An assessment of the diagnostic value of the monoclonal antibodies Leu 8, OKT9, OKT10 and Ki67 in cutaneous lymphocytic infiltrates

Biopsies from 63 patients, 40 with mycosis fungoides (MF) and 23 with other lymphocytic infiltrates, were stained with a panel of monoclonal antibodies to differentiation antigens (OKT10, Leu 8), to activation antigens (OKT9), and to an antigen expressed by all dividing cells (Ki67) to establish whether this panel was of value in the diagnosis of mycosis fungoides. None of these antibodies used in isolation was found to give a staining pattern specific for mycosis fungoides, but the phenotype Leu 3a+, OKT9+, Leu 8- of the majority of lymphocytes in the cutaneous infiltrate was strongly suggestive. The presence of Leu 3a+ Leu 8- lymphocytes is of particular interest in that in the peripheral blood both of normal subjects and of MF patients Leu 3a+ lymphocytes also are predominantly Leu 8+, were the lymphocytes in the cutaneous infiltrates of the non-MF infiltrates included in this study.     

Detection of a peripheral blood T cell clone is an independent prognostic marker in mycosis fungoides

T cell receptor gene analysis is a sensitive method for assessment of peripheral blood involvement in mycosis fungoides. This study uses polymerase chain reaction/single-strand conformational polymorphism (PCR/SSCP) analysis of the T cell receptor gamma gene and relates the results to skin stage and outcome in mycosis fungoides. Seventy-five peripheral blood samples from 66 patients were obtained from 1990 onwards and subjected to PCR/SSCP. Both Southern blot analysis and PCR/SSCP analysis were performed on 63 samples from 56 patients. Fourteen patients had T1 disease (12 IA, two IIA), 20 T2 (14 IB, five IIA, one IVA), 29 T3 (24 IIB, two IVA, three IVB, two patients tested at both T2 and T3), and five T4 (all III). The percentage of positive samples was higher with PCR/SSCP than with Southern blot analysis (29 of 63 vs eight of 63 samples, p < 0.001), and the percentage of positive samples increased with each stage (21% at T1, 35% at T2, 58% at T3, and 71% at T4). Proportional hazards analysis corrected for age, skin, and lymph node stage showed that the presence of a peripheral blood clone is associated with a worse outcome (p = 0.03, CI 1.1-6.03). These results indicate that the presence of a peripheral blood clone is an independent prognostic variable in patients with mycosis fungoides after correcting for age, skin, and lymph node stage, and that peripheral blood involvement is present in a large proportion of patients with early stage mycosis fungoides. Keywords: polymerase chain reaction/single-strand conformational polymorphism/T cell receptor gene rearrangement. J Invest Dermatol 114:117-121, 2000     

Leu-8 and Leu-9 antigen phenotypes: immunologic criteria for the distinction of mycosis fungoides from cutaneous inflammation

The distinction of mycosis fungoides from reactive cutaneous inflammation can be difficult. Unfortunately, since many reactive processes exhibit predominantly a mature helper T cell phenotype similar to that expressed by most cases of mycosis fungoides, standard immunologic marker studies have not been very helpful in differential diagnosis. To determine whether novel immunophenotypic criteria could be developed that correlate with the diagnosis of cutaneous involvement by mycosis fungoides, we studied the expression of Leu-8 and Leu-9 antigens by T cells in forty-one skin biopsy specimens from twenty-seven patients with mycosis fungoides and thirty-four skin biopsy specimens from thirty-three controls with a variety of benign cutaneous diseases. These antigens are expressed by the majority of normal T cells in the blood and lymphoid tissues but are often absent in T cell lymphomas or expressed by only a minority of tumor cells. Semiquantitative grading of the percentage of Leu-8+ and Leu-9+ T cells in our patients revealed that deficiency of these antigens (i.e., expression by less than or equal to 33% of T cells) was more prevalent among mycosis fungoides patients than among controls and became more specific for mycosis fungoides as the percentage of Leu-8+ and Leu-9+ T cells decreased. In initial biopsies, less than or equal to 33% of T cells were Leu-8+ in 82% of mycosis fungoides patients versus 15% of controls, while less than or equal to 10% of T cells were Leu-8+ in 52% of mycosis fungoides patients versus only 3% of controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood lymphocyte subpopulations studied with monoclonal antibodies in aleukaemic stages of mycosis fungoides

Peripheral blood lymphocytes from 21 patients with aleukaemic stages of mycosis fungoides were studied with monoclonal T cell antibodies. Patients with erythematous eruptions or infiltrated plaques (group I) had a higher percentage of OKT4+ cells than patients with a tumorous stage (group II) or normal subjects (p = 0.05). OKT8+ cells were decreased in group I patients whereas in group II they were usually normal or increased (p less than 0.02). These data indicate that with advancing stage of the disease a different pattern of imbalance of T-cell subsets is being established.     

Human T-cell leukemia virus in cutaneous T-cell lymphoma in Denmark. A possible association of HTLV and aneuploidy

Cutaneous T-cell lymphomas (CTCL) are neoplasias of mature T-cells and comprise Sezary syndrome, mycosis fungoides and some cases of lymphomatoid papulosis. Clinically this group of disorders differ from the more aggressive neoplasias of mature T-cells known as adult T-cell leukemia/lymphoma and T-cell lymphosarcoma leukemia which are associated with human T-cell leukemia virus (HTLV). We have found that of 68 patients from Denmark with CTCL ten were positive for HTLV antibodies and that the neoplastic T-cells from skin specimens in seven of eight HTLV-antibody positive patients studied by DNA flow cytometry exhibit DNA aneuploidy. Either one or two hyperdiploid cell clones were present. Aneuploidy was found in two patients with histologically verified mycosis fungoides, in four patients with histological non-diagnostic mycosis fungoides, and in one patient with lymphomatoid papulosis. The present data indicate that further seroepidemiologic survey studies of cutaneous T-cell lymphomas should include the early histological non-diagnostic stages, especially when aneuploidy is present.     

Leu-8/CD7 antigen expression by CD3+ T cells: comparative analysis of skin and blood in mycosis fungoides/Sézary syndrome relative to normal blood values.

Deficiencies of Leu-8 and CD7 antigens are exhibited by CD3+ T cells in the skin lesions of most patients with mycosis fungoides/Sézary syndrome. To determine whether these antigenic abnormalities are limited to involved skin, we studied Leu-8/CD7 expression in 21 skin lesions of mycosis fungoides/Sézary syndrome obtained from 16 patients and compared them with their peripheral blood leukocytes obtained concurrently. There was no correlation between Leu-8/CD7 values in skin lesions versus blood. Blood values were relatively uniform; most patients had 50% or greater of CD3+, Leu-8+ T cells and CD3+, CD7+ T cells. In contrast, skin values were highly heterogeneous; most patients lacked expression of Leu-8 or CD7 by the majority of lesional CD3+ T cells. Furthermore, Leu-8/CD7 antigen deficiency was present in lesional skin in one patient with mycosis fungoides but not in her concurrently sampled pityriasis lichenoides chronica or blood. These findings suggest that Leu-8/CD7 antigen deficiencies in skin lesions of mycosis fungoides/Sézary syndrome do not represent generalized antigenic abnormalities of CD3+ T cells in other body compartments and that within the skin, these deficiencies are disease specific within individual patients with more than one dermatosis. Comparative peripheral blood immunophenotyping of the patients with mycosis fungoides/Sézary syndrome and of the control subjects indicated that the control ranges of CD3+/Leu-8+ and CD3+/CD7+ T cells (33% or greater) extend lower than reported previously (60% or greater) and suggested that leukemic involvement in patients with mycosis fungoides/Sézary syndrome may correlate with percentages of CD3+, Leu8+ and/or CD3+, CD7+ T cells that fall below the revised control range.     

Imbalance of T helper and T suppressor cells and reduced plaque-forming cell capacity of mononuclear cells from patients with mycosis fungoides

Six patients with clinically and histologically established mycosis fungoides (stage II-IV) were investigated for the distribution of OKT₄⁺ and OKT8⁺ cells and plaque forming cells (PFC) in peripheral blood. The patients had an increased percentage of OKT8⁺ cells. They also had a markedly reduced PFC capacity for both IgM, IgG and IgA antibodies after stimulation with pokeweed mitogen (PWM). OKT8⁺ lymphocytes were partially depleted by three cycles of monoclonal antibody (OKT8) with rabbit-complement mediated lysis. The reduced OKT8⁺ percentage was followed by a significant increase in IgM-PFC among the residual cells. This suggests a normal helper activity of the residual OKT4⁺ cells. Further evidence for the helper cell activity in mycosis fungoides was shown by a continuously growing OKT4⁺ cell-line established from a patient with mycosis fungoides. Thus, the immunological competence of patients with mycosis fungoides seems to include an increased suppression of in vitro Ig secretion.     

Spectrum of p53 gene mutations suggests a possible role for ultraviolet radiation in the pathogenesis of advanced cutaneous lymphomas

There is evidence that the incidence of primary cutaneous lymphoma, like other forms of non-Hodgkin's lymphoma, is increasing, yet little is known of the pathogenetic events involved in this group of disorders. In this study we examine the frequency and spectrum of P53 gene mutations in a large series of primary cutaneous lymphomas, with particular emphasis on tumor stage mycosis fungoides, as it is in these cases that p53 overexpression has previously been reported. Sixty-six samples from 55 patients with primary cutaneous B cell and T cell lymphomas were analyzed for mutations in exons 5-9 of the P53 gene using polymerase chain reaction/single strand conformational polymorphism, and subsequent cloning and sequencing of genomic DNA. Fourteen separate P53 mutations were identified in blood, skin, and lymph node samples in 13 patients (24%). Twelve of 14 mutations occurred at dipyrimidine sites, eight resulting in C-->T transitions and one in a CC-->TT tandem base transition, a mutation spectrum strikingly similar to that reported in nonmelanoma skin cancer and characteristic of DNA damage caused by ultraviolet B radiation. In the subset of patients with mycosis fungoides, P53 mutations were identified in six of 17 patients with tumor-stage but in none of 12 patients with plaque-stage disease (Fisher's exact test p = 0.027). These data suggest a role for ultraviolet radiation in the pathogenesis of primary cutaneous lymphomas and a possible ultraviolet B-related step in the progression of mycosis fungoides from plaque to tumor-stage disease.     

The significance of Leu 8 negative T cells in lymphoid skin infiltrates: malignant transformation,selective homing or T-cell activation?

The expression of Leu 8 was studied on skin biopsies from a large group of patients with benign and malignant skin disorders and correlated with the expression of T-cell differentiation antigens and activation markers. The effect of in vitro stimulation of peripheral blood T cells and T-cell subsets on the expression of Leu 8 antigen was also determined. In all the skin diseases studied an inverse relationship was found between the proportions of cells expressing Leu 8 and HLA-DR. A deficiency of Leu 8 positive cells was not specific for mycosis fungoides, but was also found in several reactive dermatoses. Stimulation of peripheral blood cells with phytohaemagglutinin (PHA), concanavalin A (Con A), and anti-CD3-PMA resulted in a considerable decrease of Leu 8 antigen expression on day 3 in both CD4+ and CD8+ T cells. These data suggest that the low proportion of Leu 8+ T cells in mycosis fungoides and several reactive skin disorders is not related to malignant transformation or selective homing of Leu 8- T cells, but probably results from local T-cell activation.     

Epidermotropic secondary cutaneous involvement by relapsed angioimmunoblastic T‐cell lymphoma mimicking mycosis fungoides: a case report

Angioimmunoblastic T‐cell lymphoma (AITL) is frequently associated with skin lesions, but epidermotropic cutaneous involvement has never been described. A 37‐year‐old man presented with erythematous and pruriginous plaques, clinically suggestive of mycosis fungoides, distributed all over the body, 3 weeks after the last line of a polychemotherapy, given for an AITL diagnosed 1 year earlier on a lymph node biopsy. Skin biopsy showed an epidermotropic CD4⁺ T‐cell lymphoma, so that a diagnosis of mycosis fungoides was first proposed. Further investigations showed that atypical lymphocytes strongly expressed CD10 and markers of follicular helper T cells (T_FH) including PD1, BCL‐6 and CXCL13. The diagnosis of an unusual epidermotropic cutaneous localization of the AITL was finally made, supported by the presence of the same T‐cell clone in the initial lymph node biopsy and the skin. We therefore recommend performing markers of T_FH cells in patients with unusual epidermotropic cutaneous T‐cell lymphomas, particularly if they have any clinical features suggestive of AITL.     

Mycosis fungoides bullosa

A case of mycosis fungoides bullosa is presented. The results in our study confirmed that the predominant atypical lymphoid cells in the bullae, peripheral blood, and involved lymph nodes expressed the T-cell helper phenotype using immunophenotyping techniques. The literature is reviewed, confirming that our case demonstrated cells of the T-helper phenotype, not only in the skin but also in the blood and lymph node tissue. Bullous lesions in mycosis fungoides are rare.     

Expression of T-cell receptor antigens in mycosis fungoides and inflammatory skin lesions

Using immunohistologic methods, we studied the expression of the T-cell receptor (TCR)-associated antigens CD3, TCR-beta, and TCR-delta by cutaneous T cells in mycosis fungoides (MF) (36 patients) and a variety of inflammatory diseases (16 patients). Most T cells in the inflammatory diseases and patch/plaque mycosis fungoides expressed the immunophenotype characteristic of the vast majority of mature peripheral T cells: CD3+ TCR-beta+ TCR-delta-. In contrast, abnormal CD3/TCR-beta antigen expression was seen in 3 of 6 cases (50%) of tumor stage mycosis fungoides. Furthermore, we were able to document its evolution from the normal pattern present in earlier patch/plaque lesions of the two cases in which serial biopsies were available for study. Divergence of epidermal versus dermal CD3/TCR-beta antigen expression was seen in 2 of 34 (6%) of biopsies of patch/plaque mycosis fungoides but not in inflammatory controls. The TCR-delta+ cells were generally rare regardless of diagnosis. We conclude that inflammatory skin diseases and most patch/plaque mycosis fungoides are typically composed of T lymphocytes that resemble mature peripheral T cells in regard to their expression of TCR-associated antigens. In contrast, aberrant patterns of TCR-associated antigen expression can be seen in tumor stage MF, and, more rarely in patch/plaque MF.     

Adult T-Cell Leukemia/Lymphoma and Cutaneous T-Cell Lymphoma Are They Related?

Comparative studies were performed on clinical and laboratory features of four patients with different types of T-cell lymphoma of the skin; adult T-cell leukemia/lymphoma (ATLL), Sézary syndrome, mycosis fungoides, and Ki-1-positive lymphoma. All neoplastic cells studied showed a helper-inducer T-cell phenotype. A Ki-1-positive lymphoma is distinct from other types of cutaneous lymphomas because of unique morphologic and phenotypic features. Clonal proliferation of lymphocytes infected by human T-cell lymphotrophic virus (HTLV)-1 distinguishes ATLL from other T-cell lymphomas of the skin, especially in the endemic area of ATLL. From the pathogenic point of view, ATLL should not be included in a group with mycosis fungoides and Sézary syndrome.     

Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR γ-chain gene sequencing

Abstract Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30⁺ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30⁺ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR á-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells. Received: 8 September 2000 / Revised: 11 November 2000 / Accepted: 10 December 2000     

Lymphomatoid papulosis expresses immunophenotypes associated with T cell lymphoma but not inflammation

Twelve skin biopsy specimens of lymphomatoid papulosis from nine patients were studied immunohistologically. The large atypical cells morphologically resembled Reed-Sternberg cells in six cases and large cerebriform mononuclear cells in three cases. These cells expressed pan-T cell antigens (Leu-4 and/or Leu-5) and helper T cell antigen (Leu-3) in each case. They also expressed activation antigens: HLA (human lymphocyte antigen)-DR, HLA-DQ, Tac, and T9. Reactivity of many nuclei with Ki-67 indicated a high proliferative index. Phenotypic abnormality of the large atypical cells was evident by their deficiency of T cell antigens Leu-1 and/or Leu-9 in eight of nine cases. Neither Ki-1 nor Leu-M1 were reliable markers for lymphomatoid papulosis in this series, since large atypical cells were Ki-1-positive in only three of eight cases and were Leu-M1-negative in all eight cases tested. The remainder of the cutaneous infiltrate consisted of small T cells, macrophages, Langerhans cells, and granulocytes. The small T cells expressed a normal phenotype except in some cases associated with mycosis fungoides in which they were deficient in various T cell antigens. Comparison of concurrent lymphomatoid papulosis and mycosis fungoides skin biopsy specimens in two patients revealed that they were composed of phenotypically distinct T cell subpopulations. These results indicate that the large atypical cells of lymphomatoid papulosis are a proliferating population of activated helper T cells that are deficient in certain T cell antigens. Such abnormal T cell phenotypes are common in T cell lymphoma but are rarely, if ever, observed in cutaneous inflammation. In conjunction with the cytologic atypia, aneuploidy, and association with other lymphomas documented in this or previous reports, these data suggest that lymphomatoid papulosis represents a T cell lymphoproliferative disorder rather than an inflammatory disorder.     

Expression of OKM5 antigen on epidermal cells in mycosis fungoides plaque stage

To characterize and quantitate potential antigen-presenting cell subsets in the epidermis of patients with cutaneous T-cell lymphoma, epidermal cells in suspension were obtained from involved and uninvolved skin. Involved epidermis contained increased numbers of OKT6+HLA-DR+ Langerhans cells and a variable number of OKM5+ epidermal cells (ECs) in all mycosis fungoides (MF) patients tested (N = 14). The OKM5+ EC population from involved epidermis of MF patients were heterogeneous and comprised both OKM5+HLe1- keratinocytes and OKM5+HLe1+ leukocytes. Uninvolved epidermis, in 6 of 14 patients with MF, contained a small number of OKM5+ leukocytes; however, no OKM5+ keratinocytes were detected. Neither OKM5+ leukocytes nor OKM5+ keratinocytes were detected in the epidermis obtained from healthy controls. The increased number of potential antigen-presenting cells, that is, OKT6+HLA-DR+ Langerhans cells and OKM5+HLA-DR+ monocytic leukocytes, in the epidermis of patients with MF may be important for the activation of abnormal T cells contained within the epidermis of these patients. Such activated T cells may release gamma-interferon and induce expression of both HLA-DR and OKM5 antigens on keratinocytes. OKM5+ keratinocytes are present in the epidermis of patients with MF, but not in normal skin, and may thus play a role in the pathogenetic mechanisms of mycosis fungoides by recruitment of immunocompetent cells to the epidermis.     

Skin-infiltrating lymphocytes in normal and disordered skin: activation signals and functional roles in psoriasis and mycosis fungoides-type cutaneous T cell lymphoma.

T lymphocytes recruited into the skin can experience several different outcomes. On the one hand, they may be recruited by adhesion molecules and chemoattractants to enter the perivascular space, but never undergo activation. Other T cells undergo activation and further differentiation under the influence of the cutaneous milieu. These activated lymphocytes then coordinate specific and non-specific immune responses characteristic of inflamed tissue. We have explored two models for studying the activation and function of skin infiltrating T lymphocytes (SIL's). In the first model, we have identified a family of Langerhans cell-related professional dendritic antigen presenting cells that exist in the epidermis and dermis of normal skin, atopic skin, and mycosis fungoides skin. These have APC abilities to activate freshly recruited resting blood T cells that are distinct from another family of macrophage-related cells abnormally present in sunburned or psoriatic skin. In the second model, we examined the function of cells that have already been recruited into the skin of patients with psoriasis and mycosis fungoides. Lesional psoriasis and mycosis fungoides T cells exhibited a variety of T cell receptor gene rearrangements, conclusively demonstrating that heterogeneous populations of T lymphocytes exist in inflamed human skin. From psoriasis, clones were identified that were particularly effective at inducing normal keratinocytes to assume "psoriatic" phenotypic features and functions. Thus, lesional psoriatic SIL's could induce HLA-DR, ICAM, and CDw60 on normal keratinocytes. In addition, psoriatic SIL's induced increased keratinocyte proliferation and cytokine profile changes characteristic of psoriatic epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A case of classical mycosis fungoides associated with human T-cell lymphotropic virus type I

A 72-year-old male patient from north-eastern Iran developed the typical clinical and histopathological features of mycosis fungoides with lymphadenopathy, but without any other systemic involvement. Human T-cell lymphotropic virus (HTLV-I) antibodies were detected in the patient's serum by two different ELISAs and by Western blot using purified viral particles from MT-2 culture supernatants. Cultured peripheral blood lymphocytes were positive for labelling with anti-HTLV-I serum. Southern blot hybridization of DNA extracted from a skin tumour and from an involved lymph node revealed integrated proviral DNA with identical restriction patterns. This case supports a relationship between mycosis fungoides and HTLV-I and may indicate a new region of endemic HTLV-I infection.     

Mycosis fungoides. Detection of OKT6+ cells by cytofluorographic analysis in one case

An 86-year-old woman had a subjective history of pruritus and intermittent fever. The clinical diagnosis of mycosis fungoides was confirmed by cutaneous and lymph node biopsies. Immunohistochemical, cytofluorographic, and ultrastructural analysis was performed. According to immunohistochemical findings the lymphoid cells infiltrating the skin and lymph nodes were phenotypically T-helper cells. Cytofluorographic and ultrastructural analysis of the peripheral blood detected a small number of cerebriform lymphoid cells. By immunohistochemistry, these cells showed the same phenotype that was found in the skin and lymph nodes. OKT6+ cells, which are not usually present, were also found in the peripheral blood. These findings may suggest a functional relationship between the skin and the lymphoid system.     

THE MYCOSIS FUNGOIDES CELL: THE SKIN WINDOW IN MYCOSIS FUNGOIDES

SUMMARY.— The skin-window technique was applied to normal und involved skin sites of 17 patients with mycosis fungoides, 8 patients with parapsoriasis en plaque , and 6 patients with cutaneous lymphomata, and also to normal skin of 25 controls. Large mononuclear cells rapidly migrated in large numbers from plaques of mycosis fungoides and parapsoriasis. Identical cells were seen in skin windows from control sites at later intervals. No malignant cells were found in any of the skin windows of mycosis fungoides or purapsoriasis. Malignant cells were found in cases of malignant cutaneous lymphoma. Comparison of cells found on the skin windows from mycosis fungoides plaques to lymph node imprints indicates that the former are immature reticular cells without neoplastic features. We conclude that mycosis fungoides is a proliferation of these benign immature reticular cells in the skin.