Extracorporeal Photopheresis of Cutaneous T-Cell Lymphoma Is ...

Clinical and Laboratory Investigations
Dermatology 1998;196:305–308
Received: July 23, 1997
Accepted: November 10, 1997
a
b
Ch.C. Zouboulis a
M. Schmuth a
S. Doepfmer b
E. Dippel a
C.E. Orfanos a
Department of Dermatology and
Institute of Medical Statistics,
University Medical Center
Benjamin Franklin,
The Free University of Berlin, Germany
Extracorporeal Photopheresis of
Cutaneous T-Cell Lymphoma Is
Associated with Reduction of
Peripheral CD4+ T Lymphocytes
Key Words
Extracorporeal photopheresis
Cutaneous T-cell lymphoma
Lymphocyte subpopulations
Abstract
Background: Extracorporeal photopheresis (ECP) has been successfully introduced
for the treatment of cutaneous T-cell lymphoma; however, the mechanism(s)
of its action is (are) unknown. Objective: To investigate the effects of
ECP on the immune system of patients with cutaneous T-cell lymphoma. Methods:
Clinical response and changes of lymphocyte subpopulations in 20 patients
with cutaneous T-cell lymphoma under ECP monotherapy or combined regimens
were evaluated and compared after 3, 6 and 12 ECP cycles. Results: Thirteen
of 20 patients showed a ≥50% reduction of skin lesions after 6–12 ECP cycles.
An overall T-lymphocyte reduction was assessed with a balanced CD4+
T-helper and CD8+ T-suppressor cell decrease in responders. In contrast, there
was a trend of CD4+ T-helper cell increase in nonresponders which could result
from the failure of treatment to control the natural course of the disease. The
CD4+/CD8+ ratios were 1.6 at baseline and 1.4 after 12 cycles in responders,
while they increased from 1.7 to 4.1 in nonresponders, respectively (p=0.047).
In addition, there was an overall decrease in the CD57+/CD8+ T-cell subpopulation
mostly due to a reduction in the responder group. Conclusion: The
marked differences detected in certain T-cell subpopulations suggest an effect of
ECP on peripheral T lymphocytes and, especially, on CD4+ cells.
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Extracorporeal photopheresis (ECP) has been successfully
introduced for the treatment of erythrodermic cutaneous
T-cell lymphoma [1–3]. Regimens combining ECP
with interferon α (IFN-α), psoralen photochemotherapy
and/or radiation were also successful in patients with tumor
stage cutaneous T-cell lymphoma and lymph node involvement
[4]. In addition to the clinical improvement a longterm,
disease-free remission could be induced by ECP in a
minority of patients [5]. The almost complete clearing of
skin lesions and the marked diminution of extensive lymphadenopathy
in a patient with rapidly advancing Sézary syndrome
treated with a combination of ECP and low-dose
IFN-α was followed by disappearance of the malignant
T-cell clone from the peripheral blood documented by
Southern blot analysis [6]. The mechanisms of action underlying
the favorable clinical results of ECP still remain to
be elucidated.
Ultraviolet A irradiation of peripheral blood enriched by
8-methoxypsoralen was shown to induce apoptosis in malignant
lymphocytes from patients with cutaneous T-cell
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Priv.-Doz. Dr. Christos C. Zouboulis
Department of Dermatology, University Medical Center Benjamin Franklin
Hindenburgdamm 30, D–12200 Berlin (Germany)
Tel. +49 30 8445 2769, Fax +49 30 8445 4262
E-Mail zoubbere@zedat.fu-berlin.de

Table 1. Diagnosis and treatment of
patients with cutaneous T-cell lymphoma
during ECP therapy
Patients Diagnosis Treatment Result of
and stage
ECP treatment
1 MF-II ECP (12 cycles)/IFN-α response
2 MF-II ECP (12 cycles)/IFN-α nonresponse
3 MF-II ECP (12 cycles)/IFN-α response
4 MF-IV ECP (12 cycles) response
5 MF-IV ECP (12 cycles) nonresponse
6 MF-IV ECP (12 cycles) nonresponse
7 MF-IV ECP (6 cycles)/IFN-α/PUVA response
8 MF-IV ECP (6 cycles)/IFN-α nonresponse
9 MF-IV ECP (12 cycles)/PUVA nonresponse
10 MF-IV ECP (12 cycles)/IFN-α/acitretin response
11 MF-V ECP (12 cycles)/IFN-α/polychemotherapy nonresponse
12 MF-IV ECP (12 cycles)/IFN-α response
13 MF-IV ECP (12 cycles)/PUVA response
14 MF-IV ECP (6 cycles)/PUVA/irradiation response
15 MF-IV ECP (12 cycles) response
16 MF-IV ECP (12 cycles)/IFN-α response
17 MF-IV ECP (12 cycles)/PUVA response
18 SS ECP (6 cycles) nonresponse
19 SS ECP (12 cycles) response
20 SS ECP (12 cycles) response
MF = Mycosis fungoides; SS = Sézary syndrome.
lymphoma [7]. Simple photodestruction of circulating malignant
lymphocytes may, however, not be the only explanation
for the beneficial effect of ECP in cutaneous T-cell
lymphoma, since ECP is also effective in other immunological
cutaneous diseases [5].
In order to further elucidate the effects of ECP on the
immune system we have investigated the changes of lymphocyte
subpopulations in patients with cutaneous T-cell
lymphoma under therapy.
Patients and Methods
Twenty patients with the diagnosis of cutaneous T-cell lymphoma
(14 males, 6 females; median age 61.5 years, range 31–93 years) were
included in the study (table 1). All patients were shown to be HTLV-1
negative. Evaluation of disease status included histological examination
of skin biopsies, peripheral lymph nodes and bone marrow aspirates.
All patients were treated on two consecutive days at approximately
4-week intervals and completed at least 6 cycles of ECP; in 16
patients, 12 cycles had been completed. Twenty milliliters of heparinized
peripheral blood were collected before initiation of treatment
and at day 1 prior to the 3rd, 6th and 12th ECP cycle. Mononuclear
cells including monocytes were separated from 20 ml heparinized peripheral
blood by using a Ficoll-Hypaque ® density gradient. Lymphocyte
subpopulations were determined by indirect immunofluorescence
using a battery of murine monoclonal antibodies in a Becton
Dickinson FACSscan analyzer. Cell numbers were calculated as
absolute cell counts per microliter of peripheral blood. Results were
presented as median values of the absolute baseline cell numbers and
as medians of change from baseline over time in order to avoid artificial
changes of the absolute median values over time due to the reduced
number of cases. Statistical analysis was performed using the
Wilcoxon signed rank test for matched pairs of observations, comparing
the 3rd, 6th and 12th treatments with baseline. Differences were
considered to be significant when p

Table 2. Absolute baseline lymphocyte numbers (medians) and changes from baseline (medians of change) over time in patients with cutaneous
T-cell lymphoma responding and nonresponding to ECP
Before treatment After 3 ECP cycles After 6 ECP cycles After 12 ECP cycles
responders nonresponders responders nonresponders responders nonresponders responders nonresponders
(n = 13) (n = 7) (n = 13) (n = 6) (n = 13) (n = 6) (n = 11) (n = 5)
Total lymphocytes 1,805 2,432 –472 –360 –362 –31 –552 +650
(n = 7) (n = 7)
T lymphocytes 1,227 2,019 –425 –509 –286 –419.5 –328 +478
CD4+ T-helper cells 928 1,046 –157 –121.5 –101 –396 –258 +456
CD8+ T-suppressor cells 534 309 –98 –19 –90 –95.5 –111 0
CD4+/CD8+ ratio 1.60 1.70 +0.10 +1.05 –0.10 +3.70 –0.20 +2.40
(p=1.000) (p=0.726) (p=0.096) (p=0.047)
CD3+/HLA-DR+ 136 222 –63 –35 +11 +465.5 +2 +133
activated T cells
CD57+/CD8– NK cells 70 78 –34 +31 –39 –20.5 –18 +149
(p=0.035)
CD57+/CD8+ NK cells 136 113 –50 +9 –67 –66 –60 0
CD4+/LECAM-1+ 82 141 –5 +21.5 –12 +377.5 –28 +181
T-helper cells
B lymphocytes 110 49 +6 –15.5 –48 –4.5 –16 –11
Statistical analysis was performed using the Mann-Whitney U Wilcoxon rank sum test for independent samples. If not indicated, p values were >0.05.
tients without clinical improvement (nonresponders) 4 were
male and 3 were female (median age 62 years, range
57–74).
By evaluating the absolute numbers of lymphocyte subpopulations
in all patients during the course of ECP treatment,
a significant reduction of T lymphocytes was found
after 3 ECP cycles (–32.8%; p=0.042). This reduction
also persisted over the 6th (–22.1%) and the 12th cycle
(–25.1%), although no statistical significance was found
after prolonged treatment. The CD57+/CD8+ T-cell subpopulation,
representing 10% of the total T-lymphocyte
counts, was also found reduced after ECP: it decreased
from –35.4% after 3 cycles (p=0.042) to –51.5% after 6
cycles (p=0.011) and –40.4% after 12 cycles (n.s.). In the
other lymphocyte subpopulations, such as CD4+ T-helper,
CD4+/LECAM-1– T-helper, CD8+ T lymphocytes, activated
T cells (CD3+/HLA-DR+), CD57+/CD8– natural
killer cells and B lymphocytes, no significant changes were
detected.
By comparing the responder and nonresponder groups,
there were trends of reduced CD4+ and CD8+ T-cell counts
in responders (–27.8 and –20.8%, respectively, after 12 cycles)
and CD4+ T-cell increase in nonresponders (+43.8%
after 12 cycles). These changes were supported by differences
of the CD4+/CD8+ ratios: 1.6 at baseline and 1.4
after 12 cycles in responders, 1.7 at baseline and 4.1 after
12 cycles in nonresponders (p=0.047). The CD57+/CD8+
T-cell subpopulation of responders decreased by –36.8 to
–49.3% during ECP treatment, whereas there was no clear
change in nonresponders (+8.0 to –58.4%) and no significant
differences could be calculated. No differences on the
lymphocyte subpopulations were assessed regarding application
of a concomitant medication, e.g. comparing ECP
alone and ECP/IFN-α.
Discussion
ECP has been shown to cause clinical improvement and
prolonged survival in patients with erythrodermic cutaneous
T-cell lymphoma in the absence of limiting side effects
[1–5]. This finding is remarkable since it has been shown
that overall survival of patients with cutaneous T-cell lymphoma
was not altered by conventional X-ray treatment or
chemotherapy [5, 8].
It has been proposed that altered immunogeneity of irradiated
T cells may induce a therapeutically significant immunologic
reaction that targets unirradiated T-cells of the
pathogenic clone [9]. Also, treatment with ECP may alter
the cytokine patterns secreted by peripheral blood leukocytes
via alteration of peripheral blood monocytes/macrophages
leading to increased levels of tumor necrosis factor-α
[10]. In this study, we investigated the absolute
numbers of peripheral lymphocyte subpopulations before
and after treatment of cutaneous T-cell lymphoma with
ECP and/or concomitant medication over 3–12 months.
ECP and Cutaneous T-Cell Lymphoma Dermatology 1998;196:305–308
307

This time period is sufficient for evaluating ECP effects in
cutaneous T-cell lymphoma, since clinical response in the
first 6–8 months of treatment predicts long-term outcome
[5].
In 20 treated patients investigated, ECP resulted in an
overall T-lymphocyte reduction, with a balanced CD4+
T-helper and CD8+ T-suppressor cell decrease in responders.
In contrast, there was a trend of CD4+ T-helper cell increase
in nonresponders which could result from the failure
of treatment to control the natural course of the disease
[11]. The overall decreased CD57+/CD8+ T-cell subpopulation
assessed during ECP treatment was mostly due to a
reduction in the responder group. The marked differences
detected in certain T-cell subpopulations suggest an effect
of ECP on peripheral T lymphocytes; the missing significance
was probably due to the low numbers of patients
studied.
References
1 Edelson R, Berger C, Gasparro F, Jegasothy B,
Heald P, Wintroub B, Vonderheid E, Knobler
R, Wolff K, Plewig G, et al: Treatment of
cutaneous T-cell lymphoma by extracorporeal
photochemotherapy. Preliminary results. N
Engl J Med 1987;316:297–303.
2 Heald P, Rook A, Perez M, Wintroub B,
Knobler R, Jegasothy B, Gasparro F, Berger C,
Edelson R: Treatment of erythrodermic cutaneous
T-cell lymphoma with extracorporeal
photochemotherapy. J Am Acad Dermatol
1992;27:427–433.
3 Gollnick HP, Owsianowski M, Ramaker J,
Chun SC, Orfanos CE: Extracorporeal photophoresis
– A new approach for the treatment
of cutaneous T cell lymphomas. Recent Results
Cancer Res 1995;139:409–415.
4 Owsianowski M, Garbe C, Ramaker J, Orfanos
CE, Gollnick H: Therapeutische Erfahrungen
mit der extrakorporalen Photophorese: Technisches
Vorgehen, Überwachung und klinische
Ergebnisse bei 41 Hautkranken. Hautarzt
1996;47:114–123.
5 Zic JA, Stricklin GP, Greer JP, Kinney MC,
Shyr Y, Wilson DC, King LE Jr: Long-term follow-up
of patients with cutaneous T-cell lymphoma
treated with extracorporeal photochemotherapy.
J Am Acad Dermatol 1996;35:
935–945.
6 Rook AH, Prystowsky MB, Cassin M, Boufal
M, Lessin SR: Combined therapy for Sézary
syndrome with extracorporeal photochemotherapy
and low dose interferon alpha: Clinical,
molecular, and immunologic observations.
Arch Dermatol 1991;127:1535–1540.
7 Yoo EK, Rook AH, Elenitsas R, Gasparro FR,
Vowels BR: Apoptosis induction of ultraviolet
light A and photochemotherapy in cutaneous
T-cell lymphoma: Relevance to mechanism of
therapeutic action. J Invest Dermatol 1996;
107:235–242.
8 Kaye FJ, Bunn PA Jr, Steinberg SM, Stocker
JL, Ihde DC, Fischmann AB, Glatstein EJ,
Schechter GP, Phelps RM, Foss FM, et al:
A randomized trial comparing combination
electron-beam radiation and chemotherapy
with topical therapy in the initial treatment of
mycosis fungoides. N Engl J Med 1989;321:
1784–1790.
9 Perez M, Edelson R, Laroche L, Berger C:
Inhibition of antiskin allograft immunity by infusions
with syngeneic photoinactivated effector
lymphocytes. J Invest Dermatol 1989;92:
669–676.
10 Vowels BR, Cassin M, Boufal MH, Walsh LJ,
Rook AH: Extracorporeal photochemotherapy
induces the production of tumor necrosis factor-alpha
by monocytes: Implications for the
treatment of cutaneous T cell lymphoma and
systemic sclerosis. J Invest Dermatol 1992;98:
686–692.
11 Ralfkiaer E, Wolff-Sneedorff A, Thomsen K,
Vejlsgaard GL: Immunophenotypic studies in
cutaneous T-cell lymphomas: Clinical implications.
Br J Dermatol 1993;129:655–659.
308 Dermatology 1998;196:305–308 Zouboulis/Schmuth/Doepfmer/Dippel/Orfanos