|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 640-646
NEOPLASIA
High detection rate of T-cell receptor beta chain rearrangements
in T-cell lymphoproliferations by family specific polymerase chain
reaction in combination with the GeneScan technique and DNA sequencing
Chalid Assaf,
Michael Hummel,
Edgar Dippel,
Sergij Goerdt,
Hans-Henning Müller,
Ioannis Anagnostopoulos,
Constantin E. Orfanos, and
Harald Stein
From the Institute of Pathology, Consultation and Reference Center
for Lymph Node Pathology and Hematopathology and Department of
Dermatology, University Medical Centre Benjamin Franklin, The Free
University of Berlin, Berlin, Germany.
 |
Abstract |
The distinction between benign polyclonal and malignant monoclonal
lymphoid disorders by morphology or immunophenotyping is frequently
difficult. Therefore, the demonstration of clonal B-cell or T-cell
populations by detecting identically rearranged immunoglobulin (Ig) or
T-cell receptor (TCR) genes is often used to solve this diagnostic
problem. Whereas the detection of rearranged Ig genes is well
established, TCR gamma ( ) and beta ( ) gene rearrangements often
escape detection with the currently available polymerase chain reaction
(PCR) assays. To establish a sensitive, specific, and rapid method for
the detection of rearranged TCR- genes, we developed a new PCR
approach with family-specific J primers and analyzed the resulting
PCR products by high-resolution GeneScan technique. The superior
efficiency of this new method was demonstrated by investigating 132 DNA
samples extracted from lymph node and skin biopsy specimens (mostly
formalin fixed) and blood samples of 62 patients who had a variety of
T-cell lymphomas and leukemias. In all but 1 of the tumor samples
(98.4%) a clonal amplificate was detectable after TCR- PCR and the
same clonal T-cell population was also found in 15 of 18 (83%) of the
regional lymph nodes and in 7 of 11 (64%) of the peripheral blood
samples. Direct comparison of these results with those obtained
currently by the most widely applied TCR- PCR revealed an
approximate 20% lower detection rate in the same set of samples than
with the TCR- PCR method. These results indicate that the new
TCR- PCR is most suitable for a rapid and reliable detection of
clonal T-cell populations.
(Blood. 2000;96:640-646)
© 2000 by The American Society of Hematology.
| |
Introduction |
The distinction between malignant lymphomas and
reactive lymphoproliferative lesions is often difficult or
impossible to make by conventional histology alone. This holds true
especially for the identification of peripheral T-cell lymphomas
consisting predominantly of small cells such as mycosis fungoides, or
of a mixture of small and large cells like those in angioimmunoblastic
or lymphoepitheloid T-cell lymphomas. Although the availability of
several monoclonal antibodies has improved the recognition of T-cell
subpopulations, the differentiation between non-neoplastic and
neoplastic T cells still remains a problem. The configuration of the
T-cell receptor (TCR) genes has often been proposed as the most
valuable tool for identifying malignant T-cell proliferation. In normal
and reactively proliferating T cells, these genes are rearranged
differently (ie, polyclonal), whereas in T-cell lymphomas, the
neoplastic cells contain identically rearranged monoclonal TCR genes.
Initially, the Southern blot technique was used to determine the
clonality of the TCR gene rearrangements.1,2 For this purpose, the TCR- chain gene was most often analyzed, because all,
or almost all, T cells harbor functionally rearranged TCR- genes as
evidenced on the protein level.3-7 Although this assay may
detect clonal T-cell populations in the vast majority of T-cell lymphomas, several drawbacks such as low sensitivity, the need for
large amounts of fresh frozen tissue, and of radioactively labeled
probes, will exclude this technique as a general diagnostic tool.
Attempts to develop a polymerase chain reaction (PCR)-based method for
the detection of clonally rearranged TCR- genes in DNA samples
proved to be difficult, because primers capable of binding all
different 65 variable (V) and 13 joining (J)
segments8-13 require too many consensus
positions.14,15 This leads to a low detection rate of
T-cell malignancies, especially when applied to degraded DNA extracted
from formalin-fixed tissue.16,17 To overcome this problem,
most of the previous studies used RNA extracts in conjunction with
reverse transcriptase (RT)-PCR.18,19 However, this approach
is limited to the investigation of fresh frozen samples and not
applicable to formalin-fixed tissue specimens.
Therefore, various PCR assays have often been designed for the
detection of TCR- gene rearrangements at the DNA level because of
the simpler configuration of this gene and the greater homology within
the various V and J gene segments.20,21 Although the TCR- PCR is applicable to DNA extracted from formalin-fixed tissue specimens, it has the drawback that 10% to 50% of the T-cell
lymphomas escape detection, irrespective of the proportion of tumor
cells.22-30
Because of these limitations, there is a broad need for the development
of a new technique that may allow the identification of clonal TCR
rearrangements with a high detection rate, even in paraffin-embedded
tissues. In this paper, we describe a TCR- PCR that overcomes these
disadvantages by designing new family-specific J primers applied in
combination with a previously published V consensus
primer.31 With this assay, we investigated 132 samples
obtained from 53 patients with T-cell non-Hodgin's lymphoma (T-NHL)
and 9 patients with acute lymphoblastic leukemia (ALL). For control, 37 samples of reactive B-cell and T-cell lesions were included. For
comparison, the 169 samples studied were also analyzed by a TCR-
PCR, covering all known V segments, and the results of both assays
were compared using the high-resolution GeneScan technique. Most clonal
PCR products were confirmed by direct sequencing. Clonal TCR-
rearrangements were found in all but 1 of the T-cell malignancies
(98.4%) investigated, whereas clonally rearranged TCR- genes were
detectable in only 80%. The results obtained recommend the TCR-
PCR as a new valid technique for the detection of clonal T-cell
populations in patients with T-lineage lymphomas and leukemias.
| |
Patients, materials, and methods |
Patient samples and cell lines
A total of 132 samples from 62 patients with malignant T-cell
disease were selected from the files of the Institute of Pathology and
the Department of Dermatology at the Free University of Berlin. The
lymphomas were diagnosed using morphologic and immunohistologic criteria according to the revised European-American lymphoma (REAL) classification.32 The series included 96 samples (skin,
blood, lymph node) from 23 patients with primary cutaneous T-cell
lymphoma (CTCL) in advanced stages, 12 anaplastic large cell lymphomas of T-phenotype (ALCL-T), 11 peripheral unspecified T-cell lymphomas (PTCL), 9 acute T-lymphoblastic leukemias (T-ALL), 3 angioimmunoblastic T-cell lymphomas (AILD-TCL), and 11 samples from 4 patients with intestinal T-cell lymphoma (ITCL) (Table
1). Furthermore, 16 skin biopsy specimens
from patients with non-neoplastic skin disease, including 4 biopsies
from subacute or chronic dermatitis and 12 biopsies from psoriasis
vulgaris, were also investigated. The majority of the samples (102 of
148) were formalin-fixed and embedded in paraffin.
Positive controls consisted of 8 T-cell lines (Hut 102, MOLT 4, Jurkat,
DHL-1, PEER, Karpas, Del, MO-T) and 18 peripheral T-cell lymphomas with
known and sequenced clonal TCR- gene rearrangements. As negative
controls, 15 B-cell lymphomas, as well as 11 lymph node specimens from
unspecific lymphadenopathies, and 10 peripheral blood samples from
healthy donors were included (Table 1).
Immunohistology
Four-micrometer sections of paraffin-embedded tissues were
immunostained using the immunoalkaline phosphatase (APAAP)
method.33 The primary antibodies were directed against
T-cell receptor -chain (clone F1), CD3 (polyclonal CD3), CD4
(1F6), CD8 (C8-144), CD103 (Ber-ACT8), CD45RO (OPD4), CD30 (Ber-H2),
and TdT (terminal desoxynucleotidyltransferase; polyclonal TdT). With
the exception of F1, which was from T-Cell Sciences (Cambridge, MA)
and CD4 from Novocastra (Newcastle upon Tyne, UK), all other antibodies
were purchased from DAKO (Glostrup, Denmark). To unmask antigen
epitopes, deparaffinized tissue sections were subjected to
high-pressure cooking before applying primary antibody.
DNA extraction
DNA used for PCR was extracted from 25 µm paraffin sections after
dewaxing and proteinase K digestion using a QIAGEN DNA extraction kit
(Qiagen, Hilden, Germany). DNA from the mononuclear cell
fraction after Ficoll-Hypaque gradient of blood samples as well as from frozen tissue samples was extracted according to standard procedures.
Polymerase chain reaction for the detection of TCR-
rearrangements
For the detection of TCR- gene rearrangements, 200 ng of genomic
DNA was subjected to a seminested PCR. The first round of amplification
was performed as 2 separate reactions involving the same V consensus
primer31 (V pan: 5'-CTCGAATTCT(T/G)T(A/T) (C/T)TGGTA(C/T)C (G/A)(T/A)CA-3'; 200 ng) and 2 different primer sets (200 ng each set) consisting of 6 (J1 family; JFS1A) and 7 (J2 family; JFS2A) J family-specific primers, respectively (Table 2). Thirty cycles were carried out
with a primer annealing temperature of 60°C (40 seconds) for the
initial 5 cycles and 57°C (40 sec) for the remaining 25 cycles. For
reamplification, an aliquot (1%) of the first 2 reactions was used as
a template in 2 additional, separate PCRs, comprising 40 cycles each
with the same annealing temperature profile as described above. The same V primer (200 ng) was used in combination with 2 nested family-specific J primer mixes (JFS1 and JFS2; 200 ng each set; Table 2). The conditions for
denaturation (96°C, 15 seconds) and primer extension (72°C, 40 seconds) remained constant through all cycles of the first and second
PCR, whereas the concentration of MgCl2 was 2.5 mmol/L in
the first and 1.5 mmol/L in the second amplification. All reactions
were carried out in a final volume of 100 µL with 0.8 mmol/L of dNTPs
(200 µmol/L each) and 2.5 units Taq polymerase (Perkin Elmer,
Weiterstadt, Germany) in a thermal cycler (TC9600, Perkin Elmer,
Weiterstadt, Germany). It is worth noting that the application of
high-quality high-performance liquid chromatographic (HPLC)-purified
oligonucleotides is a decisive factor for successfully performing the
TCR- PCR.
Polymerase chain reaction for the detection of TCR-
rearrangements
TCR- gene rearrangements of the VI subgroup were detected by a
seminested PCR using 200 ng of genomic DNA as a template.30 The same J-specific primers were used for both rounds of
amplification, whereas 2 nested V primers were subjected to the
first and second PCR. The first amplification consisted of 2 separate
reactions (25 cycles each), one using the J primer
JGT1/223 and the other JGT323 (200 ng each), both in conjunction with
V11-8 (5'-TGCAGCCAGTCAGAAATCTTCC-3'). The
reamplification was carried out in 2 separate reactions using a nested
V primer (V21-8
5'-ACAGCGTCTTC(AT)GTACTATGAC-3') and the same
J-specific primers (JGT1/2 and JGT3). The
cycle conditions remained constant through all PCRs being 96°C, 15 seconds for denaturation; 60°C, 30 seconds for primer annealing and
72°C, 40 seconds for primer extension. The buffer conditions were
the same as those described for the reamplification of the TCR- PCR.
In those T-cell lymphoma cases in which no clonal TCR-
rearrangements were detectable with the primers for the VI subgroup, additional PCRs for the detection of rearrangements involving V
segments of the VII (V9) and VIII
(V10,11) subgroups were performed as previously
described23 with one exception: instead of using a
conventional Taq polymerase (ie, AmpliTaq;
Perkin-Elmer), we used TaqGold (Perkin-Elmer) for
amplification. We found the use of TaqGold crucial for the
elimination of unwanted additional PCR products (generated by the
V9-11 primers), which may cause erroneous detection of clonality.
GeneScan
For GeneScan analysis of the PCR products, the V and V primers
of the reamplification were replaced by fluorescence primers of the
same sequence labeled at their 5'-end with 5-carboxyflourescein (FAM). Aliquots of PCR products (1-2 µL) were mixed with loading buffer (2 µL formamide, 0.5 µL EDTA), and 0.5 µL of the internal size standard (Genescan-500) were included for precise determination of
the length of the amplificates. After denaturation for 2 minutes at
90°C, the products were separated on sequencing gel and analyzed by
automatic fluorescence quantification and size determination, using the
computer program GENESCAN 672 (ABI 373A, Applied Biosystems, Weiterstadt, Germany).
Direct sequencing
The clonal PCR products of most T-cell lymphomas and of all T-cell
lines were directly sequenced. For this purpose, reaction mixtures were
separated on 6% polyacrylamid gels and stained with ethidium bromide
(Figure 1). The most dominant band was cut out, and the gel slice was
incubated in 25 µL distilled water for at least 24 hours. Five microliters of the supernatant were subjected to
fluorescence dye terminator cycle sequencing, and the sequencing reactions were analyzed on a 377A DNA sequencer (Applied Biosystems) after removal of the unincorporated fluorescence dye. Each sequencing reaction was carried out in both directions using the primers Vpan
and JFS1 or JFS2.
| |
Results |
Primer testing, specificity, and sensitivity of the TCR- and
TCR- polymerase chain reaction
The newly designed TCR- PCR was established and optimized by
applying DNA extracted from 8 different T-cell lines. As expected, all
cell lines gave rise to single distinct PCR products that harbored the
expected DNA sequences, as confirmed by DNA sequencing (Table 3).
Similarly, all 18 peripheral T-cell lymphomas with known TCR-
rearrangements gave rise to 1 or 2 (biallelic) amplificates after
application of TCR- PCR (Figure
2). The primers
of the TCR- PCR were tested as described elsewhere,23,30
demonstrating the presence of clonal PCR products in all T-cell lines
analyzed so far.
View larger version (55K):
[in this window]
[in a new window]
| Fig 1.
Ethidium bromide stained 6% polyacrylamid gel of
representative TCR- PCR products.
(S) DNA size marker. (1) Normal tonsillar tissue. (2) Lesional skin,
CTCL (case 18). (3) Infiltrated lymph node, CTCL (case 18). (4)
Peripheral T-cell lymphoma (case 29). (5) T-ALL (case 41). (6)
Anaplastic large-cell lymphoma (case 58). (7) Intestinal T-cell
lymphoma (case 53). (8) Cell line Hut102. (9) B-cell lymphoma. PCR
products in lanes 1, 2, 8, and 9 were generated from DNA of frozen
material, and those in lanes 3, 4, 5, 6, and 7, from DNA extracted from
paraffin-embedded specimens.
|
|
View larger version (24K):
[in this window]
[in a new window]
| Fig 2.
Comparison of the fluorescence-labeled TCR- and
TCR- PCR products by GeneScan analysis.
The x-axes represent molecular size (base pairs) and the y-axes
fluorescence intensity. Samples A-D were produced by TCR- PCR and
samples E-H by TCR- PCR. (A, E) Normal tonsillar tissue. (B, F)
Cutaneous T-cell lymphoma (case 23). (C, G) Psoriasis. (D) T-cell line
HUT102 (243 bp). (H) T-cell line HUT102 (205 bp). Note: TCR- PCR
products regularly differ by 3 base pairs, indicating the presence of
functional TCR- rearrangements. TCR- PCR products differ by only
1 base pair, indicating frequent out-of-frame rearrangements.
|
|
The specificity of the TCR- and TCR- PCR technique was determined
by investigating 11 normal lymph nodes, 10 peripheral blood samples
from healthy donors, and 15 B-cell lymphomas. In addition, 16 inflammatory dermatoses were also investigated (Table 1, Figure 2). All
normal lymph node and blood samples gave rise to a polyclonal
rearrangement pattern, whereas most of the remaining samples displayed
an oligoclonal pattern sometimes comprising only a few dominant clones.
However, repeated independent PCR assays clearly demonstrated that the
dominant clones differed from each other among the various PCRs.
The sensitivity of both PCR techniques was determined by serial
dilution of T-cell lines in normal tonsillar DNA. All PCR assays were
able to detect between 0.5% and 1% of clonally rearranged T-cells in
a polyclonal background (0.5 ng to 1 ng T-cell line DNA in 100 ng of
tonsillar DNA). The use of more PCR cycles did not increase the sensitivity.
Detection of clonal T-cell populations in T-cell
lymphomas and leukemias
A total of 62 histologically and clinically well-diagnosed T-cell
lymphomas and leukemias, including 23 CTCLs, 12 ALCLs, 11 unspecified
PTCLs, 9 T-ALLs, 4 ITCLs, and 3 AILDs, were investigated with both the
TCR- and TCR- PCR techniques (Table 1). Monoclonal T-cell
populations were detectable in all but 1 of the tumor samples (61 of 62; 98%) by TCR- PCR, irrespective of the use of frozen or
formalin-fixed material (Figure 1). Moreover, the same clonal TCR-
rearrangement detected in the primary skin tumor was also found in 15 of 18 (83%) and 7 of 11 (64%) of the excised regional lymph nodes and
the peripheral blood samples of 23 patients with CTCL (Table
4). Furthermore, multiple samples
of 13 patients who had biopsies over a period of up to 3 years,
all contained the same TCR- gene rearrangement. The presence of the
same clonal TCR- rearrangement was confirmed in each case by
repeated independent analysis, including PCR, GeneScan analysis, and
DNA sequencing of most amplificates.
The application of the TCR- PCR using primers for the V subgroups
I, II, and III to the same set of samples led to the detection of
monoclonally rearranged T-cell populations in 80% (50 of 62) of
the tumor samples (Table 1, Figure
3). The
investigation of corresponding lymph nodes and blood samples of the
patients with CTCL revealed monoclonality in only 63% and 44 %, respectively (Table 4). All but 2 of the clonal TCR-
rearrangements displayed involvement of segments of the VI subgroup
(V1-8), whereas 1 of the remaining 2 patients (case 12)
had a clonal V10, and the other (case 32), a biallelic
V9/V10 rearrangement.
View larger version (23K):
[in this window]
[in a new window]
| Fig 3.
Comparison of results.
Comparison of TCR- (A-C) and TCR- (D-F) PCR results
obtained in 3 T-cell lymphomas with clonal TCR- rearrangements and
poly/oligoclonal TCR- rearrangements by GeneScan analysis. (A, D)
Anaplastic large cell lymphoma (case 53). (B, E) Peripheral T-cell
lymphoma, unspecified (case 34). (C, F) Intestinal T-cell lymphoma
(case 50).
|
|
View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
Follow-up by GeneScan analysis in a patient with
cutaneous T-cell lymphoma (case 12) by TCR- PCR.
(A) Plaque-stage at the time of first diagnosis. (B) Lymph node
involvement half year later. (C) Peripheral blood at the time of lymph
node involvement. (D) Skin lesion 2 years after chemotherapy; relapse.
Note: The same clonal TCR- rearrangement (250 bp) is detectable in
all tissue specimens, but not in the peripheral blood sample.
|
|
GeneScan analysis
Application of high-resolution separation techniques such as
GeneScan analysis appeared best suited for determining the presence of
clonal T-cell populations. The high resolution allowed the identification of PCR products that differed by only 1 base pair (bp)
in length. Furthermore, the use of internal standards enables a precise
determination of the size of the PCR products, irrespective of
gel-to-gel variations or gel shift artifacts often associated with
other techniques. This allows monitoring of lymphoma patients by direct
comparison of the results obtained at various points in time without
reanalyzing the previous samples (Figure 4).31,34,35
GeneScan analysis of normal lymphoid samples displayed a Gaussian-like
distribution of the sizes of the TCR- amplificates ranging from 234 to 261 bp (JFS1) and 237 to 264 bp (JFS2) and of TCR- PCR
products ranging from 192 to 220 bp (JGT1/2 primer set) and
from 210 to 238 bp (JGT3 primer set). Noteworthy, the products of the TCR- PCR differed by only 1 base pair in size (Figure 2E,G), indicating frequent presence of out-of-frame
rearrangements, whereas the reading frame is conserved in most TCR-
rearrangements (Figure 2A,C). T-cell lymphomas or T-cell lines were
characterized by 1 or 2 dominant peaks in the GeneScan profile,
indicating the presence of a clonal T-cell population. Application of
both TCR PCRs to reactive T-cell proliferations, especially those in
extranodal origin, resulted in some cases the appearance of few
dominant PCR products. However, in contrast to the monoclonal
amplificates encountered in T-cell neoplasias, these products were not
reproducible in independently performed PCR assays, thus representing
the presence of oligoclonal T-cell populations in these lesions.
DNA sequencing of TCR- polymerase chain reaction products
For investigation of the specificity of the TCR- PCR,
we sequenced 28 amplificates derived from various T-cell
lymphomas and T-cell lines (Table 3). Sequence information was obtained by direct sequencing without additional subcloning of the PCR products.
All sequences of the rearranged V and J segments could be
identified by comparing them with databank sequences. In contrast to
the TCR- locus the junctional TCR--CDR3 region showed extensive diversity due to the addition of N-region nucleotides between the
V-D and D-J junctions and seemed ideally suited as
clone-specific identification sequence. As an independent test of the
PCR results, a comparison of the junctional regions obtained for the
cell lines Jurkat, MOLT4, Hut 102, and PEER revealed complete agreement
with published sequences. The sizes of the sequenced CDR3 regions were in direct correlation to the length of the PCR fragments, as
demonstrated by the GeneScan analysis.
Immunohistology
In all T-cell lymphoma cases, immunohistology disclosed a T-cell
phenotype with the expression of CD3 by the atypical cells. Predominance of CD4+ or CD8+ subpopulations was
verified in several cases, underscoring the presence of a monoclonal
T-cell population. An expression of the -chain was detected in most
cases. ALC-T cases characteristically expressed CD30. ITCL cases
were characterized by the presence of atypical CD103-positive
T-lymphocytes within the epithelium. T-ALL cases, in addition to T-cell
antigen expression, showed an intranuclear positivity for TdT.
| |
Discussion |
The adjunct of PCR technology for the detection of clonally
rearranged TCR genes has greatly contributed to the distinction between
benign polyclonal and neoplastic monoclonal T-cell populations, and
thus, to the diagnosis of T-cell lymphomas. However, in a significant
proportion of malignant T-cell proliferations the tumor T-cell clone
escapes detection with currently available TCR- PCR
methods.24-30,36 Attempts to establish a technique for detection of clonally rearranged TCR- genes on DNA level suitable for investigation of formalin-fixed biopsies failed so
far.16-19,37,38 An interesting and promising attempt to
simplify the TCR- PCR was the use of highly degenerated J
consensus primers by Kneba and colleagues.15 This method
detected only large T-cell clones,15,39 and, in our hands,
produced high background and was difficult to reproduce when applied to
routine formalin-fixed biopsy specimens. However, the
results obtained encouraged us to develop a new TCR- PCR suitable
for the application in the daily diagnostic work.
To overcome the shortcomings of previous TCR- methods, we designed
family-specific J primers. These primers consist exclusively of
oligonucleotides with complete homology to their target genes therefore
obtain several advantages over degenerated consensus primers, as
already shown for the detection of clonal immunoglobulin (Ig)
rearrangements.40 As a result, application of
family-specific J primers led to amplification of largely
background-free TCR- specific PCR products. This seems especially
important for detection of small clonal T-cell populations present in
early CTCL or in minimal lymph node involvement, where polyclonal
background signals can superimpose clonality. Moreover, combination of
TCR- PCR with the high-resolution GeneScan technique enables a
precise and reliable interpretation of the PCR data and exact
monitoring between different lesions of the same
patient.30,34,35
The sensitivity, specificity, and reliability of the new TCR- PCR
assay were tested by investigating a large number of histologically and
clinically proven T- and B-cell lymphomas, as well as reactive lymphoid
lesions and normal lymphoid tissues. Monoclonal T-cell populations were
demonstrable in all but 1 of the 62 T-cell lymphomas investigated,
irrespective of the use of fresh-frozen or formalin-fixed paraffin-embedded specimens. Moreover, identical TCR- gene
rearrangements were detectable in the corresponding blood and lymph
node samples from 64% (7 of 11) and 83% (15 of 18) of the patients
and multiple lymphoma samples from 13 patients, who had biopsies over a
period of up to 3 years, contained all the same TCR- gene rearrangement.
The investigation of the same set of T-cell lymphomas with the TCR-
PCR technique using primers for the V subgroups I, II, and III
revealed a clonal TCR- gene rearrangement in only 80% of the
cases (50 of 62). This is in line with most previous
publications.24-30,41-45 No significant differences were
observed between the type of the lymphoma, its location, or the
proportion of tumor cells, with the exception of precursor
T-lymphoblastic lymphomas, where all tumors displayed clonal a TCR-
rearrangement. These results clearly indicate that the failure to
detect clonal T-cell populations in a significant proportion of cases
(12 of 62; 20%) was not due to a limited sensitivity of the TCR-
PCR, but to a true absence of appropriate TCR- rearrangements.
Therefore, other possibilities are required to explain the
nondetectability of clonal TCR- rearrangements in these cases, such
as a germ line configuration of the TCR- gene, incomplete or
deleterious rearrangement within the TCR- locus,46 or
transrearrangement between V-segments of the -chain and J-segments
of the  -chain.47 However, because the vast majority of
the rearranged TCR- genes are not expressed, these nonfunctional rearrangements are without any consequence for the survival of the
T-cells.
The specificity of the TCR- and TCR- PCR assays was determined by
investigating DNA extracted from 15 B-cell lymphomas, 11 normal lymph
nodes, and 10 normal peripheral blood samples. Neither the TCR- nor
the TCR- PCR showed clonal rearrangements in any of these samples,
confirming the specificity of both assays. Interestingly, application
of both PCR systems to inflammatory skin lesions disclosed
poly/oligoclonal gene rearrangements with clear dominance of 1 or only
a few clones in some instances (pseudoclonal). However, these dominant
PCR products were shown to possess different sizes in independently
performed PCR assays. This pseudoclonality is explainable by the
cellular composition of these lesions. T cells proliferating in the
skin are not embedded in a background of many nonclonal lymphoid cells,
leading to only a few or even single amplificates of various sizes
after TCR- or TCR- PCR. Especially for investigations of skin or
other extranodal tissue, we recommend to perform 2 independent PCRs
and to consider only those cases as clonal if complete size concordance
of the dominant PCR product occurs in both assays.
In conclusion, the TCR- PCR technique described in this article is
reliable and highly suitable for the detection of small populations of
clonal T cells, not only in frozen tissues but also in formalin-fixed
paraffin-embedded specimens. This underscores its value as a new and
highly sensitive diagnostic tool for routine biopsy material. Further,
the low background of the TCR- PCR products enables direct
sequencing. This may be useful for generating clone-specific primers
for investigation of minimal residual disease, thus allowing detection
of tumor cell specific TCR- rearrangements in the follow-up of
T-cell lymphoma/leukemia patients.
| |
Acknowledgments |
We are particularly indebted to H. Lammert and H. Hempel for excellent
technical assistance.
| |
Footnotes |
Submitted November 9, 1999; accepted February 29, 2000.
Supported by the Deutsche Krebshilfe, Grant 70-2202-Mü3.
This work contains parts of the doctoral thesis of C.A.
Reprints: Michael Hummel, Institute of Pathology, University
Medical Centre Benjamin Franklin, The Free University of Berlin,
Hindenburgdamm 30, 12200 Berlin, Germany; e-mail:
hummel{at}ukbf.fu-berlin.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Weiss LM, Hu E, Wood GS, et al.
Clonal rearrangements of T-cell receptor genes in mycosis fungoides and dermatopathic lymphadenopathy.
N Engl J Med.
1985;313:539[Abstract].
2.
Griesser H, Feller A, Lennert K, et al.
The structure of the T-cell gamma chain gene in lymphoproliferative disorders and lymphoma cell lines.
Blood.
1986;68:592[Abstract/Free Full Text].
3.
Griesser H, Feller A, Lennert K, Minden M, Mak TW.
Rearrangement of the beta chain of the T-cell antigen receptor and immunoglobulin genes in lymphoproliferative disorders.
J Clin Invest.
1986;78:1179.
4.
Weiss LM, Wood GS, Hu E, Abel EA, Hoppe RT, Sklar J.
Detection of clonal T-cell receptor gene rearrangements in the peripheral blood of patients with mycosis fungoides/sezary syndrome.
J Invest Dermatol.
1989;92:601[Medline]
[Order article via Infotrieve].
5.
Weiss LM, Picker LJ, Grogan TM, Warnke RA, Sklar J.
Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas.
Am J Pathol.
1988;130:436[Abstract].
6.
O'Connor N, Crick JA, Wainscoat JS, et al.
Evidence for monoclonal T lymphocyte proliferation in angioimmunoblastic lymphadenopathy.
J Clin Pathol.
1986;39:1229[Abstract/Free Full Text].
7.
O'Connor N, Wainscoat JS, Weatherall DJ, et al.
Rearrangement of the T-cell receptor beta-chain gene in the diagnosis of lymphoproliferative disorders.
Lancet.
1985;1:1295[Medline]
[Order article via Infotrieve].
8.
Sims JE, Tunnacliffe A, Smith WJ, Rabbitts TH.
Complexity of human T-cell antigen receptor beta-chain constant- and variable-region genes.
Nature.
1984;312:541[Medline]
[Order article via Infotrieve].
9.
Kimura N, Toyonaga B, Yoshikai Y, et al.
Sequences and diversity of human T-cell receptor beta chain variable-region genes.
J Exp Med.
1986;164:739[Abstract/Free Full Text].
10.
Tillinghast JP, Behlke MA, Loh DY.
Structure and diversity of the human T-cell receptor beta-chain variable region genes.
Science.
1986;233:879[Abstract/Free Full Text].
11.
Rowen L, Koop BF, Hood L.
The complete 685-kilobase DNA sequence of the human beta T-cell receptor locus.
Science.
1996;272:1755[Abstract].
12.
Toyonaga B, Yoshikai Y, Vadasz V, Chin B, Mak TW.
Organisation and sequences of the diversity, joining, and constant region genes of the human T-cell receptor beta chain.
Proc Natl Acad Sci U S A.
1985;82:862.
13.
Tunnacliffe A, Rabbitts TH.
Sequence of the D beta 2-J beta 2 region of the human T-cell receptor beta-chain locus.
Nucleic Acid Res.
1985;13:6651[Abstract/Free Full Text].
14.
McCarthy KP, Sloane JP, Kabarowski JH, Matutes E, Wiedemann LM.
The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders.
Am J Pathol.
1991;138:821[Abstract].
15.
Kneba M, Bolz I, Linke B, Hiddemann W.
Analysis of rearranged T-cell receptor -chain genes by polymerase chain reaction (PCR), DNA sequencing and automated high resolution PCR fragment analysis.
Blood.
1995;86:3930[Abstract/Free Full Text].
16.
Slack DN, McCarthy KP, Wiedemann LM, Sloane JP.
Evaluation of sensitivity, specificity, and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T-cell receptor genes in formalin-fixed, paraffin-embedded sections.
Diagn Mol Pathol.
1993;2:223[Medline]
[Order article via Infotrieve].
17.
Diss TC, Watts M, Pan LX, Burke M, Linch D, Isaacson PG.
The polymerase chain reaction in the demonstration of monoclonality in T-cell lymphomas.
J Clin Pathol.
1995;48:1045[Abstract/Free Full Text].
18.
Obata F, Tsunoda M, Ito K, et al.
A single universal primer for the T-cell receptor (TCR) variable genes enables enzymatic amplification and direct sequencing of TCR cDNA of various T-cell clones.
Hum Immunol.
1993;36:163[Medline]
[Order article via Infotrieve].
19.
Kono DH, Baccala R, Balderas RS, et al.
Application of a multiprobe RNase protection assay and junctional sequences to define V gene diversity in Sezary syndrome.
Am J Pathol.
1992;140:823[Abstract].
20.
Hayday AC, Saito H, Gillies SD, et al.
Structure, organization, and somatic rearrangement of T-cell gamma genes.
Cell.
1985;40:259[Medline]
[Order article via Infotrieve].
21.
Kranz DM, Saito H, Heller M, et al.
Limited diversity of the rearranged T-cell gamma gene.
Nature.
1985;313:752[Medline]
[Order article via Infotrieve].
22.
Bourguin A, Tung R, Galili N, Sklar J.
Rapid, nonradioactive detection of clonal T-cell receptor gene rearrangements in lymphoid neoplasms.
Proc Natl Acad Sci U S A.
1990;87:8536[Abstract/Free Full Text].
23.
Trainor K, Brisco M, Wan J, Neoh S, Grist S, Morley A.
Gene rearrangement in B- and T-lymphoproliferative disease detected by polymerase chain reaction.
Blood.
1991;78:192[Abstract/Free Full Text].
24.
Bottaro M, Berti E, Biondi A, Migone N, Crosti L.
Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
Blood.
1994;83:3271[Abstract/Free Full Text].
25.
Wood GS, Tung RM, Haeffner AC, et al.
Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE).
J Invest Dermatol.
1994;103:34[Medline]
[Order article via Infotrieve].
26.
Kneba M, Bolz I, Linke B, Bertram J, Rothaupt D, Hiddemann W.
Characterization of clone-specific rearrangement T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing.
Blood.
1994;84:574[Abstract/Free Full Text].
27.
Theodorou I, Delfau Larue MH, Bigorgne C, et al.
Cutaneous T-cell infiltrates: analysis of T-cell receptor gamma gene rearrangement by polymerase chain reaction and denaturing gradient gel electrophoresis.
Blood.
1995;86:305[Abstract/Free Full Text].
28.
Greiner TC, Raffeld M, Lutz C, Dick F, Jaffe ES.
Analysis of T-cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products: correlation with tumor-specific sequences.
Am J Pathol.
1995;146:46[Abstract].
29.
Ashton-Key M, Diss T, Du M, Kirkham N, Wotherspoon A, Isaacson P.
The value of the polymerase chain reaction in the diagnosis of cutaneous T-cell infiltrates.
Am J Surg Pathol.
1997;21:743[Medline]
[Order article via Infotrieve].
30.
Dippel E, Assaf C, Hummel M, et al.
Clonal T-cell receptor -chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation.
J Pathol.
1999;188:146[Medline]
[Order article via Infotrieve].
31.
Lessin SR, Rook AH, Rovera G.
Molecular diagnosis of cutaneous T-cell lymphoma: polymerase chain reaction amplification of T-cell antigen receptor beta-chain gene rearrangements.
J Invest Dermatol.
1991;96:299[Medline]
[Order article via Infotrieve].
32.
Harris NL, Jaffe ES, Stein H, et al.
A revised European-American classification of lymphoid neoplasms: a proposal from the international lymphoma study group.
Blood.
1994;84:1361[Free Full Text].
33.
Cordell JL, Falini B, Erber WN, et al.
Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).
J Histochem Cytochem.
1984;32:219 |
Top
Abstract
Introduction