with radioactive patient-specific functional
region probes. MRD negativity at the various follow-up
time points was associated with low relapse rates (3-
16%), but five to fourteen-fold higher relapse rates (41-
86%) were found in MRD positive patients. The distinct
MRD levels appeared to have independent prognostic value
(p (trend) <0.001) at all time points. Especially at the
first two time points three-fold higher relapse rates
were found in patients with high tumour loads (>_10-?) as
compared to patients with low tumour loads (s10-9). At
later time points (including the end of treatment) also
low tumour loads were associated with a high relapse
rate. Positivity in CCR patients after treatment was
rarely (<1%) observed, even when multiple sensitive PCR
analyses were performed. Finally, using the combined MRD
information of the first two follow-up time points, it
was possible to recognise a low risk group comprising 43%
of the analysed patients with a relapse rate of only 2%
and a high risk group of 15% of patients with a relapse
rate of 84%. Our MRD study unequivocally demonstrated
that monitoring of childhood ALL patients at multiple
time points gives clinically relevant insight into the
effectiveness of treatment. Combined MRD information of
the first 3 to 4 months of treatment allows identifica
tion of good prognosis and poor prognosis groups of
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substantial size, which might profit from treatment
adaptation.
However, MRD techniques need to have at least a
sensitivity of 10-4 to allow for earlier identification of
patients belonging to high and especially to low risk
groups, allowing a clinical decision process that would
allow assigning the proper treatment to the proper
patient. This would allow to reduce false-negative
results and avoid being too late with therapy when a
patient with high risk has not yet been detected as such,
and would allow reducing false positive results and avoid
cumbersome therapy in patients with low risk.
Furthermore, more sensitive MRD techniques would allow
sampling patients via routine blood sampling, instead of
by the painfull or cumbersome bone marrow aspiration
currently employed.
Replacement of BM sampling by blood sampling has been a
topic of debate in MRD studies for over a decade. Initial
immunophenotyping studies in T-ALL and acute myeloid
leukemias indicate that MRD levels in blood are generally
less than one 1°log lower than in BM. Recent PCR studies
show that also in precursor-B-ALL this difference is 1 to
1.5 1°log. This would imply that MRD techniques need to be
at least approximately one 1°log more sensitive (i.e. __<10-
5), when blood samples are monitored. Alternatively,
bigger samples (i.e. more DNA) should be analyzed but
this is too laborious and time consuming, unless a semi-
automated system is used.
If multiple BM samples are analysed during follow-up,
steady decrease of MRD levels to negative PCR results is
associated with a favourable prognosis, whereas
persistence of MRD generally leads to clinical relapse.
Low MRD levels after therapy might be associated with
late relapse, but absence of MRD at the end of treatment
is not sufficient to predict that the patient is cured. A
single time point of MRD analysis is not sufficient for
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recognition of patients with a good prognosis and
patients with a poor prognosis. However, information
about the kinetics of tumour reduction is needed for MRD-
based risk group identification and thereby provides new
openings for treatment stratification. The MRD-based low
risk patients might profit from treatment reduction. On
the other hand, the group of MRD-based high risk patients
might benefit from intensive treatment protocols,
including stem cell transplantation. Therefore, follow-up
l0 samples need to be collected during and after treatment
for obtaining insight into the kinetics of tumour
reduction and for determining the risk of relapse per
patient.
During the last decade several methods for
detection of MRD have been developed and evaluated, such
as cytogenetics, cell culture systems, fluorescence in
situ hybridization (FISH), Southern blotting
immunological marker analysis, and polymerase chain
reaction (PCR) techniques. The detection limit of most
techniques is not lower than 1-5~ malignant cells.
However, three types of techniques can detect so-called
"minimal residual disease" at more sensitive levels:
1. flow cytometric immunophenotyping, detecting aberrant
or unusual protein expression by the malignant cells; 2,
polymerase chain reaction (PCR-) based detection of
breakpoint fusion regions of chromosome aberrations; and
3. detection of clone-specific immunoglobulin (Ig) and T
cell receptor (TCR) gene rearrangements via PCR
amplification. The last technique has the broadest
applicability in lymphoid malignancies and is most
frequently used in MRD studies so far (Table 1).
Rearrangements in Ig and TCR genes result in unique
combinations of the many available variable (V),
diversity (D), and joining (J) gene segments; the
junctions between these gene segments form the so-called
"functional regions", which can be regarded as
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"fingerprint-like" sequences due to deletion and random
insertion of nucleotides during the rearrangement
process. PCR analysis of functional regions generally
reaches sensitivities of 10-4.
5 PCR analysis of a specific chromosome aberration
can use primers at opposite sites of the fusion regions
of the breakpoints e.g. TAL1 deletion, t(14;18),
t(11;14), t(1;14), and t(10;14). At the fusion regions of
breakpoints of chromosome aberrations in lymphoid
malignancies a random deletion and insertion of
nucleotides may have occurred similar to the functional
regions of Ig and TCR gene rearrangements.
Several retrospective and some limited prospective
studies indicate that MRD detection in lymphoid
malignancies has prognostic value. Sofar, the prognostic
value of MRD detection has especially been studied in
patients with non-Hodgkin lymphoma (e. g. before and after
bone marrow transplantation) and in patients with acute
lymphoblastic leukemia (ALL). Absence of MRD in ALL
patients after induction therapy is suggested to predict
good outcome. However approximately half of the ALL
patients are still positive at that time. Therefore the
level of MRD positivity was evaluated and found to have
predictive value. If multiple bone marrow samples are
repeatedly analysed during follow-up, steady decrease of
MRD levels to negative PCR results is associated with
favorable prognosis, whereas persistence of MRD generally
leads to clinical relapse. MRD data of a large prognostic
study of ALL patients show that high tumor loads (>103) at
two successive time points in the early phase of therapy
result in a RFS (relapse free survival) of only 250. In
contrast, low levels of MRD (<10-4) or MRD negativity on
both these time points result in RFS of -95~s. This
indicates that the kinetics of disappearing cells
predicts relapse and that the tumour load of a particular
patient should be identified accurately, i.e.
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quantitative MRD information is essential, therefore a
need exists for a rapid, sensitive and quantitative
method to determine the presence of malignant cells in
patient, i.e. the detection of minimal residual disease.
The present invention provides a method for the
detection of minimal residual disease by determining the
presence of malignant cells in a sample comprising
amplification of nucleic acid molecules corresponding to
common rearranged gene segments using for example in a
PCR a forward and/or reverse primer reactive with said
gene segments and further comprising the identification
of malignancy-specific nucleic acid molecules found among
those rearranged gene segments by hybridisation with a
fluorogenic probe specifically and selectively reactive
with said malignancy-specific nucleic acid molecules
(note that malignancy-specific is in most cases also
patient-specific, since a malignancy in general occurs in
a patient-specific manner).
The current MRD techniques, including immunological
marker analysis and current PCR techniques have several
disadvantages which make a prognostic assessment
inaccurate. Immunological marker analysis is only
applicable to a restricted set of patients for which
appropriate immunological tools exist. Furthermore, the
main problem with the current PCR protocols is the fact
that PCR, albeit very sensitive, is mainly qualitative
and its results can therefore not easily be related to
precise frequencies of malignant cells. Only when limited
dilution techniques are used in PCR or competitive PCR is
applied, mixing a standard nucleic acid with the sample
nucleic acid, semi-quantitative detection by endpoint
analysis of relative amounts of amplified target.
However, this is not only time-consuming and very costly
but also inaccurate and difficult to standardize.
Furthermore, the current PCR techniques are sensitive to
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contamination, require a long processing time and often
use radio-actively labelled probes.
The invention provides a method for the detection
of rriinimal residual disease by determining the presence
of malignant cells in a sample by detecting amplified
nucleic acid molecules via real-time quantitative nucleic
acid amplification (e. g. using the ABI PRISM Sequence
Detection System and the Light Cycler system), which
avoid using radio-active isotopes and contamination and
have a short processing time, and allow detection of
specifically amplified nucleic acid sequences.
Samples in which a specific nucleic acid molecule
needs to be detected are subjected to amplification (for
example by real-time quantitative (RQ-)PCR) using a
primer pair specific for said molecule. Detection of the
thus amplified molecule occurs fluorogenically in real
time (i.e. during each amplification cycle) via a
fluorogenic probe consisting of one of more
oligonucleotides. Only detection in real-time allows
reliable and reproducible quantification of MRD. RQ-PCR
makes use of data generated in the early productive PCR
cycles where the fidelity of the PCR is still high. It
enables detection with a high throughput of samples
allowing automated testing. Samples are not restricted to
bone marrow samples but can also be blood samples or
other cell samples. In a preferred embodiment of the
invention, a patient- or malignancy-specific fluorogenic
probe according to the invention is used.
One example exploits the exonuclease activity of
the Taq polymerase. The so-called TaqMan fluorogenic
probe consists of an oligonucleotide to which a reporter
dye and a quencher dye are attached. During
amplification, the probe anneals to the template molecule
somewhere between the location of the forward and reverse
primer sites. However, during the amplification process
the annealed probe is cleaved by the 5' nuclease activity
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of the polymerase. This separates the reporter dye from
the quencher dye, generating an increase in the reporter
dye's fluorescence, which finally allows real time
detection of the amplified molecule, with an increase of
the fluorescent signal per PCR cycle.
Another example is based on the proximity of two
oligonucleotides, together forming a fluorogenic probe
for fluorescence emission. The first oligonucleotide is
labelled with a donor fluorochrome that is excited by an
external light source and emits light that is absorbed by
an acceptor fluorochrome present on a.second
oligonucleotide. When the first and second
oligonucleotide are in close proximity they together form
a fluorogenic probe according to the invention which
comprises an oligonucleotide specifically and selectively
reacting with a functional or fusion region which is only
found with a specific patient and which is characteristic
for his or her malignancy. During amplification of the
target molecule the so-called fluorescence resonance
energy transfer (FRET) probe consisting of both
nucleotides hybridises to the nucleic acid between the
two primers to detect the amount of target, resulting in
emission of light by the acceptor fluorochrome. During
the annealing phase of each PCR cycle the amount of
fluorescence is a measure of the amount of PCR product
formed so far. The fluorescent signal disappears when the
probe dissociates during the extension phase of the PCR
cycle. In this way the fluorogenic probe allows real-time
quantitative detection of the PCR-target during the
annealing phases.
Samples that contain the wanted nucleic acid
molecules that are reactive with said forward and reverse
primer are thus identified by the presence of amplified
product which is detected in real time by using said
fluorogenic probe.
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Another embodiment of the invention is a method
wherein the fluorescence of said probe is detected in
real-time, during execution of the amplification,
allowing the sensitive and quantitative detection of
minimal residual disease to allow for earlier
identification of patients belonging to high or low risk
groups, allowing a clinical decision process that allows
assigning the proper treatment to the proper patient.
This allows to reduce false-negative results and avoids
being to late with therapy when a patient with high risk
has not yet been detected as such, and allows reducing
false positive results since the probe is malignancy-
specific and cross-contamination is reduced due to the
absence of post-PCR processing and avoids cumbersome
therapy in patients with low risk.
The invention also provides a fluorogenic probe
which comprises an oligonucleotide specifically and
selectively reacting with a functional or fusion region
which is only found with a specific patient and which is
characteristic for his or her malignancy. One embodiment
of the invention, exemplified in the experimental part,
is a method wherein said fluorogenic probe is linked to a
reporter dye and to a quencher dye, however in another
embodiment, said probe comprises an oligonucleotide-
linked donor fluorochrome and acceptor fluorochrome.
Another embodiment of the invention is a method wherein
said fluorogenic probe is reactive with a functional
region or fusion region of a malignancy-specific
rearranged gene segments of Ig or TCR gene rearrangements
or chromosome aberrations. Such a probe provided by the
invention and/or a method provided by the invention is
specifically used to detect and quantify the level of
MRD, for example by using the ABI PRISM sequence
detection system or the Light Cycler.
The invention also provides said probes in the
context of a diagnostic assay, comprising necessary means
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and methods to use a method provided by the invention.
Said diagnostic assays or kit may also comprise primers,
enzymes, buffers or other components that are necessary
for the amplification of the rearranged gene segments.
5 The invention is further explained in the
experimental part, which cannot be seen as limiting the
invention.
EXPERIMENTAL PART
INTRODUCTION
Thanks to advances in modern chemotherapeutic treatment
regimens during last decades complete clinical remission
can be induced in virtually all children and 75-80% of
adults diagnosed with acute lymphoblastic leukemia (ALL).
However, one third of pediatric and more than half of
adult ALL patients is suffering from disease recurrence
and further treatment intensification leads to increased
treatment-related toxicity and secondary malignancies. In
order, to further increase the survival along with an
improvement of the quality of life by preventing late
tonicities, an individualization of the treatment may be
needed. This can be realized by applying the results of
minimal residual disease (MRD) that allows the detection
of leukemia invisible to normal morphologic examination,
thereby providing more insight in the efficacy of
cytostatic treatment. MRD analysis can predict outcome by
determining the reduction of the leukemic burden during
the first months of therapy. Methods that allow sensitive
MRD detection are (i) flow cytometric detection of
leukemia-specific immunophenotypes, (ii) polymerase chain
reaction (PCR) amplification of leukemia specific
chromosomal aberrations, and (iii) PCR amplification of
clonogeneic rearrangements of immunoglobulin (Tg) and T-
cell receptor (TCR) genes. The last method has the
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broadest applicability in ALL. Using PCR techniques it is
possible to detect one leukemic cell in a background of
approximately 105 normal cells. This is about 100 to
10,000 times more sensitive than obtained with
morphology.
Immature B and T lymphocytes rearrange the V, D, and
J gene segments of their Ig and TCR genes in order to
achieve antigen diversity. A molecular fingerprint is
provided by the deletion and insertion of random
nucleotides between the joined gene segments, the so-
called functional regions. In principal, all cells from a
leukemia have the same functional region, since they
derive from one oncogenic progenitor. Thus, functional
regions of Ig/TCR gene rearrangements can be regarded as
leukemia specific DNA fingerprints. Yet, oligoclonality
of Ig/TCR gene rearrangements at diagnosis may occur,
since these rearrangements are not linked to the
oncogenic process. Furthermore, continuing rearrangements
and secondary rearrangements during the disease course
might result in the loss of the functional regions
initially identified at diagnosis. It therefore seems
important to monitor ALL patients with two or more
independent monoclonal Ig/TCR PCR targets to prevent
false-negative results during follow-up.
A patient specific PCR primer or probe is usually
designed to the sequence of the functional region in
order to detect the leukemia within the background of
normal cells that may have similar gene rearrangements
but different functional regions. Especially, the usage
of a patient-specific functional region probe has shown
to be highly effective in the detection of MRD. After PCR
amplification of the Ig/TCR gene rearrangement, the PCR
product is hybridized with the radioactively labeled
patient specific probe. For that purpose, the PCR product
is blotted after gel-electrophoresis or directly spotted
on a nitrocellulose membrane, the so-called dot-blot
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method. Alternatively, the PCR product is not fixed but
hybridized as free DNA in liquid hybridization.
Although the high sensitivity of these MRD-PCR
techniques, they provide merely semi-quantitative data
owing to the analysis of end-point results. The PCR
technique has the ability to amplify target DNA up to a
plateau, but it consequently is impossible to define
precisely the initial amount of target DNA. Strategies
to overcome the limited quantitative potential of the
PCR are the performance of competitive PCR or limiting
dilution. Quantitation by competitive PCR is performed
by comparing the PCR signal of the specific target DNA
with that of known concentrations of an internal
standard, the competitor. Quantitative estimations with
the limiting dilution assay are established by using
serial dilutions in multiple replicates of the target
DNA. The dilution endpoint defines the amount of initial
target DNA via Poisson's law. Both approaches are
laborious, require multiple PCR analysis per sample and
are difficult to perform routinely.
By several large prospective clinical MRD studies
in childhood ALL it was shown that it is important to
precisely determine the level of MRD to be able to
discriminate between low and high risk patients. This
was especially true if early remission time points were
analyzed; at later time points also low levels of MRD
have a risk of disease recurrence. By using the kinetics
of tumor reduction during the first three months of
therapy it was possible to recognize a low risk group
(~43~ of patients) with a relapse rate of only 20, an
intermediate risk group (~43%) with a relapse rate of
250, and a high risk group (~15~) with a relapse rate of
75~. In the light of these results we were aiming at a
fast method that would generate reliable quantitative
MRD data to provide the means for stratification of ALL
patients in the near future.
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Recently, a novel technology has become available,
the 'real-time' quantitative PCR (RQ-PCR). This assay
exploits the 5' - 3' nuclease activity of the Taq
polymerase to detect and quantify specific PCR products
as the reaction proceeds. Upon amplification, an internal
fluorogeneic TaqMan probe specific for the target
sequence is degraded resulting in emission of a
fluorescent signal that accumulates during the reaction.
Because of the real time detection, the method has a very
large dynamic range, over five orders of magnitude, of
initial target molecule determination. Thus, eliminating
the need for performing serial dilutions of follow-up
samples. Quantitative data can be accomplished in a short
period of time, since post-PCR processing is not
necessary.
MATERIALS AND METHODS
Patients and Cell samples
Four precursor-B-AhL (2145, 5160, 5199 and 5257)
were randomly picked for this study, based on the
presence of one or more Ig/TCR gene rearrangements as
detected by Southern blot analysis. The probe and
restriction enzyme combinations used for Southern blot
analysis comprised: for the IGH locus the IGHJH6 probe in
BglII and BamHI/HindIII digests; for the TCRD locus the
TCRDJ1 probe in EcoRI and BglII digests; fox the TCRG
locus the Jyl.3 and Jy2.1 probes in an EcoRI digest and
the J1.2 probe in a BglII digest; for the IGK locus the
IGKJS, IGKC, and the IGKDE probes in BglII and
BamHI/HindIII digest. Mononuclear cells (MNC) were
obtained from peripheral blood (PB) or BM samples at
diagnosis by ficoll density centrifugation. MNC samples
were frozen and stored in liquid nitrogen. Good quality
medium molecular weight DNA was isolated from MNC samples
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using the QIAamp kit (Qiagen Inc, Chatsworth, CA)
(Verhagen et al., manuscript in preparation).
Patient specific probes
Sequences of the functional regions were obtained by
direct sequence analysis of the Ig/TCR gene
rearrangements with the dye terminator ready reaction
cycle sequencing kit on an ABI PRISM 377 automated
sequences of PE Biosystems. The template DNA used in the
sequence reaction was either the PCR product or a homo-
(or hetero-) duplex band excised and eluted from a
polyacrylamide gel in case of a bi-allelic gene
rearrangement. Based on the obtained sequence data
patient-specific oligonucleotides were developed
complementary to the sequence of the functional region
(Table 1). Oligonucleotides that were likely to form of
secondary structures were avoided.
OLIG05.1 software (Dr. W. Rychlik: National
Biosciences, Plymouth, MN) was applied to design the
probes that were radioactively end-labeled with [a-
32p~~Tp according to standard protocols. The Tm's of the
radioactive probes ranged between 65-68°C.
Fluorochrome labeled TaqMan probes were designed
with the Primer Express software (PE Biosystems). The
TaqMan probe did not start with a G and contained more
C's than G's according to the guidelines of PE
Biosystems. The melting temperature (Tm) was around 68°C,
8-9°C above the Tm of the matching primers, to ensure
proper hybridization to the target sequence. FAM was
chosen as reporter dye at the 5' end of the TaqMan probe
and TAMRA as the quencher dye at the 3' end.
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RQ-PCR analysis
Primer design
Of two ALL patients primers and probes were designed
5 for the detection of MRD using this new technique. From
patient 1 an IgH rearrangement was applied as PCR target,
from patient 2 an incomplete TCR-delta rearrangement. The
fluorogenic probes or TaqMan~ probes were positioned at
the functional region of mentioned rearrangement; for
10 patient 1
5'-TGGTACTACTACAACCCATCCGCGCATCT-3' and patient 2:
5'-ATCCCCCAGTCCGGGCACAGTA-3'. Compatible primers at
opposite of the functional region were chosen using the
Primer Express program (Applied Biosystems, Foster City,
15 CA, USA). For amplification of the IgH rearrangement in
patient 1 the forward primer: 5'-CACGGCTGTGTATTACTGTGCAA-
3' and the reverse primer: 5'-GGTCGAACCAGTACCCAATAGC-3'
were chosen giving a PCR product of 78 base pares. For
amplification of the incomplete TCR-delta rearrangement
the forward primer: 5'-GTACTTAAGATACTTGCACCATCAGAGA-3'
and the reverse primer 5'-GAAGCTGCTTGCTGTGTTTGTC-3' were
chosen and give a PCR product of 190 base pairs.
Primers matching to the designed TaqMan probes were
developed as above using Primer-Express software (PE
Biosystems) and had Tm's of 58-60°C. IGH-1 primers:
forward, 5'-CACGGCTGTGTATTACTGTGCAA-3' and reverse, 5'-
GGTCGAACCAGTACCCAATAGC-3'. IGH-2 primers: forward, 5'-
GAGGACACGGCTGTGTATTACTGT-3' and reverse, 5'-
ACCTGAAGAGACGGTGACCAT-3'. IGH-3 primers: forward, 5'-
GAGGACACGGCTGTGTATTACTGT-3' and reverse, 5'-
AGACGGTGACCAGGGTTCC-3'. IGK primers: forward for IGK-1,
5'-AGCAGGGTGGAGGCTGA-3', for IGK-2 5'-
GGTCAGGCACTGATTTCACACT-3', and reverse for both IGK-1 and
-2 5'-AAAAATGCAGCTGCAGACTCA-3'. TCRG primers (TCRG-1 and
-2): forward, 5'-GCATGAGGAGGAGCTGGA-3' and reverse, 5'-
GGAAATGTTGTATTCTTCCGATACTTAC-3'. TCRD-1 primers:
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forward, 5'-GTACTTAAGATACTTGCACCATCAGAGA-3' and reverse,
5'-GAAGCTGCTTGCTGTGTTTGTC-3'. TCRD-2 primers: forward,
5'-GCAAAGAACCTGGCTGTACTTAAG-3' and reverse, 5'-
GTTTTTGTACAGGTCTCTGTAGGTTTTGTA-3'. The forward and
reverse primers to detect and quantify MRD in follow-up
samples of patient 5257 by were . 5'-
TGTCAGCAGTATGGTAGCTCACC-3' and 5'-AGTGGATATGGCAAAAATGCA-
3', respectively.
PCR conditions
In a reaction mixture of 50 microliters containing lx
TaqMan buffer, 300 microM dATP, 300 microM dCTP, 300
microM dGTP, 600 microM dUTP, 1.25 U TaqGold (Perkin
Elmer, Norwalk, CT, USA), 2.5 pmol probe, 300 nM forward
primer, and 300 nM reverse primer, the rearrangements
were amplified with the following cycling protocol: 10
min. 95°C, followed by 15 sec. 95°C and 1 min. 60°C for
40 to 70 cycles.
For RQ-PCR analysis the TaqManTM PCR core reagent
kit was used (PE Biosystems). Reaction mixtures of 50 ~1
contained the RQ-PCR buffer with the ROX dye as the
passive reference, 5 mM MgCl2, dNTP's: 0.3 mM dATP, 0.3
mM dCTP, 0.3 mM dGTP, and 0.6 mM dUTP, 50-900 mM primers,
1.25 U AmpliTaq GoldTM (PE Biosystems), 1 U uracil-N-
glucosidase (UNG) and 50-1000 ng of DNA. The two-step
amplification protocol consisted of a 2 minutes
incubation step at 50°C (digestion of PCR product
contaminants by UNG),10 minutes at 95°C (inactivation of
UNG, denaturation of target DNA, and activation of
AmpliTaq GoldTM), followed by target amplification via
40-70 cycles of 15 seconds at 95°C and 1 minute at 60°C.
Real time information was obtained using the ABI
PRISM 7700 Sequence Detection System containing a 96 well
thermal cycler (PE Biosystems). During the PCR the TaqMan
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probe first hybridizes to the DNA target, followed by
primer annealing. With the TaqMan probe still intact, the
emission of the reporter dye is quenched, but during the
extension phase of the reaction the TaqMan probe is
cleaved by the exonuclease activity of the Taq
polymerase. Subsequently, a fluorescent reporter signal
is generated per cycle, which is proportional to PCR
product accumulation. The fluorescence intensity is
normalized using the passive reference ROX present in the
buffer solution. Normalization corrects for fluorescence
fluctuations which are PCR independent. A real time
amplification plot is generated using the normalized
reporter signal (Rn). The PCR product yield or ~Rn is
defined as the Rn minus the baseline signal established
in the first few cycles of the PCR and is at least ten
times the standard deviation of the noise. The cycle
threshold (CT) is the PCR cycle at which a statistically
significant increase in ~Rn is first detected.
To correct for the quantity and quality of DNA in
remission follow-up samples, we used the gene encoding
albumin. Sequences for primers and TaqMan probe, were
kindly provided by Dr. E. Wensink.
All RQ-PCR experiments were performed at least in
triplicate. Before determining the sensitivity of the PCR
target, the RQ-PCR was optimized. The primer
concentration affect the Tm of the primer and can thus be
used to optimize amplification efficiency on a fixed
annealing temperature. Therefore, the amount of forward
and reverse primer was determined that resulted in the
highest yield of specific PCR product. In this primer-
matrix experiment, nine combinations of 50, 300, and 900
mM for each primer were tested in triplicate, i.e. 50/50,
50/300, 50/900, 300/50, 300/300, 300/900, 900/50,
900/300, and 900/900.
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
18
PCR target sensitivity
To determine the sensitivity of the PCR target, DNA
from the sample at diagnosis was diluted in 10-fold steps
into DNA from normal mononuclear cells (MNC), down to 10
7. To avoid skewed gene rearrangement patterns and to
obtain a bulk of the polyclonal control, the normal MNC
DNA consisted of equivalent mixtures from ten different
healthy donors. The dilution series was subjected to
(RQ)PCR analysis together with appropriate positive and
negative controls. The furthest dilution of diagnosis DNA
that gave a radioactive or fluorescent signal, in the
absence of a signal from the polyclonal control (MNC
DNA), was defined as the sensitivity threshold of the
PCR-target. The sensitivity threshold, based on the
theoretical calculations, can be 10 4 (~8 copies of the
target gene) or 10 5 (~0.8 copies of the target gene).
Conventional MRD detection techniques
PCR amplification
The primers used for the PCR analysis of the Ig/TCR
gene rearrangements were described previously. For the
amplification of IGK, TCRD, and TCRG gene rearrangements,
1 ~tg DNA of diluted diagnosis material was used and 30
pmol of each primer in reaction mixtures of 100 ~tl
containing 1 unit AmpliTaq GoldTM was used (PE
Biosystems), 1.5 mM MgCl2, 200 ~,M dNTP (Pharmacia,
Uppsala, Sweden) in a final volume of 50 ~.1. The cycling
protocol consisted of 3 minutes of initial denaturation
at 92°C, followed by 40 cycles of 95 seconds at 92°C, 90
seconds at 60°C, 2 minutes at 72°C, and a final extension
phase of 10 minutes at 72°C.
Rearrangements of the IGH gene locus were amplified
in 50 ~t1 reactions containing, 1 ~g DNA, 30 pmol of each
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
19
primer, 2 units Taq polymerase, 2 mM MgCl2, and 200 ~M
dNTP. The cycling protocol consisted of 7 minutes of
initial denaturation at 95°C, followed by 30 cycles of 30
seconds at 95°C and 45 seconds at 55°C, and a final
extension phase of 7 minutes at 72°C.
PCR products were examined after gel electrophoresis
in 1% agarose and or 6-10% polyacrylamide gels and
ethidium bromide staining.
Dot-blot hybridization
After PCR amplification of the Ig/TCR gene
rearrangements, 5 ~1 of the denaturated PCR product was
spotted in duplicate onto a Nytran N13 nylon membrane of
0.45 ~.m (Schleicher & Schuell, Dassel) and cross-linked
by UV-exposure. The filter was hybridized at 50°C for two
hours with 0.5-1.0 pmol of the radioactively labeled
patient specific probe per ml hybridization buffer.
Filters were subsequently washed for 20 minutes in 3x
SSC, 0.1% SDS at 50°C. Radioactive signals were evaluated
by phosphor imaging (STORM-820, B&L Systems, Maarssen).
Liquid hybridization
Five ~1 of PCR product was hybridized with
approximately 1 ng of the radioactively labeled probe in
2x SSC buffer for 15 minutes at 60°C after denaturation
for 10 minutes at 95°C. Subsequently, the mixtures were
size separated by electrophoresis through a 10%
polyacrylamide gel. Radioactive signals were evaluated by
autoradiography after drying of the gels.
RESULTS
Only the amplified rearrangements of the leukemic cells
in patient 1 and 2 were detected by the ABI PRISM 7700,
and not similar rearrangements in the MNC that were co-
amplified, since the TaqMan probe only hybridizes with
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
the leukemia specific functional region. For both
patients a linear correlation was found between the
initial amount of leukemic derived DNA and the threshold
cycle. The threshold cycle is that cycle where the
5 fluorescence emitted during the amplification of the
target molecule rises above a certain threshold.
Despite consumption of primers and dNTP by the
amplification of similar rearrangements in MNC, in the 10
dilution still leukemic specific rearrangements could be
10 detected, both in patient 1 and in patient 2. Thus, using
this method it was possible to detect at least one
leukemic cell in 10,000 normal cells, which is
satisfactory to obtain reliable MRD information for a
diagnostic setting. This sensitivity is comparable to
15 what is achieved with current techniques using a
radioactively labeled functional region probe that
detects the leukemic cells after PCR amplification of the
rearrangement (MRD review Tomek, Leukemia manuscript).
The advantage of the new method is that two steps
20 (amplification and identification) are taken together,
this saves time so that MRD information is earlier
available for the clinic. The absence of post-PCR
processing of amplified DNA drastically reduces the risk
of contamination which otherwise can be a major problem
for MRD detection where samples are analyzed together
with high and low tumor loads. Another advantage of the
method is the substitution of radioactive probes by
fluorescent probes. Finally, the most important advantage
of this method is the possibility of precise
quantification of MRD in blood or bone marrow samples.
Nine Ig/TCR gene targets (3 IGH, 2 TCRD, 2 TCRG, and
2 IGK) of three precursor-H-ALL were examined for their
sensitivity. For all gene rearrangements it was possible
to develop primer/probe pairs that resulted in successful
amplification and real time detection upon RQ-PCR
analysis. For 4 out of 9 PCR targets it was necessary to
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
21
design the TaqMan probe complementary to the reverse
strand of the functional region, due to the high extent
of G's (Table 2). The initial primer pair to amplify
TCRG-1 and -2 PCR targets resulted upon RQ-PCR analysis
in low sensitivities (10-2), which was unexpected because
both gene rearrangements used the rarely used Vy7 gene
segment. A new primer pair was designed, given in the
Material and Methods section, which were used in
combination with the initially designed TaqMan probes;
subsequent RQ-PCR analysis resulted in better
amplification with higher sensitivities for both TCRG
targets (Table 2). All the other primer/probe
combinations initially designed using Primer Express
resulted in efficient amplification according to the
amplification plots and sensitivities obtained.
In primer-matrix experiments different ~Rn values
were found for the different primer combinations, while
the CT value were the same. The 300/300 nM primer
combinations were used in all sensitivity experiments
performed, although other combinations sometimes had
equal ORn values.
Standard curves were established from the results of
the dilution experiments and displayed for each PCR
target a linear correlation between the CT and the
logarithm of the initial DNA target concentration (Figure
2). The dynamic range spanned over up to five orders of
magnitude. Triple or quadruple experiments had similar
values, but at low target concentrations some variation
in the CT was observed (Figure 2).
Comparison of sensitivities of the three detection
methods
For dot-blot and liquid hybridization, the same PCR
product and radioactive probes were used, but different
hybridization methods. The sensitivity results obtained
CA 02303945 2000-03-17
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22
with these conventional methods were compared with those
of the RQ-PCR analyses, which used specially designed
primer/probe combinations, but the same DNA dilutions
(Figure 3). Since RQ-PCR was performed in triplicate, the
sensitivity of RQ-PCR was based on at least two out of
three positive experiments.
The sensitivities of RQ-PCR analysis varied between
10-2 and 10-q (Table 3). The sensitivities obtained by RQ-
PCR analysis were most similar to that of the dot-blot
method (Table 3, Figure 3). Using the IGK-2 PCR target
even a higher sensitivity was found than obtained with
dot-blot analysis, but for the other PCR targets similar
or 10-fold lower sensitivities were found. In contrast,
liquid hybridization was always more sensitive, either
10-fold or even 100-fold.
The lowest sensitivity was obtained with the TCRD-2
target containing a functional region without insertion
(Table 2). For this reason, the specificity of the
patient specific probes were limited. To decrease
background signals, the liquid hybridization temperature
was increased to 64°C and an extra washing step (0.3x
SSC, 0.1% SDS at 55°C) was necessary to remove background
signal from the dot-blot. To increase the stringency in
RQ-PCR analysis we performed another experiment in which
the annealing/extension temperature was increased to 63°C
and the amount of TaqMan probe lowered to 100 nM.
However, this still resulted in a ORn for the polyclonal
MNC control, thereby limiting the sensitivity to 10-2.
3 0 l~tD analysis of follov~-up samples using RQ-PCR
A total of 12 BM follow-up samples from patient
5257, taken during and after treatment, were available
for MRD analysis. RQ-PCR analysis using a VkIII-Kde gene
rearrangement was performed in triplicate, and diagnosis
dilutions and follow-up samples were analyzed in
parallel. In addition, diagnosis and follow-up samples
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
23
were checked for the amount and integrity of DNA by
performing a albumin RQ-PCR. Quantities were determined
using a standard curve of MNC DNA diluted in milli-Q. By
dividing the diagnosis quantity by the follow-up quantity
a ratio is established that can be used to correct the
MRD level generated by the leukemia specific RQ-PCR.
With a sensitivity of 10-9 the first four time points
appeared to be MRD positive, the level of MRD slowly
decreased form 1.3 x 102 at week 5 down to 2.2 x 10-4 at
week 33 (Table 4). All further follow-up samples were MRD
negative including at the end of treatment (week 158)
until relapse that emerged four years after diagnosis
(Table 4).
DISCUSSION
The aim of this study was to test the value of RQ-
PCR analysis using the TaqManT"" technology for sensitive
and quantitative detection of MRD in follow-up samples
using rearranged Ig and TCR genes as PCR targets. In our
analysis, we have chosen for a patient specific
functional region TaqMan probe and matching primers. This
technique was applicable for all PCR targets tested, i.e.
complete IGH, IGK-Kde, TCRG, and incomplete TCRD gene
rearrangements. The majority of PCR targets tested (7 out
of 10) were derived from patient 5199. The reason for
analyzing most (7 out of 8) of the Ig/TCR gene
rearrangements of this patient was to prevent selection
of "sensitive" targets and to obtain insight into the
possibilities for finding suitable primers and TaqMan
probe combinations for RQ-PCR analysis. All functional
region specific TaqMan probes developed were able to
detect leukemia derived DNA and did not interfere with
the PCR efficiency.
The high number of PCR targets in patient 5199 might
be due to oligoclonality at diagnosis. This was not
evident from Southern blot analysis, except for the three
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
24
rearrangements of albeit equal intensity in the TCRG gene
locus. This may be explained by an extra chromosome 7 or
by two clonal populations of similar size. Two other
findings point towards oligoclonality: (i) a large
proportion of the Ig/TCR rearrangements appeared not to
be stable at relapse (data not shown) and (ii) The
sensitivities obtained by dot-blot analysis in this
patient are not representative, only 29~ (2/7) reached a
10-4, while in a series of over 300 dot-blot analysis 87°s
of the PCR targets reached a sensitivity of 10-4. We
accordingly used the PCR targets of patient 5199 for
relative comparison, but not for determining the
sensitivity of the technique. Hence, all three techniques
reached sensitivities of at least 10-4 and 10-5 : IGH, IGK,
and TCRD PCR targets in patients 2145, 5160, and 5257.
Upon comparison, the sensitivities obtained with the
RQ-PCR technique were without further optimization
similar to those obtained with the dot-blot method. The
liquid hybridization with radioactively labeled probes
appeared to be most sensitive. In principle, it should be
possible to reach even higher sensitivities with RQ-PCR
since hybridization with the TaqMan probe is also a
liquid hybridization, unless the total detection system
based on fluorescence, is less sensitive as compared to
radiography.
In another RQ-PCR application, one point mutation
appeared to be sufficient for allelic discrimination by
the TaqMan probe. The Tm of the TaqMan probe was in this
case 5 to 6°C above that of the corresponding primers.
This adaptation may also be necessary to increase the
specificity for RQ-PCR analysis of the TCRD-2 in this
study, which lacked randomly inserted nucleotides. This
would mean that for this target a new TaqMan probe should
be developed.
An alternative approach to RQ-PCR analysis of Ig/TCR
gene rearrangements might be to use a TaqMan probe
CA 02303945 2000-03-17
WO 99/14366
PCT/NL98/00542
positioned at germline sequences (V, D, or J gene
segments) in combination with one or two patient specific
functional region primers. A similar strategy has also
been used for conventional MRD detection using Ig/TCR
5 gene rearrangements as PCR targets. This RQ-PCR approach
will be more cost-effective, since it would be not
necessary to design TaqMan probes per patient but per
type of rearrangement. However, standardization will be
more difficult, since adaptation of the annealing
10 temperature may be required per case to prevent aspecific
amplification. Nevertheless, comparative studies should
demonstrate which strategy gives best sensitivity, which
may also vary per target type.
Twelve BM follow-up samples of a precursor-B-ALL
15 were analyzed for MRD by RQ-PCR, using a VkIII-Kde gene
rearrangement. The standard curve established with the
diagnosis dilution series, was used to define the initial
amount of leukemic DNA in follow-up samples based on
their CT. Subsequently, the MRD level was corrected
20 relative to the diagnosis sample based on albumin RQ-PCR
analysis. A slow decrease of tumor load was observed
during the first year of treatment. According to the
literature, this is indicative of a poor outcome; the
leukemia is relative resistant to the given treatment.
25 Indeed, this patient relapsed almost two years after
cessation of therapy and more than seven months after the
last time point analyzed, which was still MRD negative.
Except for MRD analysis of follow-up samples, RQ-PCR
may also be helpful in the identification of suitable PCR
targets at diagnosis. By precise quantification it should
be possible to discriminate between true-allelic Ig/TCR
gene rearrangements and minor gene rearrangements derived
from subclones. Subclonal rearrangements are frequently
reported in ALL, but are inappropriate PCR targets for
MRD detection. Perhaps RQ-PCR can replace Southern blot
analysis in the future, which is still considered as the
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
26
golden standard for MRD target identification but is
laborious, time-consuming and requires large amounts of
good quality DNA.
Our data show that real time PCR analysis allows
accurate definition of the level of MRD in bone marrow
follow-up samples, which is a major improvement to the
end-point PCR analysis of conventional MRD detection
methods. Similar CT values were accomplished for
quadruple or triple experiments indicating the high
reproducibility of the technique. The RQ-PCR technique
takes advantage of the first productive PCR cycles for
quantitation, where the PCR fidelity is still high;
exhaustion of reagents does not yet exists and potential
inhibitory effects are minimal. Due to the semi-automated
system it is easy to analyze more DNA per follow-up
sample. This does not only result in a more accurate
definition of the tumor load in the follow-up sample, but
simultaneously increases the sensitivity of the MRD
detection technique. The sensitivity ultimately depends
on the maximal number of cells tested; by performing the
experiment in quadruple two to four ~g of DNA may be
analyzed, corresponding to approximately 5 x 105 cells.
The here presented results demonstrate that RQ-PCR
is applicable for MRD analysis via detection of clone-
specific Ig/TCR gene rearrangements. RQ-PCR offers many
advantages over currently used techniques. The dot-blot
and liquid hybridization are dependent of using
radioactive isotopes and require individual optimization
of the hybridization or extra washing steps. In contrast
to these methods RQ-PCR analysis is simple and fast; data
can be acquired as soon as the PCR is completed without
any post-PCR handling, i.e. within 3 hours, instead of 5
days generally required for conventional methods. The
closed system minimizes the risk of PCR product
contamination, which is very important in this kind of
studies where minimal amounts of target are amplified.
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/00542
27
Most importantly, this novel technique allows accurate
quantification of MRD, essential for the discrimination
between patients with good and poor prognosis. Therefore,
we consider this sensitive and reproducible RQ-PCR
technique as an important step forward in the clinical
application of MRD studies.
CA 02303945 2000-03-17
WO 99/14366 PCT/NL98/OOS42
28
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CA 02303945 2000-03-17
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? V,
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l7 ~ C9 U ~.1d
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~
CA 02303945 2000-03-17
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31
TABhE 3: Co~mparisoa of sensitivities obtained by RQ-PCR with
those obtained by conventional I~iD detection techniques.
sensitivity
Patient PCR target
code
RQ-PCR Dot-blot Liquid
hybrydization hybzydization
219 5 IGH-1 10-q NT 10-5
5160 TCRD-1 10-4 10-4 NT
519 9 IGH-2 10-3 10-' 10-q
IGH-3 10-3 10-4 10-''
IGK-i 10 3 10 3 10'5
IGK-2 10 4 10 3 10 5
TCRD-2 10-2 IO-3 10-3
TCRG-1 10-3 10-3 10-5
TCRG-2 10-3 10-a 10-s
5257 IGK-3 10-'' 10-' NT
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TABLE 4. RQ-PCR analysis of tw~lve bone marrow sampl~s during follow-up
of patient 5257 using an VxIII-Kd~ gene r~arrangement as PCR target, with
a s~nsitivity of 10-'
Weeks after Cyale* Mean Corrected Standard
diagnosis treshold MRD MRD level deviation
level
27.14 1.0 10-Z1.3 x 10-20.25 x 10-2
x
26. 67
26.35
13 30.29 2.0 10-33.3 x 10-30.45 x 10-3
x
30.27
30.11
23 33.34 4.8 10-47.0 x 10-40.67 x 10-4
x
33.18
33.44
33 37.54 6.2 10-52.2 x 10-92.13 x 10-9
x
36.24
50.00
69/82/95/ 50.00 0 p _
110/116/ 50.00 0
145/158 50.00 0
191 2I . 4 1. 10-12 x 10-10 x 10-1
9 2 . .
x 2 13
(relapse) 21.35
21.30
* Data in triplicate
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Legends to the figures
Figure 1 Diagram of the putative relative frequencies of
leukemic cells in blood and bone marrow of acute leukemia
patients during and after treatment and during development of
relapse. The detection limit of cytomorphologic techniques as
well as the detection limit of immunologic marker analysis
and PCR techniques are indicated. I-Rx = induction treatment;
M-Rx = maintenance treatment
Figure 2 An RQ-PCR sensitivity experiment of a precursor-B-
ALL patient (2145) was performed using an IGH gene
rearrangement (VH3-JHSb; OGH-1). (A) The real time
amplification plots of the diagnosis dilutions for one series
of experiments. (B) The standard curve shows the linear
correlation between the cycle threshold (CT) and the initial
amount of DNA (tumor load) of all four experiments. With this
IGH gene rearrangement a sensitivity of 10-9 was reached in
4 out of 4 experiments.
Figure 3. The three MRD detection methods for an IGK gene
rearrangement of precursor-B-ALL 5199 (JGK-2). (A) Schematic
diagram of the VKII-Kde PCR target with a patient specific
functional region of a total of 7 nucleotides deleted and 9
nucleotides randomly inserted. Given are the sequences and
relative positions of the primers used for RQ-PCR and
conventional MRD methods, as well as the patient-specific
functional region probes. (B) Result of the dot-blot
hybridization with the radioactively labeled functional
region probe after PCR amplification of the VKII-Kde gene
rearrangements in a diagnosis dilution series. With this
technique a sensitivity of 10-3 was obtained. (C) Liquid
hybridization of the same PCR product and the same probe gave
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a sensitivity of 10-5. (D) RQ-PCR analysis of the VKII-Kde
PCR target with a different primer set and a fluorogeneic
TaqMan probe. The experiment was performed in triplicate on
the serial diagnosis dilution. Real-time information of PCR
product accumulation is given at the left. The standard curve
at the right illustrates the linear correlation between the
cycle threshold and the initial amount of DNA. With RQ-PCR
analysis a sensitivity of 10-4 was reached in 1 out of 3
experiments.
