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From the Institute for Pathology, University of Basel, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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rearrangements
and by detection of the chromosomal translocations t(14;18) and
t(11;14), require several laborious and costly PCR assays for
each of these diagnostic tests. We have developed a multiplex PCR assay
for the simultaneous determination of B- and T-cell clonality and the
detection of the chromosomal translocations t(14;18) and t(11;14) in a
single reaction, using four-color fluorescence and automated
high-resolution fragment analysis. The 26 primers combined in the
multiplex PCR correspond to the sequences of >90% of the 69 variables
and 6 join IgH genes and 100% of the T-cell receptor- variables and
join genes that could participate in the respective rearrangements. In
addition, they detect the major and the minor breakpoint
regions of the t(14;18) and the major breakpoint region of the
t(11;14), and amplify the ß-globin gene as an internal
control. The specificity of the multiplex PCR was
confirmedby analysis of 39 T-cell lymphomas and 58 B-cell
lymphomas, including 11 mantle cell lymphomas bearing the
t(11;14) and 25 follicular lymphomas bearing the t(14;18), with
known rearrangements and/or translocations. Fifteen samples of reactive
lymphadenitis remained negative.
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Introduction |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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(TCR-) locus on chromosome 7 (7p15-p14) is rearranged
in the majority of T cells. Specific chromosomal translocations are
associated with specific disorders. The most common translocations in
B-non-Hodgkin lymphoma are t(11;14) (q13;q32), associated with mantle
cell lymphoma, and t(14;18) (q32;q21), specifically found in follicle
center cell lymphoma and in a part of the diffuse large-cell lymphomas.
PCR methods for the determination of clonality by analysis of IgH and
TCR- rearrangements are widely used. Conventional methods require
several PCR assays for the analysis of the different IgH V framework
regions and for the analysis of the different TCR- V and J genes.
Similarly, singleplex PCR assays for the detection of the t(11;14) and
the t(14;18) are performed, some requiring additional laborious
procedures, eg, hybridization with specific probes. More recently,
multiplex PCR assays for the detection of TCR-
rearrangements2,3
have been reported. Some groups reported
the use of high-resolution fragment analysis (HRFA), which greatly
improves separation and visualization of PCR fragments, for the
analysis of PCR-based diagnosis of lymphoproliferative
disorders.4-11
Consensus primers and one fluorescent dye
were used to detect TCR-9,10
or IgH
rearrangements.7
Multiplex PCR for the detection of
TCR- rearrangements5,6
and IgH
rearrangements5
by separate PCR assays and using one
fluorescent dye have been reported. A method for the detection of
TCR- and IgH rearrangements and the t(14;18) by four-color
fluorescence and HRFA was reported.8
The method required
five different PCR assays, the resulting fragments could then be
combined for HRFA.
We have developed a four-color multiplex PCR assay for the simultaneous determination of B- and T-cell clonality, the detection of the most frequent and diagnostically important chromosomal translocations t(14;18) and t(11;14), and the amplification of a control gene in a single reaction. Analysis is performed using automated HRFA and GeneScan analysis. The details of these diagnostic tests are explained below.
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IgH Rearrangement |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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t(11;14) |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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t(14;18) |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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70% of follicular lymphomas and in
30% of diffuse large-cell lymphomas.22
Approximately
two thirds of the breakpoints on chromosome 18 are clustered in an area
called the major breakpoint region (Mbr), another 5% are found in the
minor cluster region (mcr). The remaining breakpoints are spread in a
larger region on chromosome 18 and are therefore not amenable to PCR
analysis. Reported frequencies of t(14;18) detected in follicular
lymphomas by molecular methods widely vary. Detection rates of
PCR-based assays between 40 and 50% seem realistic.22-25
The primers used in our multiplex PCR detect t(14;18) with breakpoints
in both the Mbr and the mcr regions. |
T-Cell Receptor-
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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locus consists of 14 variable (V) genes, 8 of them
being pseudogenes, most of which could nevertheless be rearranged, and
5 join (J) genes. The primers designed for the multiplex PCR amplify
all existing V and J genes, except the V1S1P gene, whose recombination
signal sequences with a spacer sequence of only 11 bp will not
participate in a rearrangement. T cells frequently have rearranged
TCR- loci on both alleles. This is in contrast to IgH rearrangements
in B cells, in which allelic exclusion does not allow rearrangement of
the IgH locus on the second allele.
The limited diversity of the TCR- VJ combinations and the presence
of relatively short N regions results in a small size range of the PCR
fragments derived thereof. This may cause difficulties to discriminate
clonal gene rearrangements from the polyclonal background caused by the
considerable amount of nonneoplastic T cells frequently present in
clinical samples of T-cell lymphomas. A number of methods have been
suggested that improve the separation and visualization of the PCR
fragments, eg, fragment analysis by denaturing gradient gel
electrophoresis,26-29
single-strand conformation
polymorphism analysis,30-32
temperature gradient gel
electrophoresis,33
and automated HRFA.4,6,34
The excellent resolution by HRFA will reveal clonal rearrangements in
most cases. To provide security in cases with high polyclonal
background, we have developed an additional four-color multiplex PCR
assay for the verification of clonal TCR- rearrangements. This PCR
assay further separates fragments derived from TCR- rearrangements
by different colors according to the subtype-specific V gene present,
and allows verification of clonal bands according to subtype-specific
differences in the lengths of the fragments obtained in the first
multiplex PCR assay and those obtained in the TCR--specific PCR
assay.
The 26 primers present in the multiplex PCR are labeled with three different fluorophores (5-FAM, 6-JOE, and NED) and the PCR fragments derived from the four different diagnostic tests are within defined size ranges. Excellent resolution is provided by automated HRFA performed by capillary electrophoresis and laser-induced fluorescence detection on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA), an automated instrument system capable of determining size and quantity of DNA fragments. Electrophoresis is performed in small, polymer-filled capillary tubes instead of flat gels. The dye-labeled PCR fragments electrophoresce through the polymer and separate according to size. The fluorescent dyes are excited by a laser and emit light at a specific wavelength for each dye. The light is collected and separated by a spectrograph and collected onto a charge-coupled device camera. The collected data are automatically analyzed using the GeneScan Analysis software, which sizes the DNA fragments based on a size standard labeled with a forth fluorophor (ROX). PCR fragments differing in length by 1 bp can be distinguished. Results can be displayed as electropherograms, with are shown in separate panels for each dye, and as tabular data. The DNA fragments are quantified according to the intensity of the signal, which corresponds to the peak area.
A schematic representation of the fluorescent dye labels and the size
ranges specific for the PCR fragments derived from each of the
diagnostic tests covered by the multiplex PCR is shown in Figure 1
. In Figure 1
, panel B (blue), the
fragments labeled with 6-FAM are shown. Size ranges are as follows:
fragments of IgH rearrangements amplified with primers for the V
framework region 3 (FR3) are present in a size range between 70 and 140
bp. Fragments of TCR- rearrangements appear in a size range between
180 and 280 bp. Fragments of IgH rearrangements containing the V
framework region 1 (FR1) are present in a size range between 300 and
400 bp. In Figure 1
, panel G (green), the fragments labeled with 6-JOE
are as follows: fragments derived from a t(11;14) MTC have sizes
between 160 and 300 bp. The length of the fragment of the control PCR
(ß-globin) is 352 bp. Figure 1
, panel Y (yellow), shows fragments
labeled with NED derived from the t(14;18) Mbr and mcr. The size of
these fragments is usually between 150 and 300 bp and may, in a rare
case, be up to 450 bp. Figure 1
, panel R (red), contains the fragments
of the molecular weight marker, labeled with ROX.
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Materials and Methods |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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The tissue probes used in this study were derived from surgical
specimens obtained for diagnostic purposes. Tissues were routinely
fixed in buffered formalin (4%) for at least 18 hours. They were then
embedded in paraffin and stained according to standard methods. The
diagnosis of malignant lymphoma was made according to the REAL
classification and inforced by additional immunohistochemistry using a
panel of monoclonal antibodies against line-specific antigens including
CD3, CD4, CD8, TIA, CD56, and CD57 for T lymphocytes; and CD20, CD10,
CD23, CD75, and CD79a for B lymphocytes. Additional markers were bcl2
and bcl6 for follicular non-Hodgkin lymphoma and cyclin D1 for mantle
cell lymphoma. Molecular diagnosis was performed by use of conventional
methods as follows: IgH rearrangements were analyzed by singleplex PCR
for the IgH FR1 and FR3 regions, detection of the t(11;14) MTC and the
t(14;18) Mbr and mcr was performed by nested PCR. Semi-nested multiplex
PCR was used to analyze TCR- rearrangements. Electrophoresis was
performed on 4% agarose gels. The results were confirmed by DNA
sequence analysis of the PCR fragments derived from translocations and
clonal rearrangements.
From these cases, 35 T-cell lymphomas with clonal TCR-
rearrangements and 54 B-cell-lymphomas with proven clonality, including
11 mantle cell lymphomas bearing the t(11;14) and 25 follicular
lymphomas bearing the t(14;18) were chosen to test the specificity of
the multicolor multiplex PCR. Negative controls included 11 samples of
reactive lymphadenitis. In addition, 12 samples of formalin-fixed,
paraffin-embedded tissue derived from a European proficiency test,
which had been diagnosed correctly by our conventional PCR methods,
were tested. Cultured cells of the cell lines Jurkat, DOHH2, and REC-1
were used as positive controls.
DNA Extraction
Formalin-fixed and paraffin-embedded samples were deparaffinized according to standard methods. Lymphocytes were isolated from blood and bone marrow aspirates using Ficoll gradients (Histopaque-1077, Sigma, St. Louis, MO). DNA was extracted by use of the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. If only small amounts of tissue were available (eg, tissue scratched off glass slides), carrier RNA (artificial polyC RNA; Promega) was added before DNA precipitation. Whenever possible, DNA extractions were performed in duplicate. To estimate DNA yields, OD at 260 nm was measured or gel electrophoresis was performed using 0.8% agarose gels stained with ethidium bromide.
Primer Design and Testing
The primers were newly designed by us using the GeneWorks software
(Intelligenetics), and optimized for the multiplex PCR. DNA sequences
used for primer design were obtained from the EMBL database. EMBL
accession numbers of the sequences used are as follows: TCR- locus,
AF 159056; IgH locus, V genes, AB019437, AB019438, AB019439, AB019440,
and AB019441; J genes, J00256. Bcl-1 MTC (X74150), BCL-2 Mbr (M13994),
and BCL2 mcr sequences were published by Ngan and
colleagues.35
The GCG (Genetics Computer Group Inc.)
software was used. Primer pairs of each diagnostic test were
extensively tested by monoplex and partial multiplex PCR using clinical
samples and the respective positive and negative controls before they
were included into the multiplex PCR primer mix. As the quality of the
primers is crucial to obtain clear-cut results, each primer was
examined by capillary electrophoresis and laser-induced fluorescence
detection, using a ABI 310 Genetic Analyzer (Applied Biosystems) before
use. Samples contained 0.1 pmol of each primer, 1 µl of the molecular
weight marker ROX 500 (Applied Biosystems) and 25 µl of deionized
formamide (Applied Biosystems). Primers should show one clear peak. The
peak area should be 70,000 fluorescence units, indicating efficient
labeling with fluorophores. A stock solution was then prepared
containing the primers listed in Table 1
according to the concentrations shown in Table 1
in pmol per reaction.
The primer mix stock solutions were stored in aliquots at -20°C to
avoid repeated freezing and thawing. Primers labeled with fluorophores
were protected from light.
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The reaction mix contained standard PCR buffer (Applied
Biosystems), 0.2 mmol/L each dATP, dGTP, dCTP, and dTTP, the primer mix
(Table 1)
at the appropriate concentrations, and 10 U AmpliTaq Gold DNA
polymerase per reaction. DNA (100 to 300 ng) was added and the final
reaction volume was 25 µl. PCR parameters were as follows: 95°C for
11 minutes; 36 cycles at 95°C for 40 seconds, 55°C for 40 seconds,
72°C for 60 seconds, and a final step of 20 minutes at 72°C to
complete elongation and dA addition.
Automated HRFA and GeneScan Analysis
PCR products were analyzed by capillary electrophoresis and laser-induced fluorescence detection, using a ABI 310 Genetic Analyzer (Applied Biosystems). Procedures were according to the manufacturer’s recommendations. Each sample contained 1.5 µl of the PCR sample, 1 µl of the molecular weight marker ROX 500 (Applied Biosystems), and 25 µl of deionized formamide (Applied Biosystems). Samples were denatured at 95°C for 2 minutes and immediately chilled on ice. The gel used for CE was POP-4 polymer solution (Applied Biosystems). Results were visualized using the GeneScan analysis software (Applied Biosystems).
TCR--Specific Multicolor Multiplex PCR
The multiplex PCR for the verification of TCR- fragments was
performed essentially as described above, using the primer mix listed
in Table 2
, except that the amount of
AmpliTaq Gold DNA polymerase was reduced to 5 U per reaction.
Primer concentrations were according to Table 2
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In some cases, PCR fragments derived from chromosomal
translocations and clonal IgH rearrangements could be sequenced
directly from the multiplex reaction mix. Clonal TCR- rearrangements
required the verification step to determine the family-specific primer
to be used as a sequencing primer. However, results were greatly
improved, if an additional monoplex PCR was performed with unlabeled
primers of the corresponding test. In either case, 0.5 to 1 µl of the
PCR reaction mix were used without further purification to provide the
DNA template. The corresponding unlabeled forward primers were used as
sequencing primers. DNA sequencing was performed using the dRhodamine
or Big Dye Terminator Kits (Applied Biosystems) according to the
protocols provided by the manufacturer. Sequence analysis was performed
on an ABI 310 Genetic Analyzer using the sequence analysis software
(Applied Biosystems). Procedures were according to the manufacturer’s
recommendations.
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Results |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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. Multiple signals in the expected size
range and color panel (compare Figure 1
) are created by polyclonal
populations of B and T cells, as present in reactive
lymphoproliferation and in thymus tissue (Figure 2A
, panel B). Peaks
derived from IgH FR3-J fragments appear in a range between 70 and 140
bp, those from IgH FR1-J fragments between 330 and 400 bp. Because of
the short N regions present in TCR- rearrangements, the PCR
fragments derived thereof are within a smaller size range, between 200
and 250 bp, with the bulk of fragments present in the size range
between 210 and 230 bp. In contrast to the polyclonal pattern shown in
Figure 2A
, the clonal TCR- rearrangements of the T-cell line Jurkat,
which has both its TCR- loci rearranged, are visible as two clear
peaks at 209 and 245 bp (Figure 2B
, panel B). The cell line REC-1,
derived from a mantle cell lymphoma,13
bears a t(11;14).
This results in amplification of a 229-bp fragment labeled with 6-JOE
(Figure 2C
, panel G). REC-1 cells have a rearranged IgH locus on the
other allele, as shown by amplification of both a clonal IgH FR3-J and
a FR1-J fragment. (Figure 2C
, panel B). The cell line DOHH2, derived
from a follicular lymphoma,36
harbors a t(14;18) with its
bcl-2 breakpoint in the Mbr region, as visualized by the signal at 243
bp in Figure 2D
, panel Y. DOHH2 cells also have a rearranged IgH locus
with V sequences detected by both the FR3 and the FR1 primers (Figure 2D
, panel B). The amplification of the IgH FR3-J fragment is less
efficient than that of IgH FR3-J fragment, indicating that point
mutations in the primer-binding sequences are present in the FR3
region. A peak at 352 bp in panel G is always present and shows
amplification of the control gene ß-globin. Samples lacking DNA were
always included as negative controls and did not show any signals (data
not shown).
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were
performed to test the sensitivity of the assay. Clonal IgH
rearrangements could clearly be distinguished from the polyclonal
background with 5% of REC1 or DOHH2 DNA. The ratio between the peak
height of the clonal IgH rearrangement and the highest peaks of the
polyclonal background was still 2:1. The same ratio between the peak
heights from the clonal TCR- rearrangements was obtained with 10%
DNA from Jurkat cells present in the polyclonal background. Using
standard conditions, the t(11;14) and the t(14;18) fragments were
detectable in the presence of 1% of REC1 or DOHH2 DNA, respectively,
corresponding to 1 ng of DNA or 150 cells.
All results shown below were obtained from formalin-fixed,
paraffin-embedded tissue of clinical samples of T- and B-cell
lymphomas. In clinical samples, nonneoplastic B and T cells are usually
present together with the clonal cells of the lymphoma. The signals
from the polyclonal IgH and TCR- rearrangements are often below
detection, if the majority of the probe consists of clonal B cells, or
visible as small peaks from which a clonal IgH fragment can clearly be
distinguished (Figure 3)
. In the majority
of B-cell lymphomas, signals from clonal IgH rearrangements from both
IgH FR3-J and FR1-J fragments are obtained (Figure 3A)
. This provides
additional security with respect to the result. In a few variable IgH
genes, the primers used in the multiplex PCR correspond either to
sequences of the FR3 region or to those of the FR1 region, but not to
both. More often, this will be the case in samples of lymphomas that
are subject to somatic hypermutation, eg, follicular lymphomas.
Accordingly, only one of the two IgH regions analyzed will show the
signal of a clonal IgH rearrangement. Examples are shown in Figure 3B
(FR3) and Figure 3C
(FR1). The FR1 region of seven of the variable IgH
genes can be detected by two different primers designed for the FR1
region. Clonal IgH rearrangements involving these genes may show two
peaks in the FR1-specific size range. Thus, clonal fragments from both
the FR1 and the FR3 regions or from either one only may be detected,
depending on V gene usage, and, in a rare case, two FR1 fragments of
different lengths may appear in the FR1-specific size range.
Visualization of results can be further improved, if required, by
focusing on the size range of the individual tests. In Figure 4
, the focus is on the IgH FR3-J-specific
size range. A polyclonal pattern of IgH rearrangements derived from
nonneoplastic B cells is present together with a clonal IgH
rearrangement in the sample of a B-cell lymphoma. Interestingly, the
signals obtained from the polyclonal B-cell populations are spaced by 3
bp, and the fragment lengths show, that in the majority of IgH
rearrangements the reading frame with respect to the join gene is
maintained. The specificity of the PCR fragments derived from clonal
IgH rearrangements was determined by DNA sequencing of the individual
V-N-J junctions of 22 cases. In the majority of the cases, the reading
frame of the join gene was not altered by the insertion of N sequences.
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. A clonal IgH
rearrangement (Figure 5A
, panel B) and a t(11;14) (Figure 5A
, panel G)
are present in the cells of a mantle cell lymphoma. Results obtained
from a follicular lymphoma show a t(14;18) (Figure 5B
, panel Y) and an
IgH rearrangement on the other allele of chromosome 14, whose sequences
correspond to the IgH-FR1 but not to the IgH-FR3 primers (Figure 5B
,
panel B). Polyclonal T cells are present in the same sample. A
follicular lymphoma bearing a t(14;18) with its bcl-2 breakpoint in the
mcr is shown in Figure 5C
, panel Y. In this sample, the IgH locus is
not amplified. Small amounts of polyclonal T cells are visible, signals
from polyclonal B cells are almost below detection (Figure 5C
, panel
B). DNA sequence analysis of the PCR fragments derived from 11 t(11;14)
and 25 t(14;18) was performed. The individual breakpoints of the
t(11;14) were within a range of 76 bp in the MTC (Figure 6)
. The bcl-2 breakpoints of 25
follicular lymphomas were clustered within a 122-bp region of the Mbr
(Figure 6)
. One t(14;18) had its bcl-2 breakpoint in the mcr.
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loci rearranged.
Accordingly, clinical samples of T-cell lymphomas usually show one or
two clear peaks when analyzed by the multiplex PCR assay. An example is
shown in Figure 7A
. Considerable amounts
of nonneoplastic T and B cells may be present in some samples of T-cell
lymphomas and cause a high background of polyclonal signals. A clonal
TCR- rearrangement present within the bulk of polyclonal fragments
can still clearly be distinguished, as its peak height is well above
that of the polyclonal background (Figure 7B)
. From the sample shown in
Figure 7B
, only partially degraded DNA was obtained, as is frequently
the case if formalin-fixed, paraffin-embedded tissue is used. The
control gene ß-globin is still amplified. Likewise, clonal IgH FR1-J
fragments would be amplified, but the weak signals of the polyclonal
IgH FR1-J fragments remain below detection. If results seem ambiguous
because of high polyclonal background, an additional, independent
multicolor multiplex PCR, specifically developed for the analysis of
TCR- rearrangements, can be performed. This allows confirming of the
results and to determine the subtype of the V gene present in a clonal
TCR- rearrangement. This PCR assay separates fragments derived from
TCR- rearrangements by different fluorescence colors, according to
the V gene subtype present in the rearrangement: V1 (V-I) and V5P
(VA) genes appear in panel B (blue); V2 (V-II) genes in panel Y
(yellow); and V3P (V-III), V4P (V-IV), and V6P (VB) genes in
panel G (green). In addition, a different set of V primers is used
(Table 2)
, resulting in PCR fragments that differ in their length from
those obtained in the first multiplex PCR. These differences in
fragment length are specific for each of the V gene subtypes. Expected
differences in fragment lengths are listed in Table 2
. These can vary
by a few bp, because of the denaturing power of the gel. An example is
shown in Figure 7, C and D
, respectively. Two peaks of 212-bp and
241-bp length in Figure 7C
, panel B, show the presence of two clonal
TCR- rearrangements (Figure 7C)
. The TCR--specific multiplex PCR
clearly confirms the result: A fragment of 149 bp present in Figure 7
,
panel B, shows a clonal TCR- rearrangement containing a V3P gene
(difference in fragment length, 90 bp). The 245-bp fragment present in
Figure 7
, panel G, shows a second clonal TCR- rearrangement with a
V1 gene (difference in fragment length, 33 bp).
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Discussion |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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rearrangements
(Figure 7B)
and the vast majority of the t(14;18) and the t(11;14) is
still possible, whereas IgH FR1-J fragments and larger fragments from
the translocations (which are very rare) remain below detection. The
352-bp ß-globin control fragment still allows monitoring of
successful PCR in these cases. A small peak height of the ß-globin
fragment indicates that either the DNA concentration was too low, or
the DNA is severely degraded. Thus, the status of a clinical sample with respect to B- and T-cell populations and to the most frequent chromosomal translocations occurring in B-cell lymphomas can be evaluated simultaneously in a single reaction. This provides an enormous time and labor savings when compared to current methods that need several separate PCRs for each of these diagnostic tests. In addition, using capillary electrophoresis and laser-induced fluorescence detection, hands-on time is further reduced. Routine screening for the most important molecular markers with respect to lymphoproliferative disorders is now feasible and may reveal, especially in atypical or polymorphous samples, information that would otherwise go undetected. In addition, lineage-inappropriate gene rearrangement becomes obvious. The simultaneous evaluation of B-cell clonality and the chromosomal translocations t(11;14) and t(14;18) is very useful, as the specific chromosomal translocations are demonstrable by PCR in only approximately half of the mantle cell- and follicular lymphomas. Mantle cell lymphomas frequently have a rearranged IgH locus on the other allele and do not show considerable somatic hypermutation of IgH V genes. Thus, in cases with undetectable t(11;14), a clonal IgH rearrangement can be demonstrated. In contrast, follicular lymphomas exhibit extremely high degrees of somatic hypermutation,37 which might impair primer binding. Because two different IgH framework regions are analyzed in the assay, and the IgH FR3 primers are designed to tolerate point mutations, chances to show clonality despite of somatic hypermutation are greatly increased. Indeed we found that in a number of follicular lymphomas apparently only one of the two IgH framework regions was affected by mutations and clonality could still be shown by amplification of the other.
The use of fluorescence-labeled primers and automated HRFA greatly
improves sensitivity, separation, and visualization of PCR fragments
when compared to conventional methods, eg, analysis by agarose or
polyacrylamide gel electrophoresis. The higher sensitivity allows
reducing of the number of PCR cycles, which is an asset in many
respects. The excellent separation of the PCR fragments, which allows
distinguishing of fragments differing in length by only 1 bp, is
crucial for the analysis of IgH and, even more importantly, TCR-
rearrangements. Using conventional methods, the polyclonal background
caused by nonneoplastic T and B cells frequently present in clinical
samples may obscure clonal rearrangements. Using HRFA, peaks from
clonal rearrangements usually rise clearly above those derived from
polyclonal background. The ratio between the peak height of a clonal
rearrangement and the polyclonal background depends on the amount of
reactive lymphocytes present in a sample together with the malignant
clone. The ratio between the peak height of clonal IgH rearrangements
and polyclonal background was >2:1, if 5% of the cells were clonal.
This ratio may be lower because of mutations in the primer-binding
sequences of the clone, a problem encountered mainly in follicular
lymphomas. An important control is the reproducibility of the clonal
peak. Therefore, diagnostic tests should always be performed in
duplicate. Because of their limited size range, TCR- rearrangements
may have lower clonal:polyclonal ratios. We recommend performing the
TCR--specific multiplex PCR in all cases with a possible diagnosis
of T-cell lymphoma, that do not show a ratio of at least 2:1. This
assay further separates the fragments derived from TCR-
rearrangements by different colors, and the results are confirmed by an
independent assay. In all cases with a low clonal:polyclonal ratio, the
small amount of clonal cells present in a larger polyclonal population
should be interpreted with caution. Monoclonality is not a proof of
malignancy and the diagnosis of malignant lymphoma should only be made,
if additional morphological criteria are met.
In samples with low numbers of B or T cells, eg, microdissected tissue,
evaluation of clonality should be performed with caution. Pseudoclonal
peaks of randomly amplified IgH or TCR- rearrangements can be
produced, especially if the number of cycles is increased to compensate
for the low amount of DNA present. These peaks are usually not
reproducible.
We showed, that the multicolor multiplex PCR is a useful tool for rapid screening of clinical samples with at least 5 to 10% of clonal B or T cells or 1% of cells bearing a t(11;14) or a t(14;18) present in a polymorphous background. Using our standard conditions the sensitivity, with respect to the translocations, is limited by the small amount of DNA template, 1% corresponding to 1 ng of DNA, the equivalent of 150 cells. Applications requiring higher sensitivity, eg, screening for minimal residual disease, should be performed by monoplex PCR with primers specific for the malignant clone, using higher DNA and primer concentrations. High sensitivity together with high specificity can be achieved by using one primer that corresponds to the individual sequences of the V-N-J junction of the clone-specific rearrangement or translocation (VM and AR, unpublished results). This implies DNA sequence analysis of the PCR fragments, which can be performed using the rapid methods described here. In this study, the specificity and sensitivity of the four-color multiplex PCR was shown. To establish detection rates in a series of unselected lymphomas, larger studies will have to be performed. However, as detection rates will never be absolute, it should be kept in mind, that a polyclonal or negative result, respectively, does not exclude the presence of a malignant clone.
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Acknowledgements |
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Footnotes |
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Accepted for publication September 17, 2001.
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References |
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TopAbstractIntroductionIgH Rearrangementt(11;14)t(14;18)T-Cell Receptor-{gamma}Materials and MethodsResultsDiscussionReferences |
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 C. Gobbi, M. Buess, A. Probst, S. Ruegg, P. Schraml, R. Herrmann, A. J. Steck, and S. Dirnhofer Enteropathy-associated T-cell lymphoma with initial manifestation in the CNS Neurology, May 27, 2003; 60(10): 1718 - 1719. [Full Text] [PDF]
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 L. C. Lawnicki, R. J. Rubocki, W. C. Chan, D. M. Lytle, and T. C. Greiner The Distribution of Gene Segments in T-Cell Receptor {gamma} Gene Rearrangements Demonstrates the Need for Multiple Primer Sets J. Mol. Diagn., May 1, 2003; 5(2): 82 - 87. [Abstract] [Full Text] [PDF]
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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]
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T. C. Greiner and R. J. Rubocki Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in Detecting T-Cell Receptor {gamma} Gene Rearrangements J. Mol. Diagn., August 1, 2002; 4(3): 137 - 143. [Abstract] [Full Text] [PDF]
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 R. Yao, S. A. Rich, and E. Schneider Validation of Sixteen Leukemia and Lymphoma Cell Lines as Controls for Molecular Gene Rearrangement Assays Clin. Chem., August 1, 2002; 48(8): 1344 - 1351. [Abstract] [Full Text] [PDF]
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