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The comparison of expression of cutaneous lymphocyte‐associated antigen (CLA), and Th1‐ and Th2‐associated antigens in mycosis fungoides and cutaneous lesions of adult T‐cell leukemia\lymphoma

European Journal of Dermatology. Volume 13, Number 6, 553-9, November - December 2003, Investigative report

Summary

Author(s) : Takahiro YAMAGUCHI, Koichi OHSHIMA, Takeshi TSUCHIYA, Hiroaki SUEHUJI, Kennosuke KARUBE, Juichiro NAKAYAMA, Junji SUZUMIYA, Tadashi YOSHINO, Masahiro KIKUCHI , Department of Pathology, School of Medicine, Fukuoka University, Nanakuma 7‐45‐1, Jonan‐ku, Fukuoka, 814‐0180, Japan Department of Dermatology, School of Medicine, Fukuoka University, Fukuoka, 814‐0180, Japan Department of Internal medicine, School of Medicine, Fukuoka University, Fukuoka, 814‐0180, Japan Department of Pathology, Okayama University, Okayama, 700‐8558, Japan .

Summary : Mycosis fungoides (MF) is morphologically similar to cutaneous lesions of adult T cell leukemia\\lymphoma (ATLL) of human T‐cell lymphotropic virus‐type I (HTLV‐1). In addition, the Th1 or Th2 characteristic of MF and ATLL is still controversial. In the present study, to discriminate MF and cutaneous lesion of ATLL using immunohistochemical markers, and to elucidate Th1 or Th2 dominancy in both disorders, CLA (cutaneous lymphocyte associated antigen) was expressed on epidermotrophic lymphoma cells in all early stage MF. In contrast, all ATLL were negative for CLA. CXCR3 was especially expressed in epidermotropic small lymphoma cells of MF. CCR5 was expressed in both disorders with variable sized lymphoma cells. ST2 was expressed on large transformed lymphoma cells with ATLL, but not in any MF cases. OX40 was expressed in the large transformed cell population in both disorders. These findings suggest that CLA and ST2 could be potentially useful immunohistochemical markers for discrimination of mycosis fungoides and cutaneous lesions of ATLL. And OX40 could be a useful immunohistochemical marker for the histopathological progression of both disorders.

Keywords : CLA, cutaneous lesion of ATLL, mycosis fungoides, OX40, ST2, Th1\\Th2

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ARTICLE

Auteur(s) : Takahiro YAMAGUCHI^1,2, Koichi OHSHIMA¹, Takeshi TSUCHIYA¹, Hiroaki SUEHUJI¹, Kennosuke KARUBE¹, Juichiro NAKAYAMA², Junji SUZUMIYA³, Tadashi YOSHINO⁴, Masahiro KIKUCHI¹

¹ Department of Pathology, School of Medicine, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka, 814-0180, Japan
² Department of Dermatology, School of Medicine, Fukuoka University, Fukuoka, 814-0180, Japan³ Department of Internal medicine, School of Medicine, Fukuoka University, Fukuoka, 814-0180, Japan⁴ Department of Pathology, Okayama University, Okayama, 700-8558, Japan

Article accepted on 17/9/2003

Mycosis fungoides (MF) is morphologically similar to cutaneous lesions of adult T cell leukemia/lymphoma (ATLL) of human T-cell lymphotropic virus-type I (HTLV-1). Genetic techniques such as Southern blot analysis are necessary to differentiate the two diseases. MF is a mature T-cell lymphoma presenting with skin patches/plaques and characterized by epidermal and dermal infiltration of small- to medium-sized T-cells with convoluted nuclei. The phenotypical and genotypical features are clonal proliferation of CD4 + T lymphocytes. The clinical progression of MF is very slow, from the patch and plaque stage to the dermal-based tumor stage; typically many years after the initial diagnosis. The clinical progression of MF is reported in correlation with histopathological transformation to large lymphoma cells and loss of epidermal infiltration, so called epidermotropism [1, 2].

ATLL is a human malignancy associated with HTLV-I [3]. Cutaneous lesions of ATLL consist of papules, plaques, and nodules/tumors. ATLL can be diagnosed based on clinicopathological findings and the presence of integrated HTLV-1 provirus in the DNA of tumor cells [4]. Histologically, ATLL usually exhibits the characteristics of peripheral T cell lymphoma with diffuse proliferation of atypical lymphoid cells with pleomorphic features [5]. Survival of ATLL cases with skin manifestations was reported to be significantly shorter than MF [6].

The cutaneous lymphocyte-associated antigen (CLA) recognized by the HECA-452 antibody is an adhesion molecule selectivity expressed by a subset of circulating memory T-cells, normal T cells in inflamed skin and by the vast majority of cutaneous T-cell lymphomas (CTCL) [7-9].

Chemokines are soluble proteins that regulate leukocyte migration and activation through binding to transmembrane receptors differentially expressed on lymphocyte subsets. It has recently been demonstrated that the expression pattern of chemokine receptors in normal T-cell subsets correlates with the pattern of cytokine secretion in these cells [10]. The preferential association of some chemokine receptors with human T helper (Th)1 or Th2 cells has recently been reported [10]. Th1 cells secrete interleukin (IL)-2, and interferon-γ (IFN-γ),which promote cell-mediated immunity to intracellular pathogens, whereas Th2 cells produce IL-4, IL-5 and IL-10, which are mainly involved in humoral immunity [11, 12]. CXCR3 and CCR5 are preferentially expressed on Th1 cells. CXCR3 expression is higher in cells with a prominent Th1 pattern of cytokine secretion. CCR5 has also recently been described as a surface marker of human T cells producing type 1 (Th1) cytokines [13]. ST2 is a soluble secreted form of 37.1 kD protein that lacks intracellular domain, whereas the ST2 ligand (L) is a transmembrane form of 61.5 kD protein expressed on the cell surface of Th2 cells but not on Th1 cells [14].
OX40 (CD134) is preferentially expressed on normal activated T cells. Co-stimulatory signal transduction of OX40 is involved in proliferation and cytokine production of activated T cells in vitro [15, 16].
The present study was designed to define the Th1/Th2 characteristics of MF and cutaneous lesions of ATLL. Specifically, we studied the immunohistochemical expression of the chemokine receptors, CXCR3 and CCR5. In addition, for activation of Th1/Th2 cells, we studied the expression patterns of OX40/CD134 and ST2 in 8 cases each of MF and cutaneous lesions of ATLL.

Materials and Methods

Tissue specimens

Archival skin specimens from patients diagnosed as MF or cutaneous lesions of ATLL (MF = 8, ATLL = 8) at the First Department of Pathology, Fukuoka University were selected in the present study. In all cases, clonal integration of ATLL proviral DNA was examined, using Southern blot analysis. The skin samples were fixed in buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin (H-E), Giemsa, PAS, and Gomori's for silver impregnation.

Immunohistochemistry

The paraffin embedded skin specimens were used for immunohistochemical analysis of CD20 (L26) for B cells (Dako, Glostrup, Denmark), CD45RO (UCHL-1) and CD3 for T cells (Dako), CD30 (Ber H2) (Dako), CXCR3 (PharMingen International), CCR5 (Dako), ST2 (MBL), CD134 (0X40) (PharMingen International), and CLA (Ancell Co., Bayort, MN). These staining results were evaluated semi-quantitatively by two independent observers. As described elsewhere, immunostaining was considered negative if less than 10% of the tumor cells failed to stain. In specimens considered positive, staining of the lymphoma cells was quantitated on a scale from 1 to 4 based on the percentage of positive tumor cells. The scale was structured as follows: 1 + = 10-25%; 2 + = 25-50%; 3 + = 50-75% and 4 + = > 75%. A portion of each skin specimen was kept at – 80 °C in a deep freezer and the skin specimens were examined using the monoclonal antibodies CD2, CD3,CD4, CD8, CD15, CD19, CD20 and CD30 (Coulter, Hialeah, FL; Ortho; Raritan, NJ; Becton-Dickinson; Dako).

DNA analysis

The other part of the frozen tissue was used for DNA isolation and gene analysis. Before performing DNA analysis, the samples were confirmed to contain lymphoma cells, which represented ≥ 70% of the nucleated cells, by H-E and immunohistochemical staining of the frozen samples. In the next step, the samples were examined for T cell receptor genes Cβ, Jγ, immunoglobulin heavy chain (JH) gene and proviral DNA of HTLV-1 (full length probe, including gag, pol, env, pX and LTR) by Southern blot analysis. The monoclonal integration of HTLV-1 DNA was examined by digestion with EcoRI, as described previously [17].

Morphological classification

Morphological classification was based on the new WHO classification [18, 19]. Morphologically, the two disorders were difficult to differentiate on histopathological findings including immunohistochemical findings.

Results

Clinical and histopathological findings of MF

Patients with MF included 4 males and 4 females, with a median age at the time of the initial biopsy of 50 years (range, 26-74 years). Clinically, the stage of MF in 5 patients was considered as erythematous stage with itchy erythema on the trunk and extremities, plaque stage in 2 patients with sharp-margin plaques on the trunk. These plaques were red to reddish brown and annular or serpiginous, and tumor stage in the remaining 1 patient. Histopathological examination of biopsy specimens (Fig. 1) from patients with erythema stage showed perivascular or lichenoid infiltration of small to medium-sized lymphoid cells with mild nuclear atypia. Specimens with plaque stage of MF showed lichenoid infiltration of medium-sized lymphoid cells with mild to moderate nuclear atypia, while that of MF-tumor stage exhibited nodular proliferation of large transformed lymphoma cells with severe nuclear atypia.
Phenotypically, all 8 cases were positive for CD2, CD3, CD4, CD45RO, 2 cases were positive for CD8, all cases were negative for CD30 (Table I), and all cases showed monoclonal TCR-γ and/or TCR-β gene rearrangements.

Table I. Immunological findings in MF

			Expression of chemokine receptors and adhesion molecules					Histopathological finding of H-E staining						Molecular study
Case	Age	Sex	Erup	CXCR3	CCR5	OX40	HECA	ST2	POI	SAC	NP	M	EP	PM	CD2	CD3	CD4	CD8	CD25	CD30
1	55	M	e	+ +	+ +	–	+ + +	–	p	s	+	+	+	–	+	+	+	+	–	–
2	26	M	e	+	–	–	+ +	–	l	s	+	+	+	+	+	+	+	–	–	–
3	74	F	e	+	+	+	+ +	–	l	s	+	+	+	–	+	–	+	–	–	–
4	45	F	e	–	+	–	+	–	p	m	+	+	+	–	+	+	+	–	–	–
5	40	F	e	–	+	+	+ +	–	l	m	+	+ +	+	–	+	+	+	–	–	–
6	68	M	p	–	+	–	+	–	l	m	+ +	+ +	+	–	+	+	+	–	–	–
7	53	M	p	–	+	+ +	+	–	p	m	+ +	+	+	–	+	+	+	–	–	–
8	40	F	t	–	+	–	–	–	n	l	+ + +	+ + +	–	–	+	+	+	–	–	–

Erup; eruption (e: erythema, p: plaque, t: tumor), POI; pattern of infiltration (p: perivascular, l: lichenoid, n: nodular), SAC; size of atypical cells (s: small, m: medium, l: large), NP; nuclear pleomorphism (+: Nuclei with minimal variability in size and shape, + +: Nuclei with moderate variability in size and shape, + + +: Nuclei with marked variability in size and shape), M; mitosis (+: 0-5/10 HPF, + +: 6-10/10 HPF, + + +: > 10/10 HPF), EP; epidermotropism, PM; Pautrier's microabscess; Expression of chemokine receptor and adhesion molecules; –: negative staining. +: positive staining of < 10% of cells. + +: positive staining of > 70% of cells. + + +: positive staining of all (100%) cells.

Clinical and histopathological findings of ATLL

Patients with ATLL included 2 males and 6 females with a median age at the time of the initial biopsy of 65 years (range, 52-72 years). Clinically, 2 patients had itchy erythema on the trunk, 3 patients had a few nodules on the trunk and extremities, and the remaining 3 patients had a few reddish tumors up to 2 cm in diameter on the face or extremities.
Histopathological examination of biopsy specimens (Fig. 1) from 6 patients showed dermal-based tumors consisting of large transformed lymphoma cells with bizarre-shaped nuclei, with or without prominent nucleoli. Biopsy specimens of the remaining 2 patients showed infiltration of medium sized lymphoma cells in the upper dermis. Phenotypically, all 8 cases were positive for CD2, CD3, CD4, CD5, CD25, and negative for CD8. Six cases were positive for Ki67, and only 1 case was positive for CD30 (Table II). All cases showed monoclonal TCR-β and/or TCR-γ gene rearrangements. Monoclonal HTLV-1 proviral DNA was detected by Southern blot analysis in all skin biopsy specimens.

Table II. Immunological findings in ATLL

			Expression of chemokine receptors and adhesion molecules					Histological findings of H-E staining						Molecular study
Case	Age	Sex	Erup	CXCR3	CCR5	OX40	CLA	ST2	POI	SOAC	NA	M	epd	PM	CD2	CD3	CD4	CD8	CD25	CD30	CD45RO	Ki67
1	70	F	e	–	+	–	–	–	p	s	+	+	+	–	+	+	+	–	+	–	–	–
2	72	F	e	+	+	–	–	–	p	s	+	+	–	–	+	+	+	–	+	–	–	–
3	54	F	p	–	+	–	–	+	n	m	+ +	+ + +	–	–	+	+	+	–	+	–	–	+
4	52	F	t	–	–	+ +	–	+ +	n	l	+ + +	+ +	+	+	+	+	+	–	+	–	–	+
5	68	F	t	–	+	–	–	+	n	m	+ + +	+ +	–	–	+	+	+	–	+	–	–	+
6	70	M	t	+	+ +	–	–	+	n	m	+ +	+	–	–	+	+	+	–	+	–	–	+
7	67	M	t	–	+	+ +	–	+ +	n	l	+ + +	+ +	–	–	+	+	+	–	+	–	+	+
8	72	F	t	+	+	+	–	+ +	n	l	+ + +	+ + +	+	+	+	+	+	–	+	–	+	+

Expression of CLA

CLA was extensively expressed in lymphoma cells in all 5 cases of erythema stage MF. Specifically, epidermotropic lymphoma cells showed expression of CLA (Cases 1, 2, 3, 4 and 5) (Fig. 2A). On the other hand, CLA was focally and weakly expressed in lymphoma cells in both cases of plaque stage MF (Cases 6 and 7), while the single case of tumor stage MF (Case 8) was negative for CLA. In contrast, all cases of ATLL were negative for CLA (Fig. 2B). In MF, the histopathological findings of vascular invasion and mitotic figures did not correlate with the expression of CLA.

Expression of CXCR3

CXCR3 was focally expressed in 3 of 8 cases of erythema stage MF (Cases 1, 2 and 3), and was especially expressed in epidermotropic small cell population (Fig. 2C). On the other hand, CXCR3 was focally expressed in 3 of 8 cases of ATLL with variable size of lymphoma cells (Cases 2, 6 and 8).

Expression of CCR5

CCR5 was focally expressed in 7 cases of MF (Fig. 2D) and 7 cases with cutaneous ATLL lesions with variable size of lymphoma cells. CCR5 was expressed in both disorders and there was no significant difference in the expression level between low-grade and transformed cases.

Expression of ST2

ST2 was expressed in 6 cases (Cases 3, 4, 5, 6, 7 and 8) of 8 ATLL cases. In these cases, ST2 expression was observed mainly in large lymphoma cells (Fig. 2F). In contrast, all cases of MF were negative for ST2. The large transformed cells of MF were also negative for ST2 (Fig. 2E).

Expression of OX40

OX40 was expressed in 3 of 8 cases with MF (Cases 3, 5 and 7) and 3 of 8 cases with ATLL (Cases 4, 7, and 8).The expression was observed in the large transformed cell population in both cases. In both MF and ATLL, OX40 tended to be expressed in large transformed lymphoma cells (Fig. 2G, Fig. 2H). However, in MF case 8 with tumor stage, large transformed lymphoma cells were negative for OX40.

Discussion

CLA was expressed on lymphoma cells in all 5 cases of erythematous stage MF and was weakly expressed on lymphoma cells in 2 cases of plaque stage MF. In contrast, all ATLL cases were negative for CLA expression. CXCR3 was weakly expressed in 3 of 8 cases of MF, with epidermotropic small lymphoma cells, with low-grade MF. CCR5 was expressed in all 8 cases of MF and ATLL in with variable size lymphoma cells. ST2 was expressed on large transformed lymphoma cells in 7 of 8 cases with ATLL, in contrast, all MF cases were negative for ST2. OX40 was expressed in 3 of 8 cases with each disorder in the large transformed cell population.
CLA is expressed on a subpopulation of human memory T cells and is involved in the primary step of their skin homing, and is a major ligand for E – selectin on microvascular endothelial cells in inflamed cutaneous lesions.
Herald et al. [20] demonstrated that the lymphoma cells of CTCL express the skin homing receptor of CLA + lymphocytes and that their levels in peripheral blood are increased in the erythrodermic CTCL patients rather than with minimal patch/plaque CTCL, and with CTCL in remission. The findings suggested the critical role for the skin – selective homing receptor CLA in the pathogenesis of CTCL. Borowitz et al. [21] also demonstrated an expanded peripheral blood population of CLA + T cells in patients with CTCL (including early stage CTCL).
In the present study, CLA was diffusely expressed in lymphoma cells in all 5 cases of erythematous stage MF. Specifically, epidermotropic lymphoma cells showed strong expression of CLA, while CLA was weakly expressed in lymphoma cells in both cases with plaque stage MF. Picker et al. [8] assessed the expression of CLA in 23 cases of MF. CLA was expressed in 16 of 18 cases with early stage MF. However, 5 cases of advanced tumor stage MF did not express CLA. Tumor-stage MF often showed loss of epidermotropism, wide-spread dissemination, and lack of CLA expression. These findings support the notion that the loss of CLA reactivity might precede clinical progression of patch/plaque stage disease. In the present study, cases of early stage MF also showed CLA expression, while tumor stage MF showed negative reactivity for CLA. On the other hand, all cases of cutaneous lesions of ATLL were negative for CLA. Tanaka et al. [22] reported that CLA was highly expressed in peripheral blood ATL cells compared with normal T cells. CLA expression was also significantly higher in peripheral blood ATL cells of patients with cutaneous lesions compared with cells from patients without such lesions. Further studies should be performed to confirm the mechanisms of lack of CLA expression in cutaneous lesions of ATL.
Both mycosis fungoides and ATLL are, in general, of CD4-positive T cell origin, and rarely CD8-positive. The normal CD4-positive T cell is functionally classified into functional Th1 and Th2 cells. Under normal conditions, the ratio of Th1 to Th2 cells is delicately regulated, whereas several human disorders (e.g., allergy and rheumatic disease) show distortion of this ratio. Recent studies have demonstrated that the expression pattern of chemokine receptors in normal T-cell subsets correlates with the pattern of cytokine secretion in Th1 and Th2 cells [10]. Furthermore, CXCR3 is commonly expressed on Th1 cells [10] and CCR5 expression is considered a marker of human Th1 cytokine-producing cells [10].
The surface expression of ST2L is considered to be associated with Th2 cells. On the other hand, OX40/CD134 expression is largely restricted to a subset of activated CD4-positive T cells. Strong OX40/CD134 expression has been reported in lymph node Th1 cells [15,16].
Previous studies reported the expression of CXCR3 in lymphoma cells of all 25 cases with low-grade MF, but was expressed in lymphoma cells of only 5 of 22 cases with progressive or transformed MF [23]. MF is characterized by expression of epidermal Th1-type cytokines (IL-2 and INF-γ) [24]. CXC3 is a likely candidate to mediate chemotaxis of lymphocytes into the epidermis given that its 3 chemoattractant ligands, IP-10, Mig, and I-TAC, are known to be highly expressed in the epidermis and functionally active in a wide subset of T cells [25-29]. Sarris et al. demonstrated that IP-10 appears overexpressed in lesional keratinocytes, and its expression extends to suprabasal cells in MF by immunocytochemical analysis [29]. Tensen et al. also showed that IP-10 messenger RNA is detected within keratinocytes in early – stage MF with epidermotropism, but not in the epidermis of normal human skin or in the nonepidermotropic tumor stage of MF [27]. These findings support the theory that chemokines and their receptors play a critical role in the epidermotropism of CTCL.
In the present study, epidermotropic small lymphoma cells of early stage MF were also positive for CXCR3. However, medium sized to large lymphoma cells were negative for CXCR3. These results suggest that lymphoma cells of early stage MF are suspected to be functional, probably Th1 phenotype and associated with epidermotropism.
Previous studies showed lack of OX40 expression in low-grade MF, but it was expressed in large transformed lymphoma cells of MF [16]. In the present study, OX40 was expressed in some large transformed lymphoma cells of low-grade MF, but was not expressed in large transformed lymphoma cells in the progressive nodal lesion. OX40 was expressed in the large transformed cell population of both of ATLL and MF.
In the present study, ATLL cases, but not MF, frequently expressed ST2. In these cases, ST2 was mainly expressed in large transformed lymphoma cells. ST2 ligand is expressed on the cell surface of Th2 cells. The expression of ST2 in ATLL has not been reported previously. However there is an interesting previous study that shows a Th2 dominance of ATL cells both in the peripheral blood and skin lesions. Yoshie et al. demonstrated a frequent expression of CCR4 in ATL cells by RT – PCR analysis and flow cytometric analysis, and others [30]. And they also detected strong signals for CCR4, TARC and MDC in ATL skin lesions by RT – PCR. CCR4 is the chemokine receptor for TARC and MDC, and is a selective marker of Th2. And it is known that circulating CCR4 + T cells are mostly polarized to Th2 and also contain essentially skin seeking memory T cells positive for CLA [31, 32]. These findings also support that ATL cells are suspected to be probably Th2 phenotype.
These results suggest that immunohistochemical expression might be a useful histopathological tool to discriminate MF from cutaneous lesions of ATLL, and to clarify the Th1/Th2 characteristics and transformation in MF and cutaneous lesions of ATLL.
Our results also identified OX40 expression in large transformed lymphoma cells with ATLL. Furthermore, lymphoma cells in ATLL tended to show variable reactivities for CXCR3 and CCR5. These results do not allow us to conclude the dominance of Th1 or Th2 mechanism in cutaneous lesions of ATLL.
The present study demonstrated the relationship between chemokines and chemokine receptors in MF and ATLL. CLA and ST2 could be used as immunohistochemical markers for differentiation of MF and cutaneous lesions of ATLL. However CLA was only clearly expressed in cases with erythema stage but weak or absent both in cases with plaque stage and tumor stage of MF, therefore CLA could not to be a relevant immunohistochemical marker for discrimination between advanced MF and cutaneous lesion of ATLL. Our results also suggest that OX40 could be a useful immunohistochemical marker for histopathological progression of both MF and cutaneous lesions of ATLL. However, further studies in a larger population samples are necessary to confirm our findings. n

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n ANNOUNCEMENTS n

2004

n The first international meeting on neurobiology of the skin, Münster, Germany
The European Society for Dermatological Research
13-15 February 2004
More information on
www.esdr.ch/announcements.htm
n 4^th Eden-Idea Congress, Venice, Italy
The European Society for Dermatological Research
10-12 October 2004
More information on
www.esdr.ch/announcements.htm
n The 4^th mediterranean association of dermatology will be held on 24-26 June 2004 in Montpellier, France
For all information
Le Corum, Service Congrès, BP 2200, 34027 Montpellier cedex 1, France
registration: gestion@enjoy-montpellier.fr
n The Séminaire immunologie clinique et allergologie on Allergy and anesthesia will take place on June 17-18, 2004, in Lyon, France.
For further information, please contact:
Pr Nicolas, Unité d'immunologie clinique-allergologie, CHU Lyon sud, 69495 Pierre Bénite cedex
Tel: + 33 4 78 86 15 72
Fax: + 33 4 78 86 15 28
e-mail: rose-marie.belitrand@chu-lyon.fr
n The Séminaire d'allergologie on chronic urticaria will take place on June 16, 2004, in Lyon, France.
For further information, please contact:
Pr Nicolas, Unité d'immunologie clinique-allergologie, CHU Lyon sud, 69495 Pierre Bénite cedex
Tel: + 33 4 78 86 15 72
Fax: + 33 4 78 86 15 28
e-mail: rose-marie.belitrand@chu-lyon.fr