(American Journal of Pathology. 2002;160:1035-1045.)
© 2002 American Society for Investigative Pathology
Monoclonal 
-T-Cell Receptor Rearrangement in Vulvar Lichen Sclerosus and Squamous Cell Carcinomas
Sigrid Regauer*,
Olaf Reich
and
Christine Beham-Schmid*
From the Institute of Pathology*
and Departmentof Obstetrics and Gynecology,
University ofGraz, Graz, Austria
 |
Abstract
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Risk factors for vulvar squamous cell carcinoma (SCC) are human
papilloma virus (HPV) infections and lichen sclerosus (LS). The
significance of monoclonal 
-T-cell receptor (-TCR) rearrangement
in the lymphoid infiltrate of LS and the consequence for vulvar
carcinogenesis is unknown. One hundred sixty-one biopsies of vulvar LS
and SCC, with and without LS, were examined for
monoclonal -TCR rearrangement and HPV16 expression, and for
the expression of B- and T-cell markers and fascin. Monoclonal -TCR
rearrangement was identified in 8 of 17 patients with LS and 11 of 21
patients with SCC arising in LS with only occasional HPV16 DNA
detection. None of the 19 SCC without LS showed monoclonal -TCR
rearrangement, but 14 of 19 patients had strong HPV16
detection. The lichenoid infiltrate of LS with germline configuration
consisted predominantly of T cells (CD8 > CD4), along
with numerous B cells. However, in biopsies with monoclonally
rearranged -TCR, CD4-positive T cells dominated along with B
cells and fascin-positive cells in the lichenoid infiltrate and in
deeply located lymphocyte aggregates (LAs). These LAs additionally
contained fascin-positive dendritic cells with only individual
CD8, CD57, and granzyme-positive cells. LAs in biopsies
with germline configuration demonstrated numerous T cells (CD8
>CD4), but only single peripheral B cells,
CD57, and fascin-positive lymphocytes. Our data suggest that
monoclonal -TCR rearrangement is characteristic for and limited to
LS and SCC arising in LS, raising the question for a
LS-associated antigen. We interpret B cells, CD4-positive T
cells, and fascin-expressing dendritic cells within LS as a
cellular immune response to antigen or proliferating T-cell clones. The
resulting local immune dysregulation in LS may provide a permissive
environment for the development of a SCC.
Lichen sclerosus (LS) is a skin
disease predominantly restricted to genital skin. The vast majority of
LS occurs in vulvar skin of postmenopausal women, but LS can also be
seen in younger women and even in prepubertal girls.1
Well-developed LS is characterized histologically by atrophic
epidermis, destruction of the dermoepidermal junction with basal
keratinocyte vacuolization, basement membrane homogenization, dermal
edema and sclerosis, rarefaction of vessels, and a band-like
lymphocytic infiltrate with a predominant T-cell phenotype. A recent
study on vulvar and penile LS reports a monoclonally rearranged
-T-cell receptor (-TCR) within the lymphoid infiltrate in 
50%
of the examined biopsies.2
This has been an unexpected
finding in so-called "chronic dermatitis," the preferred term for
LS in the dermatological literature. The classification and etiology of
LS is an ongoing topic of controversy.3-5
LS may truly
represent a benign chronic inflammatory skin lesion; however, vulvar LS
carries an increased risk for development of vulvar squamous cell
carcinoma (SCC).6
Patients with vulvar SCC have been
divided epidemiologically into two subgroups: 1) younger women with
human papilloma virus (HPV) infections, and 2) elderly women without
HPV risk factors but with a longstanding history of LS.7
The etiology of LS and SCC arising in LS is not well
understood.8
At present the significance of monoclonal T
cells in the lymphoid infiltrate of LS is unclear. Furthermore, the
consequence of this finding for the carcinogenesis of vulvar SCC
arising in LS is completely unknown. We investigated if and to what
extent monoclonal -TCR rearrangement can be observed in
tumor-infiltrating lymphocytes of vulvar SCC, especially in SCC arising
in LS. For this purpose, we used tissue samples of three patient groups
that we analyzed in a retrospective study: patients with LS only,
patients with SCC arising in LS (SCC/LS), and patients with vulvar SCC
without LS. All biopsies were further examined for the presence of
HPV16 DNA for correlation of our data with the known main etiological
risk factors of vulvar SCC. In addition, we analyzedthe
immunophenotypical profile of the lymphoid infiltrate in tissues with
and without monoclonal -TCR rearrangement.
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Materials and Methods
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Patient and Sample Selection
Three patient groups were investigated. The first group contained
50 biopsies from 22 patients with LS with an average age of 62 years
(range, 35 to 87 years). The second group consisted of 71 samples of 21
patients with surgical excisions of vulvar SCC/LS with an average age
of 69 years (range, 45 to 87 years) and the third group consisted of 40
biopsies of 19 patients with an average age of 69 years (range, 37 to
87 years) with surgically resected SCC unassociated with LS (for
summary see Tables 2 to 4
). Representative formalin-fixed,
paraffin-embedded material obtained during the last 6 years from the
archives of the Institute of Pathology and the Department of Obstetrics
and Gynecology of the University of Graz, Austria, were used for this
investigation (histological criteria according to Carlson and
colleagues4
). All available biopsies of the patients with
LS only were investigated. Multiple biopsies represent concomitant
biopsies. Within the SCC/LS patient group, separate tissue blocks of
the SCC and the surrounding LS obtained from the excision specimen were
analyzed. Two separate tissue blocks of carcinoma from the excision
specimens of SCC without LS were analyzed. All analyzed samples
contained representative and comparable lymphohistiocytic infiltrates.
Polymerase Chain Reaction (PCR) Analysis
Genomic DNA for detection of -TCR rearrangement and HPV16 DNA
was prepared from formalin-fixed and paraffin-embedded tissue specimens
essentially as described.9
For each paraffin block we
analyzed three separate samples. A 50-µm section of the tissue block
was cut and immediately transferred to an Eppendorf tube that was
immediately sealed. The next 50-µm section was harvested for the
second sample, and a third section was obtained for the third sample.
Between sections, the knife was cleaned.
-TCR Rearrangement Detection
The T-cell receptor gene was analyzed according to McCarty and
colleagues10
with minor modifications. DNA amplification
was performed in the same buffer as above except that
MgCl2 was 1.5 mmol/L and 1 U of Taq
polymerase was used. Primer concentration was 0.5 µmol/L for primers
A and B and 1 µmol/L for primer C (denaturation, 94°C for 60
seconds; annealing, 55°C for 90 seconds; extension, 72°C for 110
seconds). Forty cycles were performed with an annealing temperature of
55°C. All three separately obtained PCR products of each individual
paraffin block were analyzed on 6% polyacrylamide vertical gel
electrophoresis. A single band in one of three analyzed probes was
interpreted as positive for -TCR rearrangement. Specimens were not
enriched for lymphocytic infiltrates.
Detection of HPV16 DNA
A 119-bp stretch was amplified in a 50-µl reaction using primers
(5' TCA AAA GCC ACT GTC TCC TG 3', 5' CGT GTT CTT GAT GAT CTG CAA 3')
0.5 µmol/L (each), 1x buffer containing Tris-Cl, KCl,
(NH4)2SO4,
1.5 mmol/L MgCl2, pH 8.7, 0.8 mmol/L dNTP.
Taq polymerase (2.5 U; Qiagen Hot Star) were added
after an initial denaturation for 10 minutes at 95°C. Forty cycles of
denaturation (95°C for 60 seconds; annealing, 55°C for 60 seconds;
extension, 72°C for 120 seconds) were performed followed by a
10-minute terminal extension at 72°C. All three separately obtained
PCR products harvested from each paraffin tissue block were analyzed on
3% agarose gels containing ethidium bromide (NuSieve, FMC/SeaKem, FMC
2:1). A single band in one of three lanes was interpreted as positive.
Immunohistochemistry
All 161 biopsies were examined with antibodies to CD3, CD4, CD8,
CD20, CD21, CD57, perforin, granzyme B, TIA, and fascin (Table 1)
using the streptavidin-biotin complex
method, with previous trypsinization or microwave treatment as
required. For control purposes, tissues known to contain the respective
antigens were included (positive controls). Replacement of the primary
antibody by normal serum always led to negative results (negative
controls).
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Results
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Patients with LS
A total of 50 biopsies in 22 patients with LS were
analyzed (Table 2)
. Histologically, early
lesions of LS had a dense lichenoid infiltrate (Figure 1a)
with numerous intraepidermal lymphoid
cells and hyperplastic epidermis. Approximately 50% of the analyzed
samples were of longstanding LS with an atrophic or hypertrophic
epidermis with papillary edema and sclerosis with a scant or no
lichenoid infiltrate. These biopsies, however, often showed lymphocytic
aggregates (LA), either around blood vessels or hair appendages in the
deep submucosa of deeply extending biopsies (Figure 2, a and b)
. Analysis of -TCR
rearrangement was technically impossible in 10 biopsies of five
patients because of poor DNA quality. A single biopsy only was
available in four patients, one of which showed monoclonal -TCR
rearrangement. Eight of 13 patients with multiple biopsies showed
monoclonal T cells in at least one of their analyzed biopsies (Figure 3a
, patient no. 12). Intralesional and
interlesional heterogeneity was pronounced, best illustrated in a
patient with only one of six analyzed paraffin blocks demonstrating
monoclonally rearranged -TCR (patient no. 11). Only two patients
(patients no. 9 and no. 16) had monoclonal -TCR rearrangement in all
analyzed samples of their biopsies. HPV16 DNA was demonstrated in 8 of
22 patients in 19 of 50 biopsies, but only in 29 of the 150 separately
analyzed samples. However, not a single one of these biopsies displayed
morphological evidence of HPV infection.
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Figure 1. Immunohistochemical phenotype of LS with dense lichenoid
infiltrate with and without monoclonal -TCR rearrangement.
a–d: Patient no. 12
(123682/00), LS without
monoclonal -TCR rearrangement. The H&E stain shows vulvar skin with
parakeratosis, focal basement membrane thickening with dermal
sclerosis, and ectatic venules. The lymphocytic infiltrate is
predominantly dermal, dense, and band-like
(a) with scant
interface infiltrate. The majority of the lymphocytes are CD8-positive.
Note the intraepidermal presence of CD8-positive lymphocytes
(b).
CD4-positive lymphocytes are sprinkled with the dermal infiltrate or
occur in small clusters or individually
(c). Within
the dense dermal infiltrates are numerous CD20-positive B cells
(d).
e–h: Patient no. 34
(29185/99), LS with
monoclonal -TCR rearrangement. CD20-positive B cells within the
dermal infiltrate are also a feature of LS with monoclonal -TCR
rearrangement
(e). The B
cells are located in the superficial aspects of the submucosa, whereas
the T-cell population (CD3,
f) is localized in the deeper
aspects, creating a zonation effect. Within the infiltrate CD4-positive
T cells (g)
dominate over CD8-positive T cells
(h), quite in
contrast to the infiltrate without monoclonal -TCR rearrangement.
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Figure 2. Immunohistochemical phenotype of deep dermal/submucosal lymphocytic
aggregates in atrophic LS with monoclonal -TCR rearrangement.
Patient no. 21
(30665/99). The H&E stain
shows a late stage of LS with massive hyperkeratosis and focal
acanthotic epidermis. The basement membrane is massively thickened, the
papillary dermis highly sclerotic and avascular with prominent clefting
along the dermoepidermal junction. No lichenoid dermal or interface
infiltrate is present; however, beneath the sclerosis several
lymphocytic cell aggregates are present
(a and
b). These aggregates are composed of
predominately CD20-positive B cells
(c),
fascin-positive dendritic cells
(d), and
CD4-positive T cells
(e).
CD4-positive T cells dominate over CD8-positive T cells
(f), which are
not found within the B-cell fraction but at the periphery of these
lymphocytic aggregates.
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On hematoxylin and eosin-stained sections, no differences between
biopsies with and without monoclonally rearranged -TCR were
recognizable. Immunohistochemically, the intraepidermal lymphocytes of
all biopsies were CD3-, CD8-, CD57-, TIA-, and granzyme B-positive.
CD4-, CD20-, fascin-, and perforin-positive lymphocytes were not
identified within the epidermis. The lichenoid dermal band-like
lymphohistiocytic infiltrate of LS with germline configuration was
uniformly CD3-positive with a CD8 predominance over CD4 (Figure 1
; a to
c, patient no.12). A unique finding was the demonstration of numerous B
cells within the band-like lichenoid infiltrate in biopsies with a
well-developed lichenoid infiltrate (Figure 1d)
. In biopsies with
monoclonally rearranged -TCR and a dense lichenoid infiltrate,
however, CD20-positive B cells were bordered by a broad band-like
infiltrate of CD3-positive T cells (Figure 1, e and f
, patient no. 34)
creating a zonation effect. In these infiltrates, CD4 expression
dominated over CD8 (Figure 1, g and h)
. Granzyme B- and TIA-positive T
cells were numerous. Perforin expression was not observed.
CD57-positive cells were located in the epidermis and dermis alike.
Fascin-positive dendritic cells were not identified within the
lichenoid infiltrate.
Biopsies of longstanding LS with atrophic epidermis and basement
membrane homogenization showed only scant or no lichenoid infiltrate,
but deeply located submucosal LA (Figure 2, a and b)
. Differences
between concomitant biopsies of atrophic LS of the same patient with
and without monoclonally rearranged -TCR were observed within these
LAs. In biopsies with monoclonally rearranged -TCR, these LAs were
predominantly positive for CD20 (Figure 2c
, patient no.21). Numerous
fascin-positive dendritic cells created an irregular meshwork within
the B-cell population (Figure 2d)
. Fascin also cross-reacted with
endothelial cells. Numerous CD4-positive T cells were found within the
B-cell aggregates (Figure 2e)
with dominance over CD8-positive T cells,
which were identified only in the peripheral zones of the LAs (Figure 2f)
along with CD57, and granzyme-positive cells. In contrast, LA in
biopsies with germline configuration of the same patient (Figure 4, a and b)
, had only single peripherally
located B cells (Figure 4c)
, CD57, and fascin-positive lymphocytes. The
majority of lymphocytes within these LAs were T cells with the typical
CD8 dominance over CD4 (Figure 4, d and e)
. Granzyme B and TIA were
expressed, but perforin staining was absent.
Patients with SCC Arising with LS
A total of 71 separate samples were analyzed in 21 patients with
SCC arising in LS (Table 3)
. All patients
had a highly differentiated, keratinized SCC with a prominent exophytic
component. Invasion was mostly superficial. The adjacent LS was
predominantly of the classical type with epidermal atrophy, basal
keratinocyte destruction, basement membrane homogenization and dermal
edema with rarefaction of vessels, and a band-like lymphocytic
infiltrate and occasional LA. Poor DNA quality did not allow analysis
in one patient. A monoclonal -TCR rearrangement was observed in 11
of the remaining 20 patients, who showed monoclonal -TCR
rearrangement in at least one of the analyzed samples. The intersample
and intrasample heterogeneity was again pronounced (for details, see
Table 3
). Recurrent SCC, available in two patients (3 and 4 years after
primary excision, respectively) also demonstrated monoclonal
-TCR rearrangement with intralesional heterogeneity. Nine of 20
patients had no monoclonal -TCR rearrangement in all analyzed
samples of SCC and histologically classical LS. HPV16 DNA was detected
in a single biopsy only in 11 of 21 patients, mostly as a weak band in
one of three lanes without morphological evidence of HPV infection
(Figure 3b
; patient no. 34). Strong HPV16 DNA detection was observed in
only two patients in all four analyzed biopsies of SCC and LS. The
lymphoid infiltrate in LS with and without monoclonal -TCR
rearrangement was of identical immunophenotype as described above. The
tumor-infiltrating lymphocytes of the invasive SCC were a mixture of B
cells and T cells.
Patients with SCC Unrelated to LS
None of the 40 samples of 19 patients with SCC unrelated to LS
showed a monoclonal -TCR rearrangement (Table 4)
. Eighteen patients had poorly
differentiated, widely invasive SCC with scant keratinization,
significant nuclear pleomorphism, high mitotic activity, and
tumor-infiltrating lymphocytes. One patient had extensive SCC in
situ with prominent HPV-related cell changes with a dense
band-like lymphocytic lichenoid infiltrate, but no lymphocytic
aggregates. Mostly strong bands of HPV16 DNA were detected in 14 of 19
patients (Figure 2b
, patient no. 48). In five patients, all analyzed
biopsies were negative for HPV16 DNA. The tumor-infiltrating
lymphocytes were identified at the invasion front of the SCC,
underscoring the cancerous tissue. Occasionally a lymphoid infiltrate
was identified lateral to the invasive SCC underscoring the adjacent
SCC in situ component or intraepithelial neoplasia. The
infiltrate consisted of a mixture of CD4- and CD8-positive T
lymphocytes and with interspersed individual CD20-positive B cells. T
cells with perforin-containing cytotoxic granula were not identified in
any of the examined biopsies.
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Discussion
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Monoclonal -TCR-rearrangement within the lymphoid infiltrate
occurs not only in biopsies of vulvar LS but also in vulvar SCC arising
in LS, sharply contrasting the complete lack of monoclonally rearranged
-TCR in SCC unassociated with LS. We observed a pronounced
intralesional heterogeneity when multiple concomitant biopsies of a
single lesion were analyzed. Repeated PCR analysis with identical
results, however, excluded the detection of pseudo-monoclonality. The
sensitivity of the PCR method allows detection of at least 1% of
clonal T cells on a T-cell background, even within low-density
inflammatory infiltrates. On one hand, the heterogeneity may be a
result of inclusion of atrophic and longstanding LS, which often had a
rather scant lichenoid infiltrate, that probably was not sufficient for
detection of monoclonal T cells. On the other hand, we know that the
observed monoclonal -TCR rearrangement is a true focal event,
although we cannot give a clear estimate of the extent of clonal
outgrowth based on the used methods. In this study, the majority of
biopsies/excisions had several different tissue blocks analyzed, and of
each of these blocks three separately harvested individual tissue
samples were analyzed electrophoretically. Therefore, we analyzed up to
nine different areas within a single lesion of LS, SCC arising in LS,
and SCC without LS. In the majority of analyzed blocks, the monoclonal
-TCR rearrangement was detected in only one of the analyzed three
probes. We therefore believe that such focal detection of monoclonal
-TCR rearrangement is in support of a single clone buried and
proliferating within a mixed-cell infiltrate as opposed to a primary
lymphoproliferative disorder. Despite the heterogeneity, monoclonal
-TCR rearrangement seems to be specific to LS and SCC arising in LS,
although it is presently unclear at which time point in the evolution
of LS and SCC arising in LS the T-cell clones emerge. Clonal T cells in
skin diseases are rare. They occur in cutaneous lymphomas, eg, mycosis
fungoides (inclusive parapsoriasis en plaque), and in lymphomatous skin
lesions such as pityriasis lichenoides varioliformis acuta
and lymphomatoid papulosis,11-13
but among the classic
inflammatory skin diseases, such as psoriasis, lichen planus,
allergic and contact dermatitis, clonal T cells have been
demonstrated only exceptionally.2
Restricted
TCR usage may reflect prolonged exposure of the host immune system to a
local putative LS-associated antigen. Antigen-driven selection of
cytotoxic T cells suggests a relationship to
autoimmunediseases. The putative antigen seems unrelated
tomalignant tumor cells as SCC without LS show no monoclonal
-TCR rearrangement and SCC arising in LS demonstrates a high degree
of intralesional heterogeneity. We found only one previous publication
reporting a clonal expansion of T cells in carcinomas (six mucosal
oropharyngeal SCCs); this study, however, postulated an immune
reaction against tumor cell antigens.14
Interestingly, on a light microscopic level, biopsies with and without
monoclonal -TCR rearrangement were indistinguishable. The classical
histology of basal keratinocyte destruction, basement membrane
homogenization, and dermal edema with rarefaction of vessels can be
explained through the action of TIA and granzyme B, which were
identified in all biopsies. Perforin, another granular component of
cytotoxic and natural killer T cells, capable of inducing epidermal
injury15
was not demonstrated. We are the first to report
the presence of significant numbers of B cells in the lichenoid
infiltrate (either band-like or in clusters) and in deeply located LA
of vulvar LS and SCC. This supports an earlier description of
occasional small numbers of B cells in LA of pediatric penile
LS,16
but contradicts Scrimin and
colleagues,17
who could not find any B cells in the
lymphoid infiltrate of LS. These obvious discrepancies may be related
to biopsy techniques, eg, only very superficial biopsies were analyzed.
Alternatively, differences/problems with immunohistochemical techniques
and antibody choices may account for the reported lack of B cells in
LS. Biopsies with germline mutations showed the usual CD8 dominance
over CD4 in both the lichenoid infiltrate and LA. In biopsies with
monoclonally rearranged -TCR, however, we observed the reversal of
the usual CD8 dominance, with CD4-positive T cells being the
predominant phenotype. Interestingly, the CD4-positive T cells were
closely associated with the B cells. Another unusual finding was the
presence of fascin-positive dendritic cells. Fascin-expressing
dendritic cells formed an irregular mesh work in the lymphocytic
aggregates within the B cell and CD4-positive T-cell fraction in LS and
SCC/LS. Fascin, a 55-kd actin-bundling protein associated with
cell motility, is involved in antigen presentation18
and
has been demonstrated in human follicular dendritic cells, which are
the antigen-presenting cells of germinal centers.19
A
principal feature of dendritic cells located within the peripheral
tissues is antigen capture of foreign antigen and subsequent initiation
of immune responses. It is generally accepted that CD4-positive T cells
play a major role in the initial activation of resting naive B cells,
and that B-cell activation occurs within the T-cell areas of secondary
lymphoid tissues. Antigen-specific-activated CD4-positive cells
stimulate antigen-specific naive B cells to proliferate and
differentiate into germinal center founder cells. It has also recently
been suggested that interdigitating dendritic cells act as a matrix on
which antigen-specific T cells and B cells interact
efficiently20
and that they are directly involved in
regulating T-cell-mediated humoral immune responses in
humans.21
In summary, monoclonally rearranged -TCR and
the typical immunohistochemical profile (CD20, CD4 dominance,
fascin-positive dendritic cells) in biopsies of LS and SCC arising in
LS are best interpreted as a local antigen-dependent immune reaction,
raising again the question for a LS-associated antigen.
HPV16 is certainly the major risk factor for SCC unassociated with LS.
For SCC with LS, HPV16 seems to play a minor role as carcinogen; the
occasional demonstration of weak bands of HPV16 DNA in individual
samples of a biopsy is of uncertain clinical significance and may
reflect HPV exposure in the distant past without HPV-related
oncogenesis. Inadequate local host response secondary to proliferation
of selected T-cell clones in LS may facilitate the development of the
typically highly differentiated keratinized SCC in longstanding LS. In
the absence of HPV16 oncogenes as well as monoclonal -TCR
rearrangement in 10 of our 42 patients with vulvar SCC (six SCCs
arising in LS, and four SCCs unrelated to LS), however, genetic
alterations independent of HPV16 DNA or immunosuppression need to be
further investigated.
In summary, our data show that monoclonal -TCR rearrangement of the
lymphoid infiltrate is a characteristic feature of vulvar LS and vulvar
SCC arising in LS, but not of SCC unassociated with LS. T-cell clones
in such a high percentage of patients raise the question for a
LS-associated antigen. The autoimmune hypothesis is further supported
by the occurrence of B cells, CD4 dominance over CD8, and
fascin-positive dendritic cells within LA, which represent a specific
local immune reaction to an antigen or to proliferating T-cell clones.
The resulting immune dysregulation may create a permissive environment
for the development of a SCC in LS. It remains unclear what or which
antigen drives the immune system toward the observed immune reaction
and to what extent monoclonal -TCR rearrangement contributes to the
carcinogenesis of vulvar SCC arising in LS.
|
Acknowledgements
|
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We thank the technical staff of the Hematopathology Laboratory,
the staff of the Diagnostic Molecular Laboratory of the Institute of
Pathology, the Histological Laboratory of the Department of Gynecology
and Obstetrics for excellent technical support, and Mr. R. Staber and
Ms. K. Wagner for photographic help.
|
Footnotes
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Address reprint requests to Sigrid Regauer, M.D., Institute of Pathology, University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria. E-mail: sigrid.regauer{at}@kfunigraz.ac.at
Supported by the Austrian Cancer Aid/Styria (project number 04/2001).
Accepted for publication December 13, 2001.
|
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Abstract
Materials and Methods
