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NEOPLASIA
Single-cell analysis of CD30⫹ cells in lymphomatoid papulosis demonstrates a
common clonal T-cell origin
Matthias Steinhoff, Michael Hummel, Ioannis Anagnostopoulos, Peter Kaudewitz, Volkhard Seitz, Chalid Assaf,
Christian Sander, and Harald Stein
Lymphomatoid papulosis (LyP) represents an intriguing cutaneous T-cell lymphoproliferative disorder with a histologic appearance resembling malignant
lymphoma. This finding strongly contrasts with the benign clinical course of
the disease. However, in 10% to 20% of
cases, LyP can precede, coexist with, or
follow malignant lymphoma. In these
cases, the same T-cell population has
been shown to be present in the LyP as
well as in the associated lymphoma. In
most LyP cases, there is—despite the
sometimes extremely long course of the
disease—no evolution of a secondary lym-
phoma. The investigation of these uncomplicated LyP cases for the presence of
clonal T-cell receptor rearrangements has
produced heterogeneous results. This
might be explained by biologic or technical reasons arising from analyzing whole
tissue DNA extracts. To definitively clarify
whether the large atypical CD30ⴙ cells in
LyP without associated lymphoma all belong to the same clone or represent individually rearranged T cells, we analyzed
the T-cell receptor–␥ rearrangements of
single CD30ⴙ as well as of single CD30ⴚ
cells isolated from 14 LyP lesions of 11
patients. By using this approach we could
demonstrate that the CD30ⴙ cells represent members of a single T-cell clone in
all LyP cases. Moreover, in 3 patients the
same CD30ⴙ cell clone was found in
anatomically and temporally separate lesions. In contrast, with only a few exceptions, the CD30ⴚ cells were polyclonal in
all instances and unrelated to the CD30ⴙ
cell clone. Our results demonstrate that
LyP unequivocally represents a monoclonal T-cell disorder of CD30ⴙ cells in all
instances. (Blood. 2002;100:578-584)
© 2002 by The American Society of Hematology
Introduction
Lymphomatoid papulosis (LyP) occupies a highly interesting position
within the spectrum of cutaneous lymphoproliferative disorders. The
histologic picture of LyP is extremely variable and closely mimics that
of malignant lymphoma.1 Three major histologic types have been
recognized; these are designated as A, B, and C.2,3 Types A and C are
characterized by the presence of large atypical blasts including mononucleated and binucleated or multinucleated cells resembling ReedSternberg cells characteristic of Hodgkin disease. The atypical blasts
express one or more T-cell antigens as well as the lymphoid activation
antigen CD30. While in type A these cells are embedded in a dense
inflammatory background, in type C they form large sheets closely
simulating CD30⫹ cutaneous anaplastic large cell lymphoma (ALCL).
LyP type B is composed of small to medium-sized CD30⫺ T cells
showing epidermotropism, thus closely resembling classical
mycosis fungoides.
The clinical features of LyP are more uniform and usually
consist of repetitive episodes of self-healing papulonodular skin
lesions often spread over a period of several years. From 10% to
20% of LyP cases are associated with malignant lymphoma,
especially mycosis fungoides, CD30⫹ cutaneous ALCL, or Hodgkin
disease, which can precede, coexist with, or follow LyP and can
also appear in the lymph nodes.4-8 In many of these cases the same
clonal T-cell receptor (TCR) rearrangements have been found in the
LyP as well as in the associated lymphoma, identifying LyP as a
precursor lesion.6,9-12
Most LyP cases are, however, not associated with T-cell
lymphomas of various types.4,7 In these cases, clonal T-cell
populations were detectable only in a proportion of analyzed
lesions,13-16 which might, in addition, be different when taken at
various times.13 Therefore, it was concluded that LyP represents in
most instances a reactive skin disease that can, under certain
circumstances, present as a localized clonal lymphoid disorder.15
Previous investigations were performed either by Southern blot
analysis or by polymerase chain reaction (PCR) using DNA
extracts from whole-tissue samples. However, this approach is
often unable to detect small populations of clonally rearranged T
cells residing in an abundant background of polyclonal cells, as
frequently observed in LyP. Furthermore, the analysis of wholetissue DNA does not allow for the assignment of the clonal
rearrangements to a definite cell population.
To overcome these limitations, we have used an approved
single-cell technique with which we recently successfully clarified
the cellular origin and clonality of Hodgkin-Reed-Sternberg cells.17
With this technique we isolated CD30⫹ as well as CD30⫺ cells
from 14 LyP lesions of 11 patients with LyP and analyzed their
TCR-␥ rearrangements. In addition, the results of single-cell
analyses were compared with GeneScan analysis of TCR-␥ PCR
products from whole-tissue DNA extracts. With one exception,
only patients without preceding or coexisting malignant lymphoma
were included in our study. With this approach we were able to
From the Department of Dermatology, Ludwig-Maximillians—University Munich,
Germany; and Department of Dermatology, Institute of Pathology, University
Medical Center Benjamin Franklin, The Free University of Berlin, Germany.
Benjamin Franklin, The Free University of Berlin, Hindenburgdamm 30, 12200
Berlin, Germany; e-mail: [email protected].
Submitted December 11, 2001; accepted March 3, 2002. Prepublished online
as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0199.
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.
Reprints: Michael Hummel, Institute of Pathology, University Medical Center
© 2002 by The American Society of Hematology
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
CLONAL T-CELL ORIGIN IN LYMPHOMATOID PAPULOSIS
definitely answer the following questions: Do CD30⫹ cells in a
single LyP lesion represent members of a single T-cell clone? Do
CD30⫹ cells of anatomically and temporally separate lesions in the
same patient belong to the same clone or to different clones? Is
clonality exclusively restricted to the population of CD30⫹ T cells?
If not, do CD30⫺ T cells also represent members of the CD30⫹
clone or of an own separate clone?
Materials and methods
Clinical data of the 11 cases of LyP are summarized in Table 1. All 11
patients, 6 men and 5 women, presented a typical history of recurrent
self-healing papulonodular eruptions.
Tissue samples and immunostaining
Fourteen tissue samples of 11 patients with clinical and biopsy-proven LyP
were studied. The histologic diagnosis of LyP was confirmed by at least 3
independent experts (H.S., P.K., I.A.). Histologic classification was done
for all biopsies and reviewed according to established criteria.3
Immunohistochemistry was performed, as previously described, by
using the immunoalkaline phosphatase-antiphosphatase technique18 and
streptavidin-biotin technique. Antibodies against the following antigens
were applied: CD30, CD2, CD3, CD4, CD5, CD8, CD20, TCR-␤ chain,
perforin, granzyme B, and ALK1. To differentiate CD30⫺ T cells, double
staining using antibodies against CD30 and CD2 was carried out.
Isolation of single cells
Single CD30⫹ LyP cells as well as single CD30⫺/CD2⫹ cells were isolated
from immunostained frozen tissue sections using a hydraulic micromanipulation device as previously described.17 In cases of insufficient CD30/CD2
double staining, 2 to 6 single CD30⫺ cells were pooled and isolated from
tissue sections stained with antibodies against CD30. Buffer aliquots
covering the sections were aspirated as negative controls for PCR analysis.
All cases were analyzed at least twice in completely independent cell
isolation and PCR assays.
579
Single-cell PCR
Isolated cells were digested with proteinase K (1 hour, 50°C, 0.1 mg/mL)
and, after heat denaturation of the enzyme, subjected to PCR. For the
detection of TCR-␥ rearrangements, 4 different sets of primers were
established. For amplification of rearrangements involving V␥1 to V␥8
gene segments, 2 seminested PCRs were carried out in separate reactions
employing a consensus V␥1 to V␥8 primer (VG-1) in conjugation with the
J␥-specific primers JGT1/2 and JGT3, respectively. For reamplification the
VG-1 primer was replaced by a nested V␥1 to V␥8 consensus primer
(VG-2) whereas the same J␥-specific primers were used. The buffer
(TaqGold [2 U] and TaqGold buffer [Perkin-Elmer], 1.37 mM MgCl2, 0.2
mM each deoxyribonucleoside triphosphate, 200 ng each primer) and
cycling conditions (95°C for 30 seconds, 64°C [first 5 cycles] and 61°C
[remaining 35 cycles] for 30 seconds, 72°C for 30 seconds) were as
previously described.19
For the amplification and reamplification of the rearrangements involving V␥9, V␥10, or V␥11 gene segments, we created new nested V␥ primers
that were used in combination with the above-mentioned J␥-specific
primers. The annealing conditions for the detection of V␥9 rearrangements
were 5 cycles at 68°C (30 seconds) and 35 cycles at 64°C (30 seconds) for
the first round of PCR and 40 cycles at 65°C for reamplification. The
remaining buffer and cycling conditions were as described for the V␥1 to
V␥8 amplification with the exception of 100 ng of each primer and 2.0 mM
MgCl2. V␥10 and V␥11 rearrangements could be amplified using the same
annealing and cycling conditions: 35 cycles at 65°C for the first round of
amplification and 35 cycles at 60°C (30 seconds, V␥9) and 57°C (30
seconds, V␥10), respectively, for reamplification.
PCR products were separated on polyacrylamide gels (PAGE 6%)
stained with ethidium bromide.
PCR of whole-tissue DNA extracts
The data obtained from single-cell PCR were confirmed by PCR analysis of
the corresponding whole tissue DNA. For this, DNA was extracted from
frozen skin specimens by following the manufacturer’s recommendations
(QIAamp DNA Mini Kit; QIAGEN, Hilden, Germany). Whole-tissue DNA
extracts were analyzed for rearrangements of V␥1 to V␥8, V␥9, V␥10, and
V␥11 gene segments as previously described.20
Table 1. Clinical features of 11 patients with LyP A
Patient
no.
Age*/sex
Duration*
1†
62/F
1y
Site of biopsy
A: Left groin
Associated lymphoma
Remarks
—
Trunk/groin: multifocal papular/nodular skin lesions that slowly
subside; exclusion of systemic disease by computed
B: Left axilla
2†
42/F
10 y
C: Left flank
tomography of thorax and abdomen; bone marrow biopsy
—
skin lesions with partial spontaneous remission, good
D: Left thigh
3†
76/F
10 mo
4
49/F
4 mo
5
31/M
1 mo
6
57/M
?
7
82/M
30 y
E: ?
Involvement of extremities: multifocal papular/papulonecrotic
response to psoralen-UV/A therapy
—
Trunk/extremities: disseminated papular lesions
Right elbow
—
Elbows/thighs: papular/papulonecrotic lesions
Abdomen
—
Abdomen: a single nodular lesion
Right knee
—
Trunk/extremities: disseminated papular lesions/plaques;
F: Back
coexisting small-plaque parapsoriasis
?
Mycosis fungoides
Involvement of extremities: multifocal papular/papulonecrotic
skin lesions; diagnosis of mycosis fungoides 28 y after the
onset of LyP
8
7/M
3 mo
Abdomen
—
Trunk/extremities: disseminated self-healing papular lesions
9
61/M
2 mo
Right upper arm
—
Trunk/accentuation of extremities: multifocal
10
77/F
6 mo
Right shoulder
—
Accentuation of the trunk: disseminated papular lesions
11
24/M
3y
Left upper arm
—
Trunk/extremities: multifocal papular/papulonecrotic/nodular
papular/papulonecrotic lesions
skin lesions that spontaneously subside
? indicates unknown.
*At the moment of biopsy (in relation to the first biopsy).
†Investigation of 2 anatomically and temporally separate biopsies.
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580
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
STEINHOFF et al
Fluorescent fragment analysis
For GeneScan analysis, PCR amplification was carried out under the
conditions described in detail above. The amplification was performed with
5-carboxyfluorescein–labeled VG-2 primer or with 5-carboxyfluorescein–
labeled JGT1/2 primer and JGT3 primer, respectively, for amplification of
V␥9, V␥10, or V␥11 gene segments. A total of 2.0 ␮L of diluted PCR
product (dilution dependent on intensity of the amplificate) was mixed with
2.0 ␮L foramide and 0.5 ␮L 6-carboxyrhodamine dye–labeled DNA size
standard (GeneScan-500-[ROX], Applied Biosystems, Weiterstadt, Germany) and 0.5 ␮L loading buffer (applied with the size standard). After
denaturation (2 minutes at 90°C) and cooling on wet ice (3 minutes), 2.5 ␮L
was size-separated on a high-resolution polyacrylamide gel and analyzed
using an automated 373A DNA sequencer. The size of the PCR products
was determined using computer software GeneScan 672 (Applied
Biosystems).
DNA sequence analysis
Unlabeled PCR products were isolated and sequenced directly by fluorescence chain termination technique using fluorescence-labeled dideoxynucleotide triphosphates (BigDye; Applied Biosystems). The sequencing reactions were analyzed on an automated DNA sequencer (377A; Applied
Biosystems). To detect possible contamination, sequences were compared
with each other and with our own TCR-␥ sequences collected in our
institute over the last 7 years. Published germline sequences were used to
determine the type of TCR-␥ rearrangement. International Immunogenetics
database [IMGT]; (http://imgt.cines.fr.:8104; initiator and coordinator,
Marie-Paule Lefranc, Montpellier, France).
Results
Clinical characteristics
Clinical data are summarized in Table 1. All 11 patients, 6 male and
5 female, presented with a typical history of recurrent self-healing
papulonodular eruptions. In 5 of the 11 patients, biopsies taken
within 6 months after first manifestation of LyP were studied. There
was a history of lymphoma in 1 patient only (case no. 7). This
patient developed mycosis fungoides 28 years after the onset of
LyP (tissue sample of the mycosis fungoides lesion was not
available). One patient (no. 6) suffered from coexisting smallplaque parapsoriasis.
Histologic and immunophenotypical analysis
The 14 biopsies from the 11 patients with LyP included in this study
showed the features of LyP type A (Table 2). They were character-
ized by the presence of large atypical CD30⫹ cells with abundant
cytoplasm, hypochromatic nuclei and prominent nucleoli, and a
dense inflammatory background (Figure 1A). All CD30⫹ cells were
positive for at least one T-cell antigen; they were negative for the
anaplastic lymphoma kinase (ALK) protein and for B-cell antigens.
In 8 of the 14 biopsies, the cytotoxic molecules perforin and/or
granzyme B were detectable in more than 50% of the large CD30⫹
atypical cells (Figure 1B).
Amplification of the TCR-␥ rearrangements in single cells and
whole-tissue DNA extracts
A total of 387 single CD30⫹ cells were isolated from the 14 frozen
tissue specimens of 11 patients (Table 3). The cells of all but 2 cases
gave rise to TCR-␥–specific products, ranging from 3 to 17
amplificates per biopsy (total 123 PCR products). In case nos. 1 to
7, TCR-␥ PCR products revealed rearrangements using V␥ gene
segments 1 to 8 (Figure 2A), whereas in case nos. 8 and 9 TCR-␥
amplificates demonstrated a rearrangement of V␥9 and V␥10 gene
segments, respectively (Figure 2B). In the remaining 2 cases, no
PCR products could be obtained from single cells. Both cases,
however, led to the detection of unequivocal dominant PCR
products when whole tissue DNA extracts in conjugation with
GeneScan analysis were used for TCR-␥ PCR.
For comparison, GeneScan analyses were also performed from
whole-tissue DNA extracts of all other cases as well as from the
single-cell amplificates. In all of these cases a more or less
prominent dominant amplificate embedded in a varying polyclonal
background was found (Figure 3, upper panels) that was identical
in size and sequence to the corresponding single-cell amplificate
(Figure 3, lower panels). This indicates that the dominant PCR
products in the 2 cases without amplificable single cells are derived
from the clonal CD30⫹ T cells.
In 10 cases (patient nos. 1-10) lesional single CD30⫺ cells or
pooled CD30⫺ cells were isolated from 13 frozen tissue sections
(Table 3). Seventy-nine TCR-␥–specific PCR products were produced, which were different in their sizes in nearly all instances.
Sequence analysis
All PCR products were sequenced and compared with each other
and with our own and published data bank sequences (IMGT). In
each case identical sequences were found, indicating a clonal
CD30⫹ T-cell population (Tables 3 and 4). In most cases all CD30⫹
cells belonged to the same T-cell clone, whereas in 2 cases (patient
Table 2. Immunophenotypical features of 14 biopsies from 11 patients with LyP
Patient no.
CD30
CD2
CD3
CD4
CD5
CD8
CD20
␤F1
Perforin
Granzyme B
ALK1
1A
⫹
⫹
⫹
⫹
nd
⫺
⫺
nd
nd
nd
⫺
1B
⫹
⫹
⫺/⫹
⫹
⫹
⫺
⫺
⫺/⫹
⫺
E
⫺
2C
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫹
nd
⫺
2D
⫹
⫹
⫹
⫹
⫹
⫺
⫺
ni
⫹/⫺
ni
⫺
3E
⫹
⫹/⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
3F
⫹
⫺
⫺
⫹
⫺
⫺
⫺
E
⫹
⫺
⫺
4
⫹
⫹
E
E
⫺
⫺
⫺
⫺
E
⫹
⫺
5
⫹
⫹
⫹
E
⫹
⫹
⫺
⫺/⫹
⫺
⫺/⫹
⫺
6
⫹
⫹/⫺
⫹
⫹
ni
⫺
⫺
ni
⫺/⫹
⫹
⫺
7
⫹
ni
⫹
⫹
ni
⫺
⫺
ni
ni
ni
⫺
8
⫹
⫹
⫺
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫺
⫺
9
⫹
⫹
⫺/⫹
⫹/⫺
⫺
⫺
⫺
⫺
⫺
E
10
⫹
⫹
⫹
⫹
⫹
⫺
⫺
ni
ni
ni
⫺
11
⫹
⫺/⫹
⫹/⫺
⫹/⫺
⫹
⫺
⫺
ni
⫹
⫹
⫺
⫹ indicates positive; nd, not done; ⫺, negative; ⫺/⫹, less than 50% of CD30⫹ cells; E, less than 10% of CD30⫹ cells; ni, not interpretable; ⫹/⫺, more than 50% of
CD30⫹ cells.
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
CLONAL T-CELL ORIGIN IN LYMPHOMATOID PAPULOSIS
581
nos. 1 and 5) a very small portion of the CD30⫹ cells were
unrelated to the clonal population (5 [4%] of a total of 123 CD30⫹
cells). Furthermore, the TCR-␥ rearrangements of the CD30⫹ cells
isolated from biopsies of 3 patients (patient nos. 1-3), which were
taken at different times, were identical in all instances, indicating
the presence of the same T-cell clone in all lesions.
In contrast, CD30⫺ cells isolated from the same cases displayed
unrelated TCR-␥ rearrangements in all instances with only a few
exceptions (Table 3). In 4 biopsies (patient nos. 1, 2, 6, and 7) 1
single CD30⫺ cell each showed an identical TCR-␥ rearrangement
as detected in the CD30⫹ population (4 [5%] of 79 CD30⫺ cells).
Controls
To evaluate the reliability of the single-cell approach, aliquots from
the buffer covering the tissue sections were drawn after every other
single-cell isolation and were analyzed for the presence of TCR-␥
gene rearrangements. None of the 335 buffer controls gave rise to a
specific product. The PCR analysis of 78 reactive T cells isolated
from a tonsil led to the detection of nonidentical rearrangements of
the TCR-␥ gene in 22 cells (28%), whereas the 15 PCR products
obtained from 36 neoplastic cells (41%) of a peripheral T-cell
lymphoma case showed identical rearrangements without exceptions. All PCR products were sequenced.
Discussion
Figure 1. CD30 and granzyme B expression. Immunohistologic detection of CD30
expression (A; immunoalkaline phosphatase–antiphosphatase staining) and granzyme B expression (B; streptavidin-biotin staining) in 2 cases of LyP. (A) Patient no. 8;
(B) patient no 4. (Original magnification ⫻ 130.)
The nosological position of LyP is a long-standing enigma. LyP
was included in the European Organization for Research and
Treatment of Cancer classification for primary cutaneous lymphomas, taking into account its association with other lymphomas in
10% to 20% of cases.3 It was shown that LyP cases with associated
lymphomas shared the same clonal rearrangement in most instances.6,9-12 Several attempts to demonstrate the presence of clonal
T-cell populations in LyP lesions without complicating T-cell
lymphomas, however, produced conflicting results.11,13-16 This
might, besides the clinical benign course, be one reason why LyP is
Table 3. Detection of TCR-␥ rearrangements in single CD30ⴙ as well as in single CD30ⴚ cells
CD30⫹ cells
Patient no.
No. of
isolated cells
Sequences
identical/unrelated*
CD30⫺ cells
V␥
family
No. of
isolated cells
Sequences
identical†/unrelated*
1A‡
34
6;5§/3
V4/V4
18
1/7
1B‡
36
11;6§/0
V4/V4
16
0/3
2C‡
24
10;7§/0
V4/V8
12㛳
0/5
2D‡
18
5;7§/0
V4/V8
18㛳
1/16
3E‡
34
11/0
V4
16㛳
0/3
3F‡
18
5/0
V4
24㛳
0/2
4
36
15/0
V2
14
0/6
5
36
8/2
6
36
5;4§/0
V5
16㛳
0/9
V3/V8
18㛳
1/10
7
24
3/0
V8
18㛳
1/7
8
34
7/0
V10
16㛳
0/4
9
13
3/0
V9
16㛳
0/3
10
26
0/0
¶
7㛳
0/0
18
0/0
¶
387
118/5
11
Total
*Unique sequence different from the clonal CD30⫹ cells.
†Identical to clonal CD30⫹ cells.
‡Interval between the biopsies: A-B: 8 months; C-D: 52 months; E-F: 3 months.
§Biallelic rearrangement.
㛳Results from 2 to 6 pooled CD30⫺ cells.
¶No amplificate by single-cell analysis, but dominant TCR-␥ gene rearrangement demonstrated by GeneScan analysis.
0
209
—
4/75
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
STEINHOFF et al
Table 4. DNA nucleotide sequences of clone-specific TCR-␥ rearrangements
Patient
no.
V region
N region
GG
TAAGAAAC–J␥2
V␥4—CACCTGGG
TTTTCGGGGCCGAAAGAGG
GTAGTGAT—J␥P2
V␥8—CCTGGGAT
TC
ATTATTAT—J␥2
V␥4—GGATGGGC
T
GAAACTCT—J␥2
3
V␥4—CACCTGGG
TGCCC
TTATTATA—J␥2
4
V␥2—ACCTGGGA
ACCG
ATTATAAG—J␥2
5
V␥5—GGACAGGC
CGGTTG
GTGATTGG—J␥P2
6*
V␥3—CTGGGACA
CTTAGGG
2*
V␥8—CTGGGATA
Figure 2. TCR-␥ PCR of isolated single CD30ⴙ cells and single CD30ⴚ cells
(polyacrylamide gel stained with ethidium bromide, PAGE 6%). (A) TCR-␥ PCR
products obtained with primers covering V␥ gene segments 1 to 8. CD30⫹ cells
(lanes 1-5). Initial biopsy of patient no. 2 (lanes 1, 3, 4); biopsy taken 52 months later
(lanes 2, 5). Note the biallelic rearrangement in this patient. Simultaneous detection
of both rearrangements is only in lane 3, whereas lanes 1 and 2 and lanes 4 and 5
represent either 1 of the 2 rearranged alleles, respectively. Negative control (lane 6).
Reactive CD30⫺ cells of patient no. 2 (lanes 7, 8). (B) TCR-␥ PCR products obtained
with primers covering V␥ gene segment 10. CD30⫹ cells of patient no. 8 (lanes 1-5).
Negative control (lane 6). Reactive CD30⫺ cells of patient no. 8 (lanes 7, 8).
still assigned to the heterogeneous group of cutaneous pseudolymphomas, a group of benign reactive lymphoproliferative processes
that clinically and/or histologically mimic cutaneous lymphomas.21
These contradictory findings may be due to biologic and/or
technical reasons. On one hand, LyP might represent a continuum
beginning as a polyclonal and/or oligoclonal condition that in 10%
to 20% of cases becomes a clonal malignant T-cell proliferation
leading to T-cell lymphoma with high probability. On the other
hand, in previous studies, Southern blot technique or PCR technique analyzing whole-tissue DNA extracts was used. This approach often fails to detect a small number of clonal lymphoid cells
embedded in a polyclonal background. Moreover, it does not allow
for the assignment of molecular features to a morphologically
distinct cell population within the lesion. In particular, the question
of whether clonality is restricted to the population of CD30⫹ cells
or whether some CD30⫺ T cells also belong to the clone could not
be answered by this approach.
To circumvent these methodically based difficulties and to
conclusively clarify the clonality of atypical cells in LyP, we
analyzed the TCR-␥ gene rearrangements of single CD30⫹ as well
as of single CD30⫺ T cells micromanipulated from frozen tissue
sections of lesional skin.
Figure 3. Detection of TCR-␥ rearrangements by PCR and GeneScan analysis in
whole tissue DNA extracts and single CD30ⴙ cells. The x-axes represent
molecular size (base pairs) and the y-axes fluorescence intensity. (A) Whole-tissue
DNA extract (upper panel) and corresponding CD30⫹ single cell (lower panel) of
patient no. 1. (B) Whole tissue DNA extract (upper panel) and corresponding CD30⫹
single cell (lower panel) of patient no. 5.
J region
V␥4—GATGGGCA
1*
ATTATAAG—J␥2
—
ACTCTTTG—J␥2
7
V␥8—ACTGTGCC
TTTGG
GTGATTGG—J␥P2
8
V␥10–GCGTGGGA
CTACCTAGTAG
ATAAGAAA—J␥2
9
V␥9—TTGTGGGA
CGG
GAAACTCT—J␥2
*Biallelic.
Fourteen biopsies of 11 patients with characteristic clinical
features of LyP were included in our study. Five of these patients
had only a brief history of LyP (1 to 6 months), whereas the
remaining 6 patients all had a long-standing course of the disease,
ranging from 10 months to 30 years. All but 1 patient presented a
history without a complicating lymphoma. This contrasts with
previous studies that often include patients with prior or coexisting
lymphoma and therefore generates the question as to whether the
clonal lesions were true LyP or LyP-like manifestations of the
associated cutaneous T-cell lymphoma.10,13 The histologic differentiation of LyP and CD30⫹ cutaneous ALCL is, in particular, often
unreliable. Furthermore, patients with associated lymphoma were
not representative of the vast majority of patients with LyP, because
transformation into malignant lymphoma only rarely occurs.4
A total of 387 single CD30⫹ cells isolated from 14 biopsies
gave rise to 123 specific PCR products. Nucleotide sequence
analyses of all PCR products showed identical TCR-␥ gene
rearrangements within each individual case. By GeneScan analysis
of whole-tissue DNA extracts, a dominant population within a
polyclonal background could be detected in all 14 biopsies.
GeneScan analysis of the corresponding single-cell PCR product
produced an identical band showing that the dominant band
demonstrated in whole-tissue DNA is derived from atypical CD30⫹
cells. By this finding the presence of a single dominant clonal
CD30⫹ cell population within each single lesion was proven.
Repetitive attempts to produce TCR-␥–specific PCR products
in the remaining 2 cases (patient nos. 10 and 11) consistently failed.
However, a dominant TCR-␥ gene rearrangement could be demonstrated by GeneScan analysis of whole-tissue DNA extracts in
these 2 cases as well as in all other cases where the dominant PCR
product could be unequivocally assigned to the CD30⫹ population.
Therefore, we conclude that the failure to demonstrate TCR-␥
rearrangements in single CD30⫹ cells of 2 cases is not due to the
absence of rearranged TCR-␥ genes but due to a partial degradation
of the DNA, which thus became unsuitable for single-copy PCR.
This conclusion is further confirmed by the absence of detectable
TCR-␥ gene rearrangements in CD30⫺ T cells and supported by the
highly variable amplification rate of TCR-␥ gene rearrangements in
single CD30⫹ cells of the other cases (12.5%-70%), thus reflecting
the different levels of DNA degradation of the tissue samples.
Weiss et al13 described different TCR rearrangements within
separate LyP lesions of the same patient using the Southern blot
technique and suggested a multiclonal origin of the disease. The
idea of different cell clones in separate lesions that wax and wane
would also be in line with the clinical course of LyP. In our study,
however, we could demonstrate that in 3 of our patients the
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
CLONAL T-CELL ORIGIN IN LYMPHOMATOID PAPULOSIS
identical dominant CD30⫹ cell clone was also found in temporally
(up to 52 months) and anatomically separate LyP lesions. This
clearly indicates that the same clonal CD30⫹ population is
responsible for the outgrowth of papular skin lesions arising at
different points in time and/or at various anatomic sites. This
finding proves the observations of previous studies where the
same TCR rearrangement was found within separate LyP lesions of
one patient.11,16
In 2 cases (patient nos. 1 and 5) a few single CD30⫹ cells
revealed unique TCR-␥ gene rearrangements unrelated to the
clonal cell population (5 of a total of 123 CD30⫹ cells). Interestingly, the appearance of unrelated unique CD30⫹ cells was
restricted to the first of 2 biopsies in patient no. 1, whereas only
clonally related CD30⫹ cells were found in the second biopsy
obtained 8 months later. This indicates that at least in the initial
phase of the disease—as also seen 4 weeks after the onset of LyP in
patient no. 5—unrelated CD30⫹ cells can be present in addition to
the clonal population. These unrelated cells seem to disappear in
the further course of the disease, leading to a CD30⫹ population
consisting of exclusively clonally related cells.
To answer the question of whether clonality is restricted to the
population of CD30⫹ cells, we also analyzed the TCR-␥ gene
rearrangements of neighboring single CD30⫺ cells. In nearly all
instances nucleotide sequence analyses of PCR products from
isolated CD30⫺ cells were polyclonal and unrelated to the CD30⫹
cell population. In 4 cases (patient nos. 1, 2, 6, and 7), 1 single
CD30⫺ cell each displayed the same TCR-␥ gene rearrangement as
detected in the CD30⫹ population (4 of a total of 79 CD30⫺ cells).
This finding demonstrates that the clonal population is not completely restricted to CD30⫹ cells but may in addition comprise a
small number of CD30⫺ cells. It can be suggested that these cells
are either not fully activated and consequently still not CD30⫹ or
have lost their CD30 expression for unknown reasons. Technical
reasons for the presence of clonally related CD30⫺ cells such as
contaminations or erroneously picked cells are unlikely because all
controls displayed the expected results, thus demonstrating the
reliability of our approach.
In keeping with previous data,22-24 immunophenotypical analyses of the 14 reported biopsies indicated that the atypical cells in
LyP A represent CD30⫹/CD4⫹ activated helper T cells that in a few
cases show a loss of CD2, CD3, and/or CD5. Moreover, in 8 (57%)
of the 14 biopsies, more than 50% of the clonal CD30⫹ T cells
additionally express cytotoxic proteins (perforin and/or granzyme
B). In agreement with previous studies,25,26 these findings provide
583
further evidence for the derivation of atypical cells in LyP from
CD4⫹ T cells with cytotoxic activity. Today, however, the physiologic role of this subpopulation that constitutes less than 5% of
CD4⫹ T cells in peripheral blood of healthy individuals27 is still not
clear. It has been suggested that CD4-mediated cytotoxicity might
be of immunomodulatory importance by eliminating antigenpresenting cells.28 Expression of cytotoxic proteins on CD4⫹ T
cells could be induced by chronic stimulation in vitro.29,30 This
observation fits with the common hypothesis of lymphomagenesis
postulating that lymphoma development might be associated with a
persistent antigenic stimulus.
In conclusion, these results definitely demonstrate that LyP
represents a monoclonal disorder. Clonal expansion is not restricted
to an individual lesion but encloses both anatomically and temporally separate lesions. Furthermore, we could show for the first time
that clonality constitutes the population of CD30⫹ T cells. These
results indicate that the prolonged course of LyP with its typical
features of waxing and waning is due to the expansion and
regression of one single CD30⫹ cell clone that shows cytologic
signs of malignancy. By fulfilling 2 classic criteria of malignancy
(clonality and cytologic atypia) but showing a benign clinical
course, LyP takes up a highly interesting position in tumor biology.
Similar findings can be observed in monoclonal gammopathy of
undetermined significance, which is found in about 1% of the
population over 50 years of age.31 Despite a significant risk of
progression to multiple myeloma, some patients remain asymptomatic for decades. Chromosomal and gene methylation analyses of
monoclonal gammopathy of undetermined significance and multiple myeloma support the hypothesis of a multistep process for the
oncogenesis of multiple myeloma.32,33 Also, in LyP other factors
(for example, alterations in receptor signaling as previously shown)
seem to be necessary for the progression of LyP into malignant
lymphoma.34,35 The likelihood of accumulating a sufficient number
of such relevant genetic lesions is increased by the prolonged life
span of a cell clone as it is observed in LyP.
Acknowledgments
We thank D. Jahnke, H.-H. Müller, and H. Protz for their excellent
technical assistance and L. Udvarhelyi for his editorial assistance.
This paper is dedicated to Prof O. Braun-Falco on the occasion of
his 80th birthday.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2002 100: 578-584
doi:10.1182/blood-2001-12-0199 originally published online
April 17, 2002
Single-cell analysis of CD30+ cells in lymphomatoid papulosis
demonstrates a common clonal T-cell origin
Matthias Steinhoff, Michael Hummel, Ioannis Anagnostopoulos, Peter Kaudewitz, Volkhard Seitz,
Chalid Assaf, Christian Sander and Harald Stein
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