Topical application of delphinidin reduces psoriasiform lesions in the flaky skin mouse model by inducing epidermal differentiation and inhibiting inflammation.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and aberrant keratinocyte differentiation. We have shown that treatment of reconstituted human skin with delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, increased the expression and processing of caspase-14, which is involved in cornification. Delphinidin also increases the expression of epidermal differentiation marker proteins. OBJECTIVES: To determine whether topical application of delphinidin can modulate pathological markers of psoriasiform lesions in flaky skin mice and if this is associated with increased epidermal differentiation and a reduction in proliferation and inflammation. METHODS: Five-week-old female homozygous flaky skin mice (fsn/fsn) were treated topically with delphinidin (0·5 mg cm(-2) and 1 mg cm(-2) skin areas, respectively), five times a week, up to 14 weeks of age. RESULTS: Treatment of flaky skin mice with delphinidin resulted in a reduction in (i) pathological markers of psoriasiform lesions; (ii) infiltration of inflammatory cells; and (iii) mRNA and protein expression of inflammatory cytokines. Delphinidin treatment also increased the expression and processing of caspase-14, and expression of filaggrin, loricrin, keratin-1 and keratin-10. Furthermore, there was a decrease in the expression of markers for cell proliferation (proliferating cell nuclear antigen and keratin-14) and modulation of tight junction proteins (occludin and claudin-1). In addition, delphinidin treatment increased the expression of activator protein-1 transcription factor proteins (JunB, JunD, Fra1 and Fra2). CONCLUSIONS: Delphinidin could be a promising agent for treatment of psoriasis and other hyperproliferative skin disorders.

Prognostic factors, prognostic indices and staging in mycosis fungoides and Sézary syndrome: where are we now?

Mycosis fungoides is the most prevalent form of primary cutaneous T-cell lymphoma. Patients frequently present with early-stage disease typically associated with a favourable prognosis and survival of 10-35 years, but over 25% may progress to advanced disease with a median survival < 4 years, and just 13 months in those with nodal involvement. Sézary syndrome presents in advanced disease with erythroderma, blood involvement and lymphadenopathy. The Bunn and Lamberg staging system (1979) includes stages IA-IIA (early-stage disease) and IIB-IVB (advanced-stage disease) and provides prognostic information, but some patients with tumour-stage disease (IIB) have a worse prognosis than those with erythrodermic-stage (III). Conversely, patients with plaque-stage (IB) folliculotropic mycosis fungoides may have a worse outcome than those with tumour-stage (IIB). The more recent staging system of the European Organisation for the Research and Treatment of Cancer/International Society for Cutaneous Lymphoma has been designed to reflect tumour burden at different sites. However, this staging system has not been validated prospectively for prognosis. Furthermore, this staging system does not include a detailed measurement of skin tumour burden, as indicated by the modified skin weighted severity assessment tool. This assessment measures body surface area of disease and is weighted to record patch, plaque and tumour to produce a numerical value from 0·5 to 400 and is an established endpoint for clinical studies. Nor does this staging include clinicopathological features associated with a poor prognosis such as folliculotropism. Here we review the clinical, haematological, pathological and genotypic parameters outside the staging system, which may affect survival in mycosis fungoides and Sézary syndrome. Most studies are retrospective and single centre. The identification of poor prognostic factors may be used to develop a prognostic index to use alongside staging, which may be of benefit in mycosis fungoides/Sézary syndrome to identify patients with a potentially poor prognosis.

Polo-like kinase 1 (Plk1) in cutaneous T-cell lymphoma.

BACKGROUND: Polo-like kinase 1 (Plk1) has multiple functions throughout mitosis. Plk1 levels are high in a number of cancers and haematological malignancies while being low in most differentiated tissues. OBJECTIVES: To assess the immunoreactivity of Plk1 in cutaneous T-cell lymphoma (CTCL) as a potential therapeutic target, to differentiate Plk1 levels among lesion types and to compare the detection level of Plk1 in fresh frozen (f) vs. paraffin-embedded (p) tissue. METHODS: Immunohistochemical staining of CTCL skin lesions with anti-Plk1 antibody was performed in a total of 65 biopsies from 49 patients with CTCL. Both f and p tissue was available for comparison in 46 biopsies. RESULTS: Tumour-stage CTCL lesions displayed significantly more Plk1 (mean f 7·7%, p 8·8%) than patch (mean f 0·7%, p 2·0%) and plaque-stage lesions (mean f 1·1%, p 2·0%) (P < 0·05). Plk1 ranged from 0% to 18% in f and 0% to 24% in p samples. p tissue revealed a higher mean Plk1 detection rate of 4·4% compared with 2·9% in f tissue with no statistical significance. CONCLUSIONS: Our results indicate that in CTCL, Plk1 is increased mainly in advanced lesions. Several Plk1 inhibitors have already shown promising results in preclinical and clinical phase I and II trials for different types of cancers with low adverse effects. Immunohistochemical detection of high Plk1 levels in patients with CTCL could help select individuals who might benefit from treatment with small molecule Plk1 inhibitors.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-caused tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin by a polyphenolic fraction isolated from green tea.

Our laboratory has been studying cancer chemopreventive effects of polyphenolic fraction isolated from green tea (GTP). In prior studies we have shown that (a) GTP possesses antigenotoxic effects in various test systems; (b) topical application of GTP protects against UV radiation and chemical carcinogen-induced tumorigenesis in murine skin; and (c) feeding of GTP in drinking water p.o. to mice protects against carcinogen-induced forestomach and lung tumorigenesis. Recently, we showed that in a dose-dependent manner GTP inhibits tumor promoter-caused induction of epidermal ornithine decarboxylase activity in SENCAR mice (R. Agarwal et al., Cancer Res., 52: 3582-3588, 1992). In the present study, we assessed the effect of GTP on TPA-induced skin tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated SENCAR mouse. Topical application of varying doses of GTP (1-24 mg) 30 min prior to that of each TPA application resulted in highly significant protection against skin tumor promotion in a dose-dependent manner. The animals pretreated with GTP showed substantially lower tumor body burden such as decrease in total number of tumors per group, number of tumors per animal, tumor volume per mouse, and average volume per tumor, as compared to the animals that did not receive GTP. Since TPA-induced epidermal cyclooxygenase and lipoxygenase activities and edema and hyperplasia are conventionally used markers of skin tumor promotion, we also assessed the effect of preapplication of GTP on these parameters. As quantitated by the formation of prostaglandin and hydroxy-eicosatetraenoic acid metabolites from, respectively, cyclooxygenase- and lipoxygenase-catalyzed metabolism of arachidonic acid, skin application of GTP to SENCAR mice resulted in significant inhibition of TPA-caused effects on these 2 enzymes. Prior application of GTP to mouse skin also resulted in 30-46% inhibition of TPA-induced epidermal edema and hyperplasia. The results of the present study suggest that GTP possesses anti-skin tumor-promoting effects, and that the mechanism of such effects may involve inhibition of tumor promoter-induced epidermal ornithine decarboxylase, cyclooxygenase and lipoxygenase activities, edema, and hyperplasia. Further studies are in progress to define which component present in GTP is responsible for its anti-skin tumor-promoting effects.

Genomic instability in radial growth phase melanoma cell lines after ultraviolet irradiation.

BACKGROUND/AIMS: Although ultraviolet (UV) irradiation, apoptosis, and genomic instability are all potentially involved in the pathogenesis of melanoma, in vitro studies investigating these changes in the radial growth phase of this neoplasm are still lacking; therefore, this study was designed to investigate these changes. METHOD: An in vitro system consisting of three radial growth phase Wistar melanoma cell lines (WM35, WM3211, and WM1650) was established. Cells were UV irradiated (10 mJ/cm2 for UVB and 6 J/cm2 for UVA), harvested after UV exposure, and evaluated for viability and apoptosis using Trypan blue and terminal deoxynucleotidyl transferase mediated dUTP digoxigenin nick end labelling assays, respectively. Polymerase chain reaction based microsatellite assays were used to examine the cell lines for the presence of microsatellite instability (MSI) using 21 markers at the 1p, 2p, 3p, 4q, 9p, and 17p regions. RESULTS: Exposure to UV initiated progressive cell death associated with pronounced apoptosis, with UVA having a greater effect than UVB. MSI was found in UVB (WM35 and WM3211) and UVA (WM35) irradiated cell lines at 1p, 9p, and 17p, but not in non-irradiated cells. The prevalence of MSI was higher after UVB irradiation (14%) than UVA irradiation (4.7%), and was most frequently found at D1S233. CONCLUSIONS: The ability of erythemogenic UV irradiation to induce both apoptosis and MSI in radial growth phase melanoma cells is suggestive of its role in melanoma pathogenesis. This instability may reflect a hypermutability state, oxidative stress induced DNA damage, replication infidelity, or a combination of these factors.

Lymphocyte activation in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a neoplasm of CD4+ T cells that includes mycosis fungoides and its leukemic variant, Sezary syndrome. The phenotype of CTCL cells and their predilection for localizing in the skin and regional lymph nodes indicate that CTCL is a neoplasm of mature, memory helper T cells belonging to the skin-associated lymphoid tissue. Experimental evidence suggests that the mechanisms of lymphocyte activation in CTCL may involve both T-cell receptor-dependent and -independent pathways. Furthermore, recent studies of the consequences of this activation have yielded several important findings. First, a dominant T-cell clone is generally present in CTCL specimens, including the earliest histologically diagnosable cutaneous patch lesions. Second, some cases of apparent chronic dermatitis harbor dominant T-cell clones. Such cases, known as "clonal dermatitis," have been observed to develop into overt CTCL. This suggests that patients with clonal dermatitis may be at increased risk for subsequent CTCL. Third, diseases associated with CTCL, such as lymphomatoid papulosis, large-cell lymphoma, and Hodgkin disease, arise as subclones of the original CTCL tumor and thereby share its clone-specific T-cell receptor gene rearrangements. Development of these secondary lymphoproliferative disorders appears to involve somatic mutations leading to deregulation of lymphoid activation/proliferation pathways. Fourth, CD8+ tumor-infiltrating lymphocytes present within lesions of CD4+ CTCL express a phenotype consistent with activated, major histocompatibility complex-restricted, cytotoxic T-cell differentiation. They tend to be more plentiful in early-stage disease and their proportion appears to correlate positively with improved survival, suggesting that they may exert an anti-tumor host response.

Human Langerhans cells express a novel form of the leukocyte common antigen (CD45).

CD45 is a family of transmembrane glycoproteins that function as protein tyrosine phosphatases. All isoforms exhibit common CD45 epitopes, whereas the restricted CD45 epitopes (RA, RB, and RO) are each limited to one or more isoforms. In prior studies, we showed that human Langerhans cells in normal epidermis express a novel CD45 phenotype. They express common CD45 epitopes but are characteristically RA- RB- RO-. This suggests that Langerhans cells can express a novel form of CD45. In order to clarify this issue further, mRNA extracted from enriched Langerhans cell preparations was reverse transcribed into cDNA. The 5' portion of CD45 cDNA was then amplified using polymerase chain reaction primers complementary to exon 2 and exons 9-10, which flank the CD45 variable exon region (exons 4-6). Cloning and sequencing of the dominant 441 bp polymerase chain reaction product revealed the following exon configuration for the 5' translated region of Langerhans cells CD45: exon 3/7/8/9/10. This is the same exon configuration associated with the 180 kd CD45 isoform expressed by memory T cells and monocytes/macrophages; however, these cell types are RO+ whereas normal Langerhans cells are RO-. The RO epitope is known to be an oligosaccharide with a terminal sialic acid moiety. Therefore, we determined the expression of a related epitope, OPD4, by Langerhans cells. This is another terminal sialic acid moiety expressed by the 180 kd CD45 isoform of memory T cells but not by monocytes/macrophages. Langerhans cells were OPD4-. Our data suggest that memory T cells, monocytes/macrophages, and Langerhans cells all express a common CD45 transcript lacking exons 4-6; however, this transcript appears to undergo lineage-specific, post-translational glycosylation to create three distinct CD45 glycoproteins: RO+ OPD4+, RO+ OPD4-, and RO- OPD4-, which are expressed typically by memory T cells, monocytes/macrophages, and Langerhans cells, respectively. Because these epitopes are located extracellularly, they are postulated to allow differential responses to extracellular stimuli by creating differential ligand specificity.

Detection of low-level tumor cells in allergic contact dermatitis induced by mechlorethamine in patients with mycosis fungoides.

Two patients with histologically proven mycosis fungoides, a malignancy of phenotypically mature T cells, received a topical challenge with mechlorethamine to areas of clinically uninvolved skin to exclude possible hypersensitivity reactions to this chemotherapeutic agent. In both patients, allergic contact dermatitis (ACD) developed at the sites of the application and resolved completely after withdrawal of mechlorethamine. The lesions were biopsied and analyzed for the presence of clonal T-cell receptor (TCR)-gamma gene rearrangements using two polymerase chain reaction (PCR)-based assays involving denaturing gradient gel electrophoresis (PCR/DGGE) and ribonuclease protection analysis (PCR/RPA). The former method has a clonal detection threshold of 10(-3)-10(-2), while the latter has a sensitivity of 10(-5). In both cases, the ACD lesions were polyclonal by PCR/DGGE. In contrast, PCR/RPA detected tumor-specific TCR-gamma gene rearrangements in these same lesions. This indicates that the ACD lesions contained tumor cells at a density within the 10(-5)-10(-2) range. Analysis of peripheral blood mononuclear cells from both patients failed to detect the malignant clone and showed the same result as blood from four normal individuals. The normal skin from one skin patient also lacked detectable TCR-gamma gene rearrangements. These results indicate that mycosis fungoides tumor are present within ACD lesions induced in mycosis fungoides patients and that this phenomenon does not appear to be due to the ubiquitous presence of detectable levels of these tumor cells in the blood or skin. These findings might be explained by nonspecific recruitment of malignant T cells to sites of local inflammation mediated by non-neoplastic antigen-specific T cells. Alternatively, they might be due to the local proliferation of very rare tumor cells in apparently normal skin in response to cytokines generated during the ACD reaction. In either case, the present study offers evidence that the malignant cells in myosis fungoides retain at least some capability of responding in vivo to physiologic stimuli.

Molecular staging of cutaneous T-cell lymphoma: evidence for systemic involvement in early disease.

Biopsies of various tissues from eight patients with confirmed cutaneous T-cell lymphoma were analyzed for lymphomatous involvement using V-J junctional sequences in rearranged T-cell receptor-gamma genes as specific molecular markers for the malignant clone. The patients included one stage IA, one stage IB, and six stage IVA. Twenty-five specimens were analyzed including 14 skin, five lymph node, four blood, and two bone-marrow samples. Ten skin samples and four lymph node samples were histologically positive for lymphoma. The other specimens were morphologically uninvolved. An assay involving polymerase chain reaction (PCR) amplification of T-cell receptor-gamma gene rearrangements and denaturing gradient gel electrophoresis was used to identify the tissue specimen containing the greatest tumor clone density in each case. This specimen was then used to generate a tumor-specific RNA probe that was used to molecularly stage each patient by means of an assay involving PCR gene amplification and RNase protection analysis (PCR/RPA). This assay detected malignant cells in all available biopsies, including morphologically uninvolved extracutaneous tissue samples (two blood, one lymph node, and one bone marrow) obtained from the two patients in pathologic stage I. Microscopic examination and the less sensitive PCR/denaturing gradient gel electrophoresis technique failed to detect lymphomatous involvement in 11 (44%) and eight (32%) of these 25 specimens, respectively. We conclude that molecular biologic staging using PCR/RPA is able to demonstrate morphologically occult dissemination of cutaneous T-cell lymphoma in early disease. In addition, PCR/RPA may be able to monitor tumor response to therapy and detect early recurrence of malignant lymphomas during clinical remission.

Enrichment of murine and human Langerhans cells with solid phase immunoabsorption using pan-leukocyte monoclonal antibodies.

Using a solid phase immunoabsorption (panning) technique, we have employed pan-leukocyte monoclonal antibodies to enrich and deplete murine and human Langerhans cells from cell suspensions of normal skin. Langerhans cell-enriched fractions contained 80-99% mononuclear cells, almost all of which had the ultrastructural features of Langerhans cells. These results are comparable to those achieved by panning for human Langerhans cells with anti-Leu-6(T6) antibody. Similarly, less than 1% of these cells were detectable in Langerhans cell-depleted fractions and such fractions were incapable of stimulating allogeneic lymphocytes in the skin cell-lymphocyte reaction. We conclude that panning with pan-leukocyte antibodies is an effective means of enriching or depleting Langerhans cells from heterogeneous skin cell suspensions and can yield results similar to those achieved with more Langerhans cell-specific reagents such as anti-Leu-6(T6). These findings are of particular significance to the enrichment and depletion of murine Langerhans cells since they express no known correlate of the human Leu-6(T6) antigen.

Lymphomatoid papulosis and associated cutaneous lymphoproliferative disorders exhibit a common clonal origin.

Determination of the clonal relationship among multiple lymphoproliferative disorders occurring in individual patients has been hampered by dependence on molecular biologic techniques that require analysis of advanced lesions containing high tumor clone densities to isolate dominant, clonal antigen-receptor gene rearrangements. Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) involves the amplification of T-cell receptor (TCR)-gamma gene rearrangements followed by their electrophoresis in denaturing gradient gels. This method detects dominant TCR-gamma gene rearrangements at tumor clone densities as low as 0.1%, making this assay suitable for analysis of early as well as late lesions. Using this approach, we analyzed skin lesions of lymphomatoid papulosis and either CD30+ large-cell lymphoma or early patch/plaque mycosis fungoides that developed in three patients. In each case, the dual specimens exhibited an identical band pattern by PCR/DGGE analysis, suggesting a common clonal origin. To confirm this clonal relationship, the dominant TCR-gamma gene rearrangements were eluted, amplified, cloned, and sequenced. In each case, they showed identical junctional sequences. These findings are significant for several reasons: 1) they demonstrate the common clonal origin of lymphomatoid papulosis and CD30+ large-cell lymphoma or mycosis fungoides occurring in individual patients; 2) they confirm that co-migrating PCR/DGGE bands exhibit identical nucleotide sequences; and 3) they provide a method for determining the sequence of a tumor-derived TCR-gamma gene rearrangement in early lesions containing a low tumor clone density. This latter feature should allow the prospective molecular staging of early cutaneous lymphoproliferative disorders.

Langerhans cells react with pan-leukocyte monoclonal antibody: ultrastructural documentation using a live cell suspension immunoperoxidase technique.

Langerhans cells are generally regarded as members of an Ia+ dendritic cell system capable of potent accessory cell function in immune responses. While it has been shown that murine Langerhans cells are bone marrow-derived, the ontogenic relationships among human Langerhans cells, other dendritic cells, macrophages, and leukocytes in general have yet to be fully clarified. Recently, several pan-leukocyte monoclonal antibodies have been produced which react with the human leukocyte common antigen. This antigen resembles the murine T200 antigen and is expressed by all leukocyte subtypes but not by nonhematopoietic cells. Using an immunoperoxidase technique for staining suspensions of live skin cells, we have documented Langerhans cell reactivity with pan-leukocyte monoclonal antibody L3B12 at the ultrastructural level. Reactivity with this highly sensitive and specific pan-leukocyte marker supports the concept of the human Langerhans cell as a specialized form of bone marrow-derived mononuclear leukocyte and defines an immunologic feature common to dendritic cells, macrophages, and leukocytes that is not shared by other cell types. This finding is discussed in the context of other recent data concerning the immunologic phenotype of Langerhans cells. Since the immunoultrastructural method employed does not require cell fixation of any kind prior to immunologic staining, it should prove particularly useful for studying cell surface antigens that are adversely affected by fixation.

Allergic contact dermatitis: novel immunohistologic features.

Leu-8 is a novel antigen expressed by the majority of mature T cells and certain other cells. Recent studies of the allogeneic mixed leukocyte reaction indicate that both Leu-8+ and Leu-8- subsets of Leu-3+ T cells have important functions in cell-mediated immunity in vitro. In order to determine whether Leu-3+8+ and/or Leu-3+8- T cells are present in cell-mediated immune reactions in vivo, we studied the immunohistology of allergic contact dermatitis in 8 biopsies from 8 patients with positive patch tests and 3 biopsies from 2 patients with Rhus dermatitis. Both Leu-3+8+ and Leu-3+8- T cells were present in each biopsy. Only 1 case had a definite minority of the Leu-3+8+ subset. These results suggest that, analogous to in vitro systems, both Leu-8+ and Leu-8- subsets of Leu-3+ T cells are involved in cell-mediated immunity in vivo. HLA-DR+ keratinocytes were present in only 3 of 11 biopsies at days 3-7. No HLA-DQ+ keratinocytes were identified. We also confirmed prior findings that Leu-3+ cells are the predominant T-cell population, Langerhans cells are increased, and B cells and NK cells are rare. Furthermore, Tac and Ki-67 expression by T cells and Leu-3 expression by Langerhans cells tended to increase over time.

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.

Detection of clonal T-cell receptor gene rearrangements in the peripheral blood of patients with mycosis fungoides/Sezary syndrome.

Involvement of the peripheral blood in mycosis fungoides/Sezary syndrome (MF/SS) has a significant impact upon prognosis, but it is often difficult to distinguish circulating cells of MF/SS from atypical reactive lymphocytes. We compared the standard morphologic method of identifying leukemic cells, the Sezary preparation, to a genotypic method using Southern blot analysis of T-cell receptor gene rearrangements in concurrent blood samples. We studied 26 MF/SS patients, five of them in remission, together with five controls from cases of various non-MF/SS skin diseases. Six of 26 MF/SS patients had morphologically atypical circulating leukocytes (3%, 4%, 5%, 14%, 16%, 19%). Seven of 26 MF/SS patients had clonal T-cell receptor gene rearrangements, including the four patients with the greatest percentages of atypical cells and three patients lacking atypical cells. Six of seven patients had skin disease at the time of sampling, including three with erythroderma, two with generalized thick plaques, and one with generalized patches, while one patient was in clinical remission. All five controls lacked morphologic and genotypic evidence of atypical or clonal T-cells. Relative to genotyping, in our series the Sezary preparation was less sensitive and less specific. There were three apparent false negative results in the Sezary preparations, and two potential false positive (patients with 3% and 4% atypical leukocytes); however, there was agreement between the two techniques in most cases. We conclude that gene rearrangement studies may provide an effective test with which to assess the peripheral blood of MF/SS patients.

No evidence of KSHV/HHV-8 in mycosis fungoides or associated disorders.

The recently discovered human virus known as Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) has been associated with body-cavity-based lymphomas in AIDS patients. It is most closely related to two other herpesviruses, the Epstein-Barr virus and herpesvirus saimiri, which are known to be associated with lymphomas in humans and nonhuman primates, respectively. To determine whether KSHV/HHV-8 is involved in the pathogenesis of mycosis fungoides (MF) and related disorders, we used a genomic PCR assay followed by confirmatory Southern blot analysis with a nested oligonucleotide probe to analyze cases for the presence of this virus. The specimens studied included fresh-frozen lesional tissues obtained from 16 patients with MF, seven with lymphomatoid papulosis, seven with primary cutaneous CD30+ large cell lymphoma of T-cell lineage, and five with Hodgkin's disease. Two T-cell tumor lines were also studied: MT4 (derived from a patient with adult T-cell leukemia/lymphoma) and Jurkat (derived from a patient with T-cell acute lymphoblastic leukemia). All cases were uniformly negative for KSHV/HHV-8, whereas Kaposi's sarcoma-positive controls and human beta-globin DNA integrity controls were appropriately positive. These findings provide strong evidence against a role for KSHV/HHV-8 in the pathogenesis of MF or associated lymphoproliferative disorders.

Expression of Leu-8 antigen, a majority T-cell marker, is uncommon in mycosis fungoides.

Predominance of mature helper T cells with the Leu-1+, 2-, 3+, 4+, 5+ phenotype was confirmed in 22 biopsy specimens of mycosis fungoides from 15 patients. Dissection of the T helper/inducer cells into phenotypically distinct subsets was performed with the use of a new monoclonal antibody, anti-Leu-8. One might predict a predominance of Leu-8+ in mycosis fungoides, as the known ratio of Leu-8+/Leu-8- cells is approximately 70/30 in the peripheral blood. Unexpectedly, a deficiency of Leu-8 was demonstrated in 18 of the 22 specimens from 13 of 15 patients. This finding could not be attributed to an artifact of the staining method or to therapy, and was present in early- as well as late-stage disease. Whether neoplastic cells in mycosis fungoides derive from Leu-8-subset of T cells at risk for malignant transformation, or whether there is antigen loss with malignant transformation remains to be determined. Implications of our finding with regard to etiopathogenesis of mycosis fungoides are discussed.

Selective enrichment of human epidermal cell subpopulations using monoclonal antibodies.

In studying the mechanisms that regulate the growth and differentiation of the human epidermis, it would be helpful to obtain relatively pure populations of the different epidermal cell types. We have used a solid-phase immunoabsorption method termed "panning" to positively select two types of epidermal cells: Langerhans cells and the keratinocytes found in the basal cell layer (basal cells). To attach basal cells to a goat anti-mouse IgG-coated plastic surface, we used murine monoclonal antibodies (VM-1 or VM-2), which were recently produced in our laboratory and bind specifically to antigens on human basal cells. Using antibodies VM-1 or VM-2, we panned for basal cells and obtained a yield of about 40 percent (an enrichment of about 2.5-fold). The cells enriched for basal cells demonstrated much better growth and DNA synthesis than did the cell fraction depleted of basal cells. For positive selection of Langerhans cells, we used OKT6, a murine monoclonal antibody that binds specifically to Langerhans cells in the epidermis. We determined that of those cells preincubated with OKT6 and adherent to an antibody-coated petri dish surface, about 70 percent demonstrated OKT6 binding by fluorescence microscopy. This represents a 15- to 20-fold enrichment for Langerhans cells. The nonadherent cell fraction contained less than 1 percent OKT6-positive cells. Ultrastructural studies showed that the cells thus separated were Langerhans cells. The OKT6-positive but not the OKT6-negative cells were capable of stimulating allogeneic lymphocytes in the skin-cell-lymphocyte reaction. Thus the panning technique is an effective method for obtaining greatly enriched subpopulations of viable epidermal cells.

Transformation of mycosis fungoides: T-cell receptor beta gene analysis demonstrates a common clonal origin for plaque-type mycosis fungoides and CD30+ large-cell lymphoma.

It is well recognized that patients with classical mycosis fungoides (MF) may develop a large-cell lymphoma (LCL), a phenomenon known as "transformation." An unresolved issue regarding the transformation of MF is whether MF and LCL represent two separate lymphomas or whether they are derived from the same T-cell clone. We report the clinicopathologic, immunophenotypic, and immunogenotypic analysis of MF and LCL in a white male. He developed a rash at age 51 that was diagnosed at age 56 as clinical stage IA patch/plaque MF. After topical nitrogen mustard and total skin electron beam therapy for progressive generalized CD3+CD4+ patch/plaque lesions, he developed nodules of Ki-1+ (CD30+) T-LCL at age 72. Southern blot analysis of DNA digested with Bg/II or BamHI and probed with a T-cell receptor (TCR)-beta gene J beta 1/J beta 2 probe showed a single, identical rearranged band in both the MF and LCL skin lesions that had been obtained 4 years apart. V beta gene family--specific gene amplification assays demonstrated dominant V beta 6 PCR products in both types of lesions. These PCR products and lesional cDNA exhibited a monoclonal pattern when amplified with consensus TCR-beta gene VDJ joint primers and electrophoresed under conditions that allowed the resolution of small differences in size. Furthermore, sequence analysis of the V beta 6 PCR products amplified from both the MF and LCL lesions showed an identical nucleotide sequence involving V beta 6.4, D beta 1.1, J beta 1.2, and C beta 1. These findings indicate that both the MF and the LCL in this patient arose from the same T-cell clone and that these diseases developed at a stage in the clone's differentiation subsequent to rearrangement of the TCR-beta gene.

Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE).

We used a gene amplification strategy to analyze T-cell receptor (TCR) gene rearrangements in 185 specimens, including mycosis fungoides/Sezary syndrome (MF/SS), other cutaneous neoplasms, inflammatory dermatoses, reactive lymphoid tissues, and normal skin. Genomic DNA was extracted from lesional tissues and rearrangements of the TCR-gamma chain gene were amplified using the polymerase chain reaction (PCR) with primers specific for rearrangements involving V gamma 1-8 or V gamma 9 gene segments. The resulting PCR products were then separated according to their nucleotide sequence as well as size by denaturing gradient gel electrophoresis (DGGE). Dominant clonal TCR-gamma gene rearrangements were detected in 61 of 68 MF/SS cases by PCR/DGGE. This sensitivity of 90% compared to a sensitivity of only 59% when dominant clonality was sought in 17 of these same cases by Southern blot analysis of TCR-beta gene rearrangements. This difference in sensitivity was greatest in early, minimally infiltrated skin lesions. PCR/DGGE was also more sensitive than Southern blot analysis for detecting peripheral blood involvement in two cases of early MF. Among 12 additional specimens of suspected MF/SS, nine (75%) showed clonal TCR-gamma gene rearrangements by PCR/DGGE including six of eight cases with a previously confirmed diagnosis of MF/SS and three of four cases without prior known MF/SS. Among 105 non-MF/SS specimens, dominant TCR-gamma gene rearrangements were detected in only six cases (6%). Four were diagnosed as chronic dermatitis and two were diagnosed as cutaneous lymphoid hyperplasia. We conclude that the large majority of MF/SS cases, including patch phase disease, possess dominant clonal TCR-gamma gene rearrangements. PCR/DGGE is more sensitive than Southern blot analysis for detecting dominant clonality and staging disease in patients with a confirmed diagnosis of MF/SS. However, because PCR/DGGE is sensitive enough to detect dominant TCR-gamma gene rearrangements in a subset of patients with chronic dermatitis, it cannot be used as the sole criterion for establishing a diagnosis of T-cell lymphoma. As with other molecular biologic clonality assays, clinicopathologic correlation is essential. Nevertheless, the detection of dominant clonality in some cases of histologically nonspecific dermatitis allows the identification of a previously unrecognized subset of patients, i.e., those with "clonal dermatitis." It will be important to determine the long-term risk of MF/SS among these patients because our study indicated that MF/SS can sometimes present with lesions indistinguishable from clonal dermatitis.