Peripheral blood involvement in a mycosis fungoides patient with limited skin lesions: phenotypical features and homing molecule pattern

ARTICLE

Peripheral blood involvement occurs in a percentage of mycosis fungoides (MF) patients ranging from 3.6 to 19% [1-4]. It is more frequent in the advanced stages (tumoral lesions or lymph node involvement), whereas it is extremely rare in early stage disease.

Herein, a case of a MF patient with limited skin lesions and peripheral blood involvement is reported.

Case report

A female patient aged 72 years presented to our clinic with a 2-year history of erythematous pruritic macules and patches on her shoulders, upper chest and thighs. Her medical history was unremarkable. Physical examination revealed (Fig. 1) scattered slightly scaly, indurated, round or oval patches, ranging in diameter from 5 to 12 centimeters. She had no superficial palpable adenopathies or hepatosplenomegaly. Histological examination revealed a band-like dermal infiltrate with single-cell epidermotropism (Fig. 2). The infiltrate was mainly constituted by clusters of small to medium atypical lymphoid cells, intermingled with numerous macrophages and dermal dendritic cells. Routine staging procedures (chest X rays, abdomen ultrasound, thoracic and abdominal CT scans) were unremarkable. Morphological examination of circulating white blood cells revealed the presence of circulating atypical lymphoid cells (Sézary cells, SC), with the characteristic cerebriform nuclear profile confirmed at electron microscopy (Fig. 3). The SC population accounted for 36% of circulating leukocytes with absolute values at diagnosis of 1,224/mm³. T cell clonality was confirmed by the detection of a dominant TCRgamma gene rearrangement in a heteroduplex analysis in both skin and peripheral blood. A bone marrow biopsy specimen showed no SC infiltration. The final diagnosis was therefore MF stage IB (T₂N₀M₀) with peripheral blood involvement (B₁) according to the Mycosis fungoides Cooperative Group Classification [5].

The patient was treated with fludarabine 25 mg/m² 5 days a month for up to 4 cycles, achieving a complete hematological response, whilst the cutaneous lesions remained unchanged. After chemotherapy was discontinued due to persistent bone marrow toxicity (neutropenia and thrombocytopenia), a hematological early relapse occurred. The patient therefore underwent extracorporeal photochemotherapy treatment (ExP, 5 cycles for 2 consecutive days each month); at the time of writing, no response has been achieved.

Materials and methods

Immunohistochemistry and flow-cytometry

Immunohistochemistry was performed on cryostatic sections stained with an avidin-biotin-peroxidase complex method (LSAB-Plus Peroxidase K0690 Kit, DAKO Corporation, CA 93013 USA). In addition, biopsy fragments were dissociated using an automated mechanical disaggregation device (Medimachine, CONSUL^TS, Italy, distributed by Becton-Dickinson, S. José, CA, USA, and Dakopatts, Denmark) and three color flow cytometry was performed simultaneously on cellular suspension, as reported [6]. Two-, or three-color immunofluorescence peripheral blood lymphocytes (PBL) analysis was performed simultaneously using FITC-, PE-, and PerCP-conjugated antibodies, according to standard procedures. A wide panel of MoAbs directed against T cell antigens, as well as activation and proliferation markers, were tested (table I). KM-93 antibody (indirect immunofluorescence using a FITC-conjugated rabbit anti-mouse IgM, Southern Biotechnology Associates, Inc., Birmingham, AL, USA), is directed against the sialyl LewisX antigen (sLe^x) [7, 8], even if it has not yet been clustered as CD15s like other sLe^x-reactive MoAbs. Although no data are reported in literature as to KM-93 expression on cutaneous infiltrates, Priest et al. showed that KM-93 is highly expressed on HUT78 cells, a skin-homing CTCL line, and that is capable of blocking the binding of HUT78 cells to E-selectin [9]. The clonal rearrangement of the T cell receptor (TCR) was identified using a panel of 28 MoAbs directed against the variable regions of the beta chain (Serotec Ltd., Oxford, UK; Beckman Coulter Company, Marseille, France; Endogen, Cambridge, MA, USA; Pharmingen, S. Diego, CA, USA).

Soluble interleukin-2 receptor assay

Soluble interleukin-2 receptor (sIL-2R) levels were determined with enzyme-linked immunosorbent techniques (ELISA) using a commercially available kit (Genzyme; Cambridge, MA, USA), as previously reported [10].

Results

The phenotype of the cutaneous infiltrate is shown in table I. Three color flow cytometry performed on cellular tissue suspensions showed that the majority of lymphoid cells (75%) expressed a single variable region of the TCR beta-chain (vbeta17); clonal T cells were phenotypically CD3⁺CD4⁺CD45R0⁺CD7^-CD26^-. Activation markers (CD25, CD38, CD71) were expressed on less than 30% of the cell infiltrate. L-selectin/CD62L and the sLe^x antigen KM-93 were expressed on 20% of the cell infiltrate. CD103 expression was nearly restricted to epidermotropic cells.

Circulating cells showed a similar phenotype, expressing the same variable region of the TCR-beta chain (vBeta 17). All the TCR vbeta17⁺ cells were CD4 positive and CD26 negative (Fig. 4). The kinetics of the CD4⁺ vbeta17⁺ and CD4⁺CD26^- subpopulations during follow-up are shown in Figure 5a. A decrease which paralleled the reduction in the SC count, was observed during the fludarabine treatment, followed by a progressive increase during ExP corresponding to the early relapse after the hematological remission. The sIL-2R kinetic was similar, showing a decrease from baseline levels (4,984 U/mL) down to normal values, followed by a further increase (Fig. 5b). No significant modifications in the KM^-93⁺, CD62L and CD103 expression on PBL could be found during follow-up. In fact, the percentage of CD4⁺CD26^-KM^-93⁺ cells remained between 7 and 18% during all the follow-up period, without relation to the number of phenotypically atypical lymphoid cells. Similarly, the percentage of the CD4⁺CD26^-CD62L⁺ cells within the lymphoid population remained between 10 and 32% (Fig. 5c).

Discussion

This paper reports on a female MF patient stage IB (T₂N₀M₀) with peripheral blood involvement. In particular, clonality in the peripheral blood was demonstrated by the expansion of a single restricted variable region of the TCR beta-chain (vbeta17), which was the same expressed in the cutaneous infiltrate. Atypical lymphoid cells in the skin and peripheral blood showed a similar CD4⁺CD7^- T helper memory phenotype, with a lack of CD26, which we have recently shown as a common characteristic feature of atypical circulating lymphoid cells in both SS [11, 12] and MF patients with peripheral blood involvement [12].

We feel that the interest of our case lies in the presence of peripheral blood involvement in a MF patient with such limited patch lesions, in as much as it is commonly associated with cutaneous nodular lesions and/or extracutaneous involvement. Schecter et al. [2] found peripheral blood involvement in 27% of T₃ patients and in 9% of T₂; Vonderheid et al. [1] reported similar results (13.6% in T₃ patients and 3.1% in T₂ patients); any of these authors found peripheral blood involvement in T₁ patients. Toro et al. [3] did not find peripheral blood involvement in their stage I or II MF patients. On the other hand, using PCR amplification, a higher rate of peripheral blood involvement was demonstrated, with up to 57% positivity [13, 14].

Even if the presence of peripheral blood involvement does not affect the stage according to the present TNM classification [5], a series of papers suggested that it is associated with a worse prognosis [1-4, 14]. Schechter et al. [2] reported a 22% 5-yr survival in T₂-T₃ MF patients with peripheral blood involvement, with respect to 70% in patients without peripheral blood involvement. More recently, Fraser-Andrews et al. [14] showed that the presence of a peripheral blood clone is an independent prognostic variable after correcting for age, skin, and lymph node stage. On the basis of these data, our patient was treated with a more aggressive approach than would have been usual for her cutaneous objectivable disease. The complete hematological remission obtained by monochemotherapy with fludarabine was associated with the normalisation in sIL-2R levels, confirming the usefulness of this measurement in the monitoring of CTCL patients [10]. However, the hematological clearing was not associated with a parallel cutaneous response and an early hematological relapse occurred, associated to a further sIL-2R level increase.

The biological mechanisms responsible for haematological spreading are as far as we know not completely defined, even if the literature reports clearly show that homing molecules may be involved in these processes [15-17]. In fact, it is conceivable that the low sLe^x/CLA expression [15] in the cutaneous lymphoid infiltrate could give rise to a higher recruitment in the peripheral blood. The persistent low CD62L/L-selectin expression on circulating cells could be related to the absence of lymph node involvement, as suggested by Borowitz et al. [17]. The finding of a low KM-93 expression on circulating cells is in keeping with previous reports [16-18], showing that the levels of CLA⁺ circulating cells correlate with the stage and the extent of cutaneous involvement. Moreover, our results showed that the percentage of sLe^x/KM-93⁺ CD4⁺CD26^- circulating cells did not depend on the whole CD4⁺CD26^- levels, in as much as an increase in the CD4⁺CD26^- subpopulation was not associated with a parallel KM-93⁺ amount. It could therefore be hypothesised that the increase in the peripheral blood tumour burden could be related in our case to the persistent low expression of cutaneous homing molecules on the PBL surface.

CONCLUSION

Article accepted on 8/5/01

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