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Multiparametric image cytometry in mycosis fungoides.

Eighteen cases of early mycosis fungoides were compared with 18 cases of eczematous dermatitis by multiparametric image cytometry. A minimum of 100 lymphocytes was measured in each case. A large number of measurements was acquired for each lymphocyte, characterizing nuclear DNA content, area, shape, and texture. There were significant differences between the two groups, especially in nuclear DNA content and texture. These differences allowed the two groups of nuclei to be distinguished with 78% accuracy. The two groups of lesions were distinguished with 94% accuracy, using neural network analysis.

Cell Nucleus↗

Progression of mycosis fungoides is associated with increasing cutaneous expression of interleukin-10 mRNA.

Cytokines are believed to play an important role in the pathogenesis of cutaneous T cell lymphoma. Data regarding the local cytokine pattern in mycosis fungoides (MF) are partly conflicting. Recent studies have suggested a shift from type 1 to type 2 cytokine pattern because IL-4 and IL-5 mRNA have been more frequently detected in lesions of advanced stages. Another study has described a type 1 cytokine pattern in MF lesions. None of the previous studies of cytokine mRNA expression in MF, however, used quantitative methods, and therefore only the presence of a cytokine, but not the level of expression, could be determined. To gain better insight into the development of cytokine pattern during tumor progression we used semiquantitative reverse transcriptase-polymerase chain reaction to analyze cytokine mRNA expression in MF skin lesions at different stages. Biopsies from patients with patch (n = 11), plaque (n = 6), and tumor (n = 3) stage MF were compared with biopsies from patients with pleomorphic T cell lymphoma (n = 5), psoriasis (n = 7), atopic dermatitis (n = 5), and nonlesional skin (n = 8). MF progression was associated with significantly higher IL-10 and lower interferon-gamma mRNA expression. Moreover, the stage-dependent increase in IL-10 mRNA expression was also found in paired samples from individual patients. Unlike in pleomorphic T cell lymphoma, however, typical T helper 2 cells did not seem to be the source of increasing IL-10 in advanced MF, because stage-independent IL-4 mRNA was rarely detected, suggesting contribution of nonlymphoid cells to local IL-10 production. The overexpression of IL-10 in MF may be of importance for tumor progression, because this immunosuppressive cytokine might be involved in downregulation of immunologic tumor surveillance.

Disease Progression↗

Localization of human T-cell lymphotropic virus-1 tax proviral sequences in skin biopsies of patients with mycosis fungoides by in situ polymerase chain reaction.

The histopathologic diagnosis of mycosis fungoides (MF), even when clinical manifestations of the disease seem convincing, is often tenuous. The observation that practically all patients with MF harbor human T cell lymphotropic virus type I (HTLV-I) proviral sequences in their circulating lymphocytes raised the possibility that such viral footprints could be detected in their cutaneous infiltrates. Application of in situ polymerase chain reaction (PCR) to skin biopsies of 11 of 12 patients demonstrated this assumption to be correct. In addition, cells suspected to be keratinocytes were also positive. None of 10 skin biopsies from a variety of sources used as controls, nor 3 lymph node biopsies from patients with B-cell lymphomas, showed any HTLV proviral sequences on in situ PCR. On the basis of these observations, it is concluded that in situ PCR carried out on skin biopsies of patients with presumptive MF may help to established the diagnosis.

Genes, pX↗

T-cell receptor gene rearrangement in canine mycosis fungoides: further support for a canine model of cutaneous T-cell lymphoma.

Canine cutaneous T-cell lymphoma (CTCL) is a morphologic and immunophenotypic simulant of human mycosis fungoides (MF) characterized by an infiltrate of atypical, hyperconvoluted, epidermotropic T cells. To further support our hypothesis that canine MF is a useful model for the study of human CTCL, we have used Southern blotting to search for clonal T-cell proliferations in canine MF. Cellular DNA was extracted from normal dog buffy coat cells (n = 8), lesional canine MF skin (n = 8), canine MF buffy coat cells (n = 7), normal dog skin (n = 3), and normal human buffy coat cells (n = 5), digested with a panel of restriction enzymes and Southern blotted onto nylon membranes. All cases of canine MF were also immunophenotyped with anti-canine monoclonal antibodies to CD4, CD8, CD18, CD45RA, canine class II, T-cell activation antigens, and pan-B-cell antigens. Normal dogs gave reproducible digestion patterns in blood and skin, which differed from the human germline patterns when probed with a human T-cell receptor (TCR), beta chain constant region (C beta) cDNA. Common germline bands between the species included the 3.5-kb Eco RI, 3.4-kb Bam HI, 5.4-kb Sac I. These results confirmed that the TCR-beta gene is evolutionarily conserved between dog and man. Immunostaining revealed that 3/7 cases were CD4+ canine CTCL and 4/7 were CD8+ canine CTCL. Rearranged bands, deletion of germline bands, as well as minor alterations in electrophoretic mobility were observed in lesional DNA from seven of eight cases of canine MF, with at least two restriction digests in each case. Dog rearrangements were best detected with Bgl II, Eco RI, Eco RV, and Sac I, whereas deletions were detected with Bgl II, Sac I, Eco RV, and Bam HI. These studies demonstrate the presence of clonal TCR rearrangement in canine MF, further supporting the similarity of this tumor to human MF and its role as an animal model of CTCL.

Animals↗

Differences in IL-2 receptor levels between mycosis fungoides and cutaneous type adult T-cell leukemia/lymphoma in the early stages of the disease.

Skin lesions in mycosis fungoides (MF) and cutaneous-type adult T-cell leukemia/lymphoma (c-ATL) are sometimes difficult to differentiate. Our previous investigations showed that the alpha-chain of interleukin-2 receptor (IL-2R alpha) secretion is enhanced in both acute and chronic type ATL. This finding suggested increased levels of IL-2R alpha in c-ATL that might differentiate it from MF. In this study, we report several observations on IL-2R alpha levels in MF and c-ATL. 1) Plasma levels of IL-2R alpha determined by enzyme-linked immunosorbent assay were higher in patients with c-ATL (tumor group, 17,571 +/- 5,919 U/ml; non-tumor group, 4,546 +/- 1,788 U/ml) than those of patients with MF (tumor group, 2,352 +/- 861 U/ml; plaque group, 530 +/- 174 U/ml). 2) The percentages of IL-2R alpha-positive cells in skin lesions of c-ATL were 30.5 +/- 13.4% in the non-tumor group and 62.7 +/- 12.0% in the tumor group, which were significantly higher than those of MF, plaque group (12.7 +/- 5.9%), and tumor group (3.4 +/- 2.3%). 3) Further, there was a significant correlation between plasma IL-2R alpha levels and the number of IL-2R alpha-positive cells in skin lesions in patients with c-ATL (r = 0.654, p < 0.0001) and MF (r = 0.537, p < 0.01) on an individual basis; whereas no increased IL-2R alpha levels in peripheral blood mononuclear cells were observed in patients with either MF or c-ATL. 4) Skin lesions of c-ATL had much higher levels of IL-2R alpha genomic RNA than those of MF in their initial stages when measured by DNase/reverse transcription/polymerase chain reaction and liquid hybridization analysis. These findings suggest that increased plasma IL-2R alpha levels in patients with either MF or c-ATL may be linked to cellular IL-2R alpha levels in the skin lesions, and thus reflect the difference between MF and c-ATL.

Adult↗

Mycosis fungoides exhibits a Th1-type cell-mediated cytokine profile whereas Sezary syndrome expresses a Th2-type profile.

We determined T-cell cytokine profiles in the epidermis, dermis, and blood of cutaneous T-cell lymphoma to differentiate whether unique cytokine profiles were associated with mycosis fungoides (MF) versus Sezary syndrome. Punch biopsy specimens from plaque stage MF (n = 7) were compared to Sezary skin (n = 3) after undergoing rapid heat-saline separation of epidermis from dermis. Normal adult skin (n = 11), neonatal foreskin (n = 4), untreated psoriatic plaques (n = 6), and normal donor peripheral blood leukocytes (n = 3) were studied as controls. Total RNA was extracted from all skin specimens, as well as peripheral blood leukocytes from MF (n = 3) and Sezary patients (n = 7), and was converted to cDNA by reverse transcriptase. Polymerase chain reaction amplification of cDNAs using interleukin 2 (IL-2), IL-4, IL-5, IL-10, and interferon gamma-specific primers was used to differentiate Th1-type responses (IL-2+ and interferon gamma +) from Th2-type responses (IL-4+, IL-5+, and IL-10+). beta-actin specific primers were included as a positive control for mRNA integrity. All MF specimens contained mRNAs for IL-2 and interferon gamma limited to epidermis but not IL-4, IL-5, or IL-10. In contrast, Sezary skin and blood showed a cytokine mRNA pattern dominated by IL-4, IL-5, and IL-10. MF blood showed a pattern similar to normal peripheral blood T cells with mixed detection of all T-helper cell cytokine mRNAs. All psoriasis samples contained mRNAs for IL-2 and interferon gamma in both epidermis and dermis with no IL-4 or IL-10 in either compartment. These findings demonstrate that the cutaneous lesions of MF are characterized by an epidermal Th1-type cytokine profile, whereas both the blood and skin of patients with Sezary syndrome is characterized by a Th2-type profile. This work suggests that differences in cytokine production may be related to the pathophysiology and clinical presentation in cutaneous T-cell lymphoma.

Base Sequence↗

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.

Base Sequence↗

Electron microscopic identification of Langerhans cells in the dermal infiltrates of mycosis fungoides.

The importance of the physical interaction between epidermal Langerhans cells and malignant cells in mycosis fungoides (MF) has been stressed recently. To further evaluate this concept, electron microscopic study was carried out to identify whether or not Langerhans cells are also present in the dermal infiltrates of MF. The cells with characteristic Langerhans cell-granules were found in the dermal infiltrates, from early patch to tumor lesions of MF. No vacuolous change suggestive of cell degeneration was found in these cells. These Langerhans cells revealed only a small number of specific granules, and nuclear cleavage and chromatin pattern similar to those of malignant lymphoid cells, thus hardly differentiating 2 cell types in a low power view. Similar to epidermal MF lesions, however, a close apposition of dermal Langerhans cells with malignant lymphoid cells was seen. In one occasion, one Langerhans cell was present at the epidermodermal junction, appearing as being in the process of crossing over the basal lamina. Inasmuch as malignant cells in MF have been shown to possess the functional properties of helper T cells and receptors for Ia (immune associated antigen), expressed in the epidermis solely on Langerhans cells, our findings may further suggest that these malignant T cells bind to Langerhans cells through a "lock and key" mechanism involving membrane-bound Ia.

Cytoplasmic Granules↗

Lymphocyte function and chromosome aberrations in patients with early mycosis fungoides and parapsoriasis en plaques.

Thirteen patients with stage I or II mycosis fungoides (MF) and 10 patients with large-plaque parapsoriasis en plaques (PEP) were examined for immunologic and cytogenetic disturbances. Total lymphocyte counts and immunoglobulin concentrations in the blood were normal. In vitro lymphocyte responses to polyclonal activators and various antigens in standard concentrations were normal. However, titration of phytohemagglutinin and concanavalin A (ConA) disclosed significantly lowered responses to suboptimal concentrations in the patient group, most pronounced in patients with MF II. ConA-induced leukocyte migration inhibitory factor (LIF) production, tested in an indirect leukocyte migration inhibitory assay, was low in the patient group. Furthermore spontaneous LIF production in vitro and small amounts of serum LIF were demonstrated in a few patients. The chromosomal banding pattern, sister chromatid exchange, and break frequency were within normal limits except for 3 translocations in the MF group. It is concluded that even in early-stage MF a pathologic function of blood lymphocytes can be demonstrated, when sensitive methods are applied. The findings might be important for monitoring disease activity and effect of treatment.

Adult↗

Mycosis fungoides: clinical course and cellular abnormalities.

Mycosis fungoides is a lymphoma that appears to begin in skin. Although variable in its clinical course, it tends to affect middle-aged people, is present for a median of 4 yr before diagnosis, and generally results in death of the patient 4 to 5 yr after diagnosis. Cutaneous tumors and palpably enlarged lymph nodes are associated with shortened survival, and each of these frequently is accompanied by extracutaneous dissemination of the disease. The malignant cells are T lymphocytes with highly infolded nuclei, and they frequently express a helper cell function. Their chromosome complement has been found to be abnormal in karyotyping studies, and measurement of the amount of DNA by cytophotometry may permit diagnosis of the disease before the histological characteristics are detectable by light microscopy.

Adult↗

A technique for immunoultrastructural identification of T6-positive Langerhans cells and indeterminate cells in mycosis fungoides.

An immunoelectron microscopic method was developed using T6 antiserum in an immunoperoxidase technique to label the dendritic cells of the epidermis in mycosis fungoides (MF). The technique allows simultaneous identification of intracellular Birbeck granules and T6 membrane positivity. Ultrastructural examination of the epidermal infiltrate of MF, using this method, confirmed the T6-positive nature of Langerhans cells and indeterminate cells. All Sézariform and mature lymphocytes were T6-negative and a small population of T6-negative histiocytic cells were also observed.

Horseradish Peroxidase↗

Association of HLA-DR5 with mycosis fungoides.

Mycosis fungoides (MF) and Sézary syndrome (SS) are uncommon neoplasms of the lymphoreticular system with distinct clinical, histologic, and immunologic features. Based on the thymus-derived nature of the neoplastic cells, MF and SS are both classified as cutaneous T-cell lymphoma. While substantially greater understanding of MF and SS has been made possible, the exact mechanism for the initiation of either disease is still unknown. The possible involvement of environmental factors as well as viral etiology, i.e., retroviruses, has been suggested. In order to investigate the possible role of HLA-associated variations in genetic susceptibility, 74 patients with histologically documented MF were typed for HLA-A, -B, and -C antigens. Half of these patients were also typed for HLA-DR antigens. An increase in DR5 was the only statistically significant deviation in HLA antigen frequencies in these patients (53% in MF as compared with 20% in controls). An increased frequency of HLA-DR5 has also been associated with scleroderma and juvenile rheumatoid arthritis both of which have immunologic alterations. Also HLA-DR5 has been associated with renal cell carcinoma and Kaposi's sarcoma. The association of MF with DR5 suggests that some individuals with the DR5 antigen may be at higher risk for virally initiated and/or neoplastic diseases possibly through an HLA-linked defect in the immune system.

Adenocarcinoma↗

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.

Blotting, Southern↗

Phenotypic and functional characterization of tumor infiltrating lymphocytes in mycosis fungoides: continuous growth of CD4+ CD45R+ T-cell clones with suppressor-inducer activity.

Tumor-infiltrating lymphocytes (TIL) were obtained by mechanical release from a solitary rapidly grown tumor of a patient with mycosis fungoides. The preparations separated by density gradient centrifugation contained a major portion of CD3+ CD8+ WT31+ CD5- large T-cell blasts and a minor portion of non-blastic TIL predominantly of the CD3+ CD4+ phenotype. Using cDNA-probes for the constant region of the T-cell receptor beta-genes, the large cell fraction was identified as tumor by its distinct monoclonal rearrangement. TIL were expanded by culture in recombinant interleukin 2 and cloned by limiting dilution. Phenotypic analysis of expanded TIL and two clones further analyzed in more detail showed CD3+, CD4+, CD8-, and 2H4+ (CD45R+) expression. Cloned and uncloned TIL showed no NK and LAK activity, no proliferative response, and no cytotoxic activity against autologous tumor cells. These cells were unable to suppress the proliferative response of alloreactive T-cell clones stimulated by B-lymphoblastoid cell lines (i.e., they had no suppressor-effector activity), but strongly suppressed proliferation responses in allogeneic mixed lymphocyte culture (i.e., they most likely had suppressor-inducer activity). This was not the case when irradiated tumor cells were added. The present results demonstrate continuous in vitro growth of CD4+ and 2H4+ T-cell clones with suppressor-inducer activity obtained from TIL, and indicate that a subpopulation of TIL may down-regulate immune responses which may lead to suppression of antitumor immunity.

Antigens, CD↗

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) induces apoptosis in Mycosis fungoides/Sézary syndrome cells.

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) can serve as a ligand for the peroxisome proliferator activator receptor-gamma (PPAR-gamma) and has been shown to inhibit cell proliferation, and to induce differentiation and apoptosis in tumor cell lines. Bexarotene is an RXR-selective retinoid that can induce apoptosis of mycosis fungoides (MF) and Sézary syndrome (SS) cells. Since the PPAR-gamma and RXR receptors can form heterodimers, we studied the effects of CDDO and its synergism with bexarotene on apoptosis in MF/SS cell lines (MJ, Hut78, and HH) and freshly isolated peripheral blood lymphocytes (PBL) from SS patients with circulating atypical T cells (CD4+CD26-). CDDO treatment at 1-5 microM for 48 h caused a concentration-dependent apoptosis in three MF/SS cell lines and patients' PBL compared to vehicle controls. Bexarotene augmented CDDO-induced apoptosis in these cells. PPAR-gamma was expressed but decreased by 47% in MJ, 42% in Hut78, and 77% in HH cells following CDDO treatment. The anti-apoptotic protein bcl-xL, but not bcl-2, was decreased by 69% in MJ, 31% in Hut78, and 59% in HH cells and caspase-3 was activated following CDDO treatment. Interestingly, the PPAR-gamma antagonist T007 did not block CDDO-induced apoptosis, and the more potent PPAR-gamma agonist rosiglitazone required much higher concentrations (>50 microM) than CDDO to induce apoptosis in MF/SS cells. In summary, CDDO induces apoptosis that is further enhanced by bexarotene and decreases the PPAR-gamma and bcl-xL proteins in MF/SS cells. CDDO's effects on MF/SS cells may be at least partly mediated through a PPAR-gamma-independent mechanism. Our findings suggest the rationale for further investigation of the clinical potential of CDDO, either alone or in combination with bexarotene for MF/SS patients.

Anticarcinogenic Agents↗

Establishment of a mouse xenograft model for mycosis fungoides.

Mycosis fungoides (MF) is the most frequent variant of cutaneous T-cell lymphomas (CTCLs). MF primarily involves the skin initially with patches and plaques. In later stages, cutaneous tumors develop and tumor cells may spread to lymph nodes and finally to visceral sites. Here, we describe an animal model for MF in immune-deficient nude mice, using the CTCL cell line MyLa. Subcutaneous transplantation of MyLa cells leads to the formation of cutaneous tumors in 80% of the mice (50/60 total). Spread of tumor cells to visceral sites was detected by immunohistochemistry and polymerase chain reaction (PCR)-based detection of specific T-cell receptor-gamma rearrangement. MyLa cells were found circulating in the blood, lymph nodes, and in blood vessels of heart, kidney, lung, and liver. In lung and liver tissue, tumor cells presented perivascular invasion, but no large secondary tumors developed. The nude mouse model described here will be a valuable test system for new therapeutic approaches for the treatment of MF and opens the unique opportunity to study the disease in vivo.

Animals↗

Cutaneous T cell lymphoma differing from classical mycosis fungoides and Sézary syndrome: clinical, histological and immunohistochemical studies of six cases.

Six cases of cutaneous T cell lymphoma (CTCL), differing from classical mycosis fungoides (MF) and Sézary syndrome (SS), were studied. Initial skin eruptions were erythematoplaques, papules and tumors. Some cases showed frequent spontaneous regressions without any specific therapy; however, the prognosis was not always good. Surface phenotypes of neoplastic cells were markedly heterogeneous from the onset of initial skin eruption. Three cases expressed the T helper/inducer (Th/i) phenotype, one case had both the Th/i and T suppressor/cytotoxic phenotypes, and the other two cases lacked both surface markers. These results suggest that CTCL other than MF and SS displays marked heterogeneous clinical behavior and diverse morphological and immunophenotypical characteristics of the postthymic T cells.

Aged↗

Towards a better understanding of cellular events in Mycosis fungoides: an immunohistochemical study.

In the present work, we have attempted to pay special attention to epidermal and dermal cellular events in Mycosis fungoides (MF). In the epidermis, the two dendritic cell populations, CD1+ and OKM5+, HLe-1+, the adhesion molecules OKM5 and DR on the surface of epidermal cells (Ecs), and cytoskeletal proteins were studied. CD1+ dendritic epidermal cells were generally more abundant than OKM5+, HLe-1+ ones with no interrelationship in their presence. OKM5+, HLe-1+ dendritic cells prevailed in the second stage plaques. The staining pattern with anti-spectrin polyclonal antibody gradually presented increased intensity from Pre-MF to tumor stage. The activation-proliferation related immunophenotype was also examined, as well as other useful markers, in an attempt to correlate their presence in different stages of MF. Special emphasis was given to the staining pattern with the epidermal growth factor receptor (EGF-R) monoclonal antibody against three targets: epidermal, endothelial, and lymphoid cells. The presence of Ki-67 proliferation marker in suprabasal epidermal layers and lymphoid cells in the dermal infiltrate was also of interest.

Cell Division↗