Follow-up of the T-cell clonality in Sezary patients treated by extracorporeal photopheresis using a new assay: the immunoscope technique

1. Introduction 

Sezary’s 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. Sezary cells present a typical cerebriform nucleus. The count of cells in peripheral blood usually exceeds 1000 per mm3 (10% of total circulating leukocytes). The topical treatments of SS include the use of nitrogen mustard or PUVAtherapy, but have most often proved unable to control the clonal component of the peripheral blood. Systemic treatments of SS such as extracorporeal photochemotherapy (ECP), interferon alfa or antineoplastic polychemotherapy often induce a transient improvement of the clinical signs.

However, the efficiency of either group of treatments is low. ECP may induce complete remission in certain cases of SS [1]. However, a recent study showed that the mean survival time with ECP was not longer than that induced by other treatments [2]. Recently, it has been suggested that the association of interferon alfa with ECP was more effective than ECP alone, on the clinical course of the disease [3], [4].

The search on the best treatment of SS is impeded by the lack of unequivocal biological parameters to be monitored. The quantitative molecular follow-up of the malignant clone is most probably the best method to evaluate the effect of the treatments. Southern blotting has been used in few studies to monitor the evolution of the clonal component in the blood of SS patients treated with interferon [5] or ECP [6]. The latter study showed the disappearance of the peripheral blood clonal component in 2/10 patients with a partial remission of the disease. Because of the limitations of this radioactive and rather unsensitive technique, the 10 times more sensitive but not quantitative PCR techniques, using α, β and γ TCR genes, have been developed for the diagnosis of CTCL. Indeed PCR-γ [7] PCR–DGGE-γ [8], [9], PCR–PAGE-γ [10] PCR–SSCP-γ [11] PCR-β [12], [13] have been developed whereas these techniques could not been used for the semi-quantitative follow up of the clonal component in SS. In others experiments the clonal population could be followed with a clonotypic specific probe [14].

In order to investigate the T-cell clonality in the peripheral blood of patients with SS and to monitor its evolution in patients treated with ECP, we used a semi-quantitative and highly sensitive (0.01%) RT-PCR based method. This technique called “Immunoscope” has been widely used in the monitoring of specific T-cell clones in various physiological and pathological situations [15], [16], [17], [18].

2. Patients and methods 

2.1. Patients 

Eight patients consisting of 7 men and 1 woman were included in the present study. Age ranged from 59 to 75 years (mean 65). The characteristics of the 8 patients and the different treatments received are listed in Table 1. The diagnosis of SS was established on the basis of clinical criteria (exfoliative erythroderma, pruritus, palmoplantar keratoderma, lymphnodes), and biological parameters including the presence of typical circulating Sezary cells (number>1000/mm3), histological data (cutaneous epidermotropic T-cell lymphoma), and detection of a T-cell clone in the blood and in the skin by qualitative PCR DGGE. All these patients were treated with ECP, 4 patients according the protocol described by Edelson and 4 patients according our own intensive protocol as described thereafter. The T-cell clonal component was monitored in the peripheral blood during the clinical evolution of the disease. For 3 patients, we were able study the T-cell clone in the cellular product before and just after UVA irradiation with a delay of 30–45 min between both sample collection.

Table 1. Clinical characteristics of the 8 patients

	Name	Sex (M/W)/age (yr) at diagnosis	Symptoms	Delay of diagnosis	Previous treatment
	V	M/68	Exfoliative erythroderma, PPK, inguinal lymphadenopathy	4 months	Chlormetine, BCNU
	Sa	M/62	Pruritus, erythroderma	18 months	pUVA
	Vd	M/66	Pruritus, exfoliative erythroderma, PPK, nail dystrophy, lymphadenopathy	3 years	0
	Bra	M/70	Pruritus, reticular erythema	A few months	Chlormetine, CS, pUVA then pUVA+, chlorambucil,
	Ciz	M/62	Exfoliative erythroderma,PPK	1 year	CS, chlormetine, chlorambucil, IFN α, MTX
	Sy	W/75	Pruritus, xerosis, annular erythematous lesions, then exfoliative erythroderma	1 year	pUVA
	Sv	M/60	Exfoliative erythroderma, pruritus, PPK, nail dystrophy	6 years	cytapheresis, chlormetine, pUVA, IFN α
	R	M/59	Pruritus, erythroderma, axillar lymphadenopathy	3 years	pUVA, chlormetine

PPK, palmoplantar keratoderma; pUVA, pUVAtherapy; CS, corticosteroids; IFN, interferon.

The clinical course was followed using clinical cutaneous criteria (pruritus, erythroderma) and systemic clinical criteria (fever, weight loss, abnormal nodes). Sezary cells were counted.

The mean follow-up was 30 months (range 18–64). The average number of blood samples studied during the follow-up period was 4 per patient (range 3–6). The date of sampling corresponded to evolution in the clinical course of the disease i.e., relapse or improvement.

3. Results 

3.1. Analysis of the complete BV repertoire in PBLs of each patient 

The TCRB repertoire analysis showed the presence of an expanded peak belonging to one BV family in the blood samples of the eight studied patients, associated with gaussian like usage of the other BV families. This peak was indicative for an oligo- or monoclonal expansion of T cells sharing the usage of the same BV and CDR3 length. There was no preferential or common BV usage accounting for the T-cell expansions. We observed a large diversity of rearranged BV regions, as summarized in Fig. 1. For 6 patients (patients Sa, Vd, Bra, Ciz, R and Sv), the direct sequencing of the CDR3 of the dominant clone was carried out. All CDR3 sequences were found different and no common CDR3 was shared by the different sequences (data not shown). The deduced aminoacid sequences of the CDR3 regions of the clones were extensively analyzed using advanced BLAST software and no homology with any already published T-cell clone was detected. In addition to the dominant clones present during the evolution of the disease, we observed transitory peaks corresponding to expanded T-cell population varying with time.

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Fig. 1. The complete TCRB repertoire analysis showed the presence of an expanded peak in one BV family in the blood samples of the eight studied patients (in black), associated with gaussian like usage of the other BV families (in white).

3.2. T-cell clonality and responsiveness to ECP 

In patient Sy, we observed a decrease of the relative frequency of the clone from 15.6% to close to 0%, paralleling a complete clinical remission and a disappearance of the circulating Sezary cells (Fig. 2). In the seven other cases, ECP was found inefficient and the clinical condition of the patients worsened, sometimes after a stable or a short partial improvement period. In these seven cases, the relative frequency of the clone paralleled the clinical condition, except in one case at the onset of ECP treatment (patient R). The results of the 8 patients treated with ECP are summarized in Table 2. As example, the evolution for patient Vd is given on Fig. 3. Surprisingly, the relative frequency of the clone did not always follow the Sezary cells count evolution (Table 2).

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Fig. 2. The relative frequency of the clone in patient Sy decreased from 15.6% to close to 0%.

Table 2. Clinical and molecular evolution of the patients

	Patient	Clinical course	Evolution of Sezary cells	Evolution of the relative frequency of the clone
	V	Improvement	7→2%	↓
				9→0→0.5%
	Sa	Stable then worsening	10→31%	↑
				9→30%
	Vd	Progressive worsening	42→26%	↑
				9→18%
	Bra	Improvement then worsening	16→19%	↓ then ↑
				84→8→37→20%
	Ciz	Partial efficiency then worsening	7→5%	Fluctuations
				18→12%
	Sy	Complete remission	27→0%	↓
	Sv	Worsening	67→88%	Fluctuations
				18→10→43%
	R	Stable than worsening	2→9%	↓ then ↑
				20→2→8.5%

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Fig. 3. Evolution of T-cell repertoire in patient Vd during PCE. The clonal population is defined by the usage of BV6B.

3.3. T-cell clonality in the cellular product 

PBL were harvested just before UVA irradiation and 30 min after UVA irradiation from the cellular product. The T-cell repertoire was analyzed with “Immunoscope”. In 3/3 patients, we observed a decrease of the relative frequency of the expanded clone after irradiation (Table 3).

Table 3. Relative frequency of the T-cell clone in the cellular product before and 30 min after UVA irradiation

	Patient	Before irradiation (%)	30 min after irradiation (%)
	Sa	17	4
	Bra	19	8
	Ciz	20	15

4. Discussion 

In order to determine the BV usage of the clonal Sezary cells, the complete BV repertoire was determined in PBLs of 8 patients. Using the high sensitive Immunoscope technique we were able to detect the presence of a well defined dominant T-cell clone in 8/8 patients. These results contrast with a previous published study using less sensitive technique that detected oligoclonal or polyclonal expansion in CTCL patients [20]. In addition we did not find a preferential or common BV usage shared by all patients ruling out the hypothesis of a superantigenic dependent T-cell stimulation. These results confirm earlier reports on the high diversity of the BV usage by Sezary cells [12], [21], [22]. We neither found preferential usage of BJ segment. Moreover, no homology of the CDR3 chain sequence with already published T-cell clone was found.

In all patients, the evolution of the disease has been monitored using 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 in time. The significance of such clonal populations remains to be elucidated but they might reflect non-tumoral reactive lymphocytes. In previous works, it has been found that the BV T-cell repertoire is polyclonal and present a gaussian distribution in normal blood [23]. The sensitivity of the technique was previously determined by dilution experiments with a clonal T-cell line. T-cell clone with a frequency among PBMC of 5×10−4 can be detected [24].

We wondered if the dominant T-cell clones observed in peripheral blood corresponded to tumor cells. The context of malignancy and the persistence of the same clone in several months interval collected samples strongly support that this clonal population belongs to the malignant Sezary cells compartment. In some Sezary patients, we used antibodies directed against the BV segments found associated to clonal expansions to study skin biopsies (data not shown). The expression of the BV chains was found predominantly associated to lymphocytes showing the phenotype of tumor cells. Monoclonal antibodies directed against BV regions of the TCR β chain have been previously used by other authors and resulted in the staining of the majority of the skin lymphocytes in 11/15 patients with CTCL (mycosis fongoides and Sezary syndrome) whereas no reactivity was observed in patients with benign dermatoses [25]. It can thus be concluded that the persistant and expanded clones correspond to the malignant T-cell population. However in our study, the Sezary cell count was not correlated with the relative frequency of the monoclonal population. The relative frequency is likely to be more accurate than the optical counting of Sezary cells, which may be subjective [26], and be an unfaithful reflection of the tumoral population because all tumoral lymphocytes do not have the typical aspect of Sezary cells and Sezary cells may also be reactive cells that can be found in benign dermatoses [27].

In order to determine the effect of PCE on the tumoral population, the relative frequency of the clone was determined, immediately before and after UVA irradiation in the cellular product. We observed a decrease of the relative frequency of the expanded clone 30 min after UVA irradiation. A direct cytotoxicity of the association 8MOP+UVA might induce an immediate decrease in the clone frequency [28].

In conclusion, we have shown that Immunoscope technique could be used to monitor accurately the evolution of the Sezary cells throughout ECP. It could also be used in patients treated with antineoplastic polychemotherapy or interferon. In all ECP treated patients, a parallelism between the relative frequency of the T-cell clone and the 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 the UVA irradiation, a finding suggestive of direct cytotoxicity of the treatment onto Sezary cells. Finally “Immunoscope” appears to be an efficient semi-quantitative assay to appreciate the amount of tumoral cells and monitor the evolution of the clonal component in the Sezary syndrome. Moreover this technique could thus be used to evaluate the efficiency of a treatment, but also to monitor the anti-tumoral T cell response. In the present study, we have shown that molecular monitoring is correlated to the clinical evolution. Additional studies are required to analyse the clonal component in other T-cell cutaneous lymphomas and pseudolymphomas.