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IMMUNOBIOLOGY
From the Institut de Recherche sur la Peau, Institut
National de la Santé et de la Recherche Médicale (INSERM)
U532, Hôpital Saint-Louis, Paris, France; Institut Pasteur,
Unité de Biologie Moléculaire du Gène, INSERM U277,
Département d'Immunologie, Paris; Unité de therapie
cellulaire et de clinique transfusionelle, Hôpital Saint-Louis,
Paris, France.
Sézary syndrome is a leukemic form of epidermotropic
cutaneous T-cell lymphoma related to the malignant proliferation of clonal CD4+ T cells. Extracorporeal photochemotherapy may
induce a transient improvement of the clinical signs, but its
efficiency is discussed. To investigate the frequency of the T-cell
clone in the peripheral blood of patients with Sézary syndrome
and to monitor its evolution in patients treated using extracorporeal
photopheresis or chemotherapy, we used the immunoscope technique. In
one patient, we observed a decrease of the relative frequency of the
clone from 15.6% to 0%, paralleling a complete remission of the
clinical disease and a disappearance of the circulating Sézary
cells. In the other cases, the evolution of the relative frequency
paralleled the initial improvement of the clinical status and the
absence of long-term efficiency in patients treated with extracorporeal
photopheresis or chemotherapy. We observed a quick-acting direct
cytotoxicity of the association 8MOP + UVA on the T-cell clone.
The immunoscope technique appears to be an efficient tool to appreciate
the amount of tumoral cells and to monitor the evolution of the clonal
component in the Sézary syndrome.
(Blood. 2002;100:2168-2174) Sézary syndrome (SS) is characterized
by exfoliative erythroderma, palmoplantar keratoderma, partial
alopecia, abnormal lymph nodes, and pruritus. SS is a leukemic form of
epidermotropic cutaneous T-cell lymphoma (CTCL) related to the
malignant proliferation of clonal CD4+ T cells.
Sézary cells present a typical cerebriform nucleus. The count of
cells in peripheral blood usually exceeds 1000 per microliter (10% of
total circulating leukocytes). Topical treatments of SS include the use
of nitrogen mustard or PUVA therapy, but they have most often proved to
be unable to control the clonal component of the peripheral blood.
Systemic treatments of SS such as extracorporeal photochemotherapy
(ECP), interferon alfa (IFN- However, the efficiency of either group of treatments is
low and controversial. Indeed, the rate of complete remission
(CR) with IFN- The search for the best treatment of SS is impeded by the lack of
unequivocal biologic parameters to be monitored. Quantitative molecular
follow-up of the malignant clone is probably the best method to
evaluate the effect of the treatments. Southern blot analysis has been
used in a few studies to monitor the evolution of the clonal component
in the blood of SS patients treated with IFN7 or
ECP.8 The latter study showed the disappearance of the
peripheral blood clonal component in 2 of 10 patients with partial
remission of the disease. Because of the limitations of this
radioactive and insensitive technique, polymerase chain reaction (PCR) techniques To investigate the frequency of the T-cell clone in the
peripheral blood of patients with SS and to monitor its evolution in
patients treated with ECP or chemotherapy, we used a semiquantitative and highly sensitive (0.01%) RT-PCR-based method. This technique, called immunoscopy, permits determination of the CDR3 length
and of the BV and BJ segments of the expanded clones, and it permits monitoring of the expanded population of interest. CDR3-length spectratyping has been used by other authors to describe this technique.17 Immunoscopy has been widely used in the
monitoring of specific T-cell clones in physiological and pathologic
situations.18,19
Patients
The clinical course was followed using clinical cutaneous criteria
(pruritus, erythroderma, nodules, palmoplantar keratoderma, alopecia)
and systemic clinical criteria (sweat, fever, weight loss, abnormal
nodes, and other visceral involvement). Sézary cells were counted.
Mean follow-up was 28 months (range, 6-64 months). Average number of
blood samples studied during the follow-up period was 4.5 per patient
(range, 3-6 samples). The date of sampling corresponded to changes in
the clinical course of the disease (ie, relapse or improvement).
Extracorporeal photopheresis protocols
cDNA and PCR reaction Peripheral blood lymphocytes (PBLs) were isolated from heparinized venous blood samples by centrifugation over Ficoll-Hypaque (Eurobio, Paris, France). Four-millimeter punch skin biopsy samples were taken and snap-frozen in liquid nitrogen for subsequent analysis.Total RNA was extracted using the phenol-chloroform technique. Three micrograms total RNA was reverse transcribed according to Clontech (Palo Alto, CA) recommendations. For each patient, the resultant cDNA was PCR-amplified for 40 cycles (94°C at 30 seconds; 60°C at 30 seconds; 72°C at 30 seconds) using 24 BV-specific primers and one BC-specific primer,24 Taq polymerase from Promega (Madison, WI), 10 mM dNTP (Boehringer Mannheim), and 25 mM Mg2+ (Promega). The 24 BV-BC amplifications were individually performed. After checking the quality of amplification on a 2% agarose gel, we submitted each of the 24 BV-BC PCR-resultant products for 3 cycles of a run-off elongation reaction (same PCR conditions as above) using a nested BC primer labeled at its 5' end with a fluorescent dye Fam (Eurogentec Oligold). Immunoscope analysis Immunoscope analysis has been described in detail elsewhere.18 Briefly, after the addition of 10 mL solution of 20 mM EDTA (ethylenediaminetetraacetic acid)-formamide, each of the 24 resultant runoff products was loaded onto a 6% acrylamide sequence gel and analyzed using an automatic sequencer and the Immunoscope software package (Applied Biosystems, Palo Alto, CA). The intensity of fluorescence of each band was determined. Each BV-BC runoff product appeared as a family of peaks characterized by the usage of a definite CDR3 length. A polyclonal distribution that reflects the absence of expanded T-cell clone(s) generated a Gaussian-like pattern, whereas clonal expansion of a T-cell clone was revealed by a distortion of the Gaussian pattern. The 24 BV-BC profiles were automatically inserted into a TCR- repertoire sheet.18 The relative
frequency of the T-cell clone was calculated by dividing the
fluorescence intensity of the studied peak by the sum of fluorescence intensities of all peaks of the 24 BV families as previously
described.25 Direct sequencing of the CDR3 region of the
clonal expansion was performed using the Sequenase kit (Amersham).
Immunohistochemical analysis Immunohistochemical analysis of frozen 4-mm punch skin biopsy specimens was performed for 3 patients. Immunohistochemical staining was performed using antibodies directed against CD3 (total T cells), BV regions (Ultratech; Immunotech, Marseilles, France), and the Ki67 protein for the detection of tumor cells. Biotinylated antibodies and peroxidase-labeled streptavidin (Ultratech HRP streptavidin-biotin universal detection system; Immunotech). Horseradish peroxidase enzyme activity was revealed using amino-ethyl-carbazole substrate. Results were expressed as the percentage of the positively stained cells in the cutaneous infiltrate. In one patient, the double-staining procedure was performed as follows: in the first step, sections were incubated successively with anti-BV17 mAbs (1 of 5, 45'; Immunotech) and by goat fluorescein-conjugated anti-mouse IgG (1 of 40, 30'; Immunotech), then with anti-Ki67 mAbs (30'; Immunotech) revealed by goat cyanine 3-conjugated anti-mouse IgG (1 of 200, 30'; Sigma, St Quentin Fallavier, France). Sections were examined on a light fluorescence microscope.
The complete BV repertoire was determined in PBLs from each
patient. TCR-
Expression of the BV chains found expanded in the blood was analyzed in
the skin. Expression of the expanded BV chains was analyzed using
immunoscopy and immunohistochemistry in the lesional skin samples of 3 patients. To normalize the amount of lymphoid RNA obtained from skin
biopsy samples, the amount of infiltrating T cells was assessed by
immunohistochemistry using anti-CD3 antibodies before immunoscope
analysis. Monoclonal antibodies directed against expanded BV regions of
the TCR-
Relative frequency of the dominant T-cell clone was determined for each sample taken from each patient In patient Sy, we observed a decrease in the relative frequency of the clone from 15.6% to close to 0%, paralleling a complete clinical remission and a disappearance of the circulating Sézary cells (Figure 4). In the 7 other patients, ECP was found inefficient and the clinical condition of the patients worsened, sometimes after a period of stability or of short partial improvement. In these 7 patients, the relative frequency of the clone paralleled the clinical condition, except in one patient at the onset of ECP treatment (patient R). Results for the 8 patients treated with ECP are summarized in Table 2, and the evolution for patient Vd is given in Figure 5. Surprisingly, the relative frequency of the clone did not follow the Sézary cell count evolution (Table 2).
To determine the effect of chemotherapy on the clonal T-cell
population, we included 2 Sézary patients (D, V) treated with CHOP. In patient D, CHOP was the first-line treatment because of a
serious systemic extension of the disease (intestinal, hepatic, pulmonary). He entered partial remission after the first cycle of
treatment. The relative frequency of the clone decreased from 43% to
6.7%. The description of the complete TCR- Relative frequency of the clone was determined, before and immediately after UVA irradiation PBLs were harvested just before UVA irradiation and in cytapheresis products 30 minutes after UVA irradiation. The T-cell repertoire was analyzed using immunoscopy. In 3 of 3 patients, we observed a decrease in the relative frequency of the expanded clone. Analysis of the T-cell repertoire 4 months after treatment showed that the relative frequency of the clone had increased to the previously determined level before irradiation (Table 3).
To determine the BV usage of clonal Sézary cells, the
complete BV repertoire was determined in PBLs of 15 patients. Using the
highly sensitive immunoscope technique, we were able to detect the
presence of a well-defined, dominant T-cell clone in 15 of 15 patients.
Our results confirm that SS is related to the proliferation of clonal T
cells. These results contrast with a previous published study using
less sensitive techniques that detect oligoclonal or polyclonal
expansion in CTCL patients.22 The V In 10 patients, disease evolution was monitored using the immunoscope
technique. The expanded clone persisted throughout the survey period,
including the treatment. In addition to the dominant clones, we
observed variable peaks corresponding to expanded clones varying with
time. The significance of such clonal populations remains to be
elucidated, but they might reflect nontumoral-reactive lymphocytes. In
previous works,24,28 we have found that the BV T-cell
repertoire is polyclonal and presents a Gaussian distribution in normal
blood. The sensitivity of the technique was previously determined by
dilution experiments with a clonal T-cell line. A T-cell clone with a
frequency among peripheral blood mononuclear cells of
5 × 10 We sought to determine whether the dominant T-cell clones
observed in peripheral blood corresponded to tumor cells. The
persistence of the same clone in collected samples after an
interval of several months strongly indicated that this clonal
population belongs to the malignant Sézary cell compartment.
Moreover immunoscopy of skin biopsy specimens and of PBLs showed that
the same We have used the above-defined indicator to monitor the evolution of the T-cell clones in 8 patients treated with ECP. The relative frequency of the dominant T-cell clone was determined. In patient Sy, we observed a decrease in the relative frequency of the clone from 15.6% to 0%, paralleling a 4-year complete remission of the clinical disease and disappearance of the circulating Sézary cells. In the 7 other patients, the relative frequency of the clone paralleled the clinical status. The evolution of the relative frequency paralleled the initial improvement of the clinical status and the absence of long-term efficiency in patients treated with CHOP. To ascertain the effect of PCE on the tumoral population, the relative frequency of the clone was determined immediately before and after UVA irradiation. We observed a decrease in relative frequency of the expanded clone 30 minutes after UVA irradiation. A few months after treatment, in vivo analysis revealed that the relative frequency of the clone had increased to the level achieved before UVA irradiation. Direct cytotoxicity of the association 8MOP + UVA might induce an immediate decrease in clone frequency.33 However, cytotoxicity must have been transient because the relative frequency of the clone had increased again in samples taken 4 months later. In conclusion, we have shown that immunoscopy could be used to monitor accurately the evolution of the Sézary cells throughout ECP and during polychemotherapy treatments. In all ECP- and CHOP-treated patients, parallelism between the relative frequency of the T-cell clone and clinical evolution was observed. This finding argues for the role of the T-cell clone in the phenotypic expression of the disease. In addition, the relative frequency of the clone was found to decrease between the beginning and the end of UVA irradiation, a finding suggestive of direct cytotoxicity of the treatment onto Sézary cells. Finally, immunoscopy appears to be an efficient tool to determine the amount of tumoral cells and to monitor the evolution of the clonal component in Sézary syndrome. Because treatments used in patients with Sézary syndrome are likely to induce T-cell death in the tumoral and nontumoral population, the immunoscopy technique allows semiquantitative evaluation of tumoral and nontumoral compartments during treatment. Moreover, this technological strategy could be used to evaluate the efficiency of a treatment and to monitor the antitumoral T-cell response. In the present study we have shown that molecular monitoring is correlated with clinical evolution but that follow-up of the clonal population could not replace clinical monitoring of the disease. Additional studies are required to analyze the clonal component in other T-cell cutaneous lymphomas and pseudolymphomas.
We thank Drs I. Moulonguet and F. Mouly for clinical assistance and Profs D. Charron, and F. Sigaux and Dr C. Rabian for PBL storage.
Submitted November 20, 2001; accepted May 23, 2002.
Supported by grants from INSERM and the Association de la Recherche en Transfusion.
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: Philippe Musette, Institut de Recherche sur la Peau, INSERM U532, Hôpital saint Louis, 75475 Paris Cedex 10 France.
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© 2002 by The American Society of Hematology.
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  N. Ortonne, D. Huet, C. Gaudez, A. Marie-Cardine, V. Schiavon, M. Bagot, P. Musette, and A. Bensussan Significance of circulating T-cell clones in Sezary syndrome Blood, May 15, 2006; 107(10): 4030 - 4038. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), or antineoplastic polychemotherapy
(methotrexate, CHOP) often induce a transient improvement of the
clinical signs.
10 times more sensitive but not quantitative
T-cell-receptor (TCR) genes have been developed for the diagnosis of CTCL. Indeed PCR-
4 could be detected.