Genetic markers associated with progression in early mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is a rare, but potentially devastating malignancy. It classically presents with cutaneous patches and plaques and can progress to tumours on the skin with lymph node, blood and visceral involvement. While most patients with MF have a relatively benign disease course, a subset of patients will develop progressive disease that is often fatal. OBJECTIVE: The aim of this study was to identify genetic markers in early MF limited to the skin (stages IA-IIA) that distinguish those patients who will have progressive disease from those who will not, so that early appropriate treatment may be instituted. METHODS: The study includes 18 patients who were diagnosed with early stage MF at the time of biopsy and had follow-up to determine which patients developed progressive disease. RNA was extracted from skin biopsy specimens and analysed for expression of CD3, FOXP3, IFNγ, Interleukin (IL)-4, IL-13, KIR3DL2, MICB, PLS3 and STAT4 by quantitative real-time polymerase chain reaction. RESULTS/CONCLUSIONS: Reduced expression of FOXP3 and STAT4 and increased expression of IL-4 relative to CD3 expression levels were significantly associated with MF progression. Further studies will be needed to fully assess the usefulness of these genetic markers to predict disease progression and guide treatment options in patients diagnosed with early MF.

Safety of a new, single, integrated, closed photopheresis system in patients with cutaneous T-cell lymphoma.

BACKGROUND: Extracorporeal photopheresis (ECP) is an approved palliative treatment for cutaneous T-cell lymphoma (CTCL). The THERAKOS CELLEX (continuous flow separation) system (Therakos, Exton, PA, U.S.A.) has been developed from the current THERAKOS UVAR XTS system. It is designed to reduce treatment times and extracorporeal volumes, and allows the use of either a single- or a dual-needle configuration. OBJECTIVES: To assess the safety of the THERAKOS CELLEX system to provide ECP for patients with CTCL. METHODS: Patients received ECP with the THERAKOS CELLEX system for up to 6 months. The treatment schedule was defined by their current treatment and response to ECP. At least 150 treatments were required to assess safety of the new system. Safety was assessed using reports of adverse device effects (ADEs) and unanticipated ADEs (UADEs), device malfunctions and defects, vital signs, laboratory parameters and physical examinations. RESULTS: Thirteen patients were enrolled and 12 completed the study; 155 ECP treatments were initiated and 153 completed. There were no ADEs or UADEs reported during the study. The mean treatment time was shorter for patients who received dual- compared with single-needle treatments (74.4 vs. 103.0 min, P < 0.0001) and the extracorporeal volume was lower (216 vs. 266 mL). CONCLUSIONS: This new ECP system provides lower extracorporeal volumes, faster treatment times, and flexibility to use either single- or dual-needle access, while not being associated in this study with any ADEs, and therefore having a positive benefit-risk ratio for patients with CTCL.

Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing.

To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16 promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target the epigenetically silenced tumor suppressor genes.

Association of the human type C retrovirus with a subset of adult T-cell cancers.

To determine whether the human T-cell lymphoma-leukemia virus (HTLV) is associated with particular cancers, patient sera were surveyed for HTLV-specific antibodies. An association was seen with aggressive cancers of mature T-cells, specifically Japanese adult T-cell leukemia (ATL) and T-cell lymphosarcoma cell leukemia (TLCL), a similar cancer of Caribbean blacks. Ninety to 100% of these patients possessed HTLV-specific antibody. Forty-seven and 20% of relatives of ATL and TLCL patients, respectively, and 12 and 4% of healthy donors from ATL and TLCL endemic areas were also antibody positive. Visceral organ involvement, hypercalcemia, and skin manifestation, features of ATL and TLCL, were often seen in other antibody-positive patients. Childhood cancers, most cutaneous T-cell and all non-T-cell leukemias and lymphomas, myeloid leukemias, Hodgkin's disease, and solid tumors were not associated with HTLV. Healthy United States donors and European patients with non-malignant diseases were antibody negative. HTLV is thus associated with a subtype of adult T-cell leukemia-lymphoma, clustered in viral endemic areas, with apparent racial and geographic predilection.

Aberrant cytokine production by Sezary syndrome patients: cytokine secretion pattern resembles murine Th2 cells.

Sezary syndrome (SzS), the leukemic stage of cutaneous T-cell lymphoma (CTCL), is typically a CD4+ T-cell lympho-proliferative disease characterized by numerous immunologic abnormalities, including decreased T-cell responses to antigens and mitogens, decreased natural killer and lymphocyte-activated killer cell activities, eosinophilia, and increased levels of immunoglobulins, particularly IgE and IgA. Because this constellation of abnormalities is reminiscent of the pleiotropic in vitro activities of IL-4 and IL-5, we examined the possibility that malignant T cells in SzS may be producing increased amounts of IL-4 with a concomitant decrease in IL-2 and IFN-gamma production. Such a cytokine secretion pattern would be similar to that produced by murine Th2 cells. Serum IL-4 enzyme-linked immunosorbent assay measurements revealed that 33% of SzS patients (n = 21) had levels of IL-4 significantly higher (mean, 7.2 pg/ml; range, 0-48, p less than 0.05) than normal controls (mean, 1.59; range, 0-3.1). Although the majority of tested patients had elevated serum IgE, no direct correlation between serum IL-4 levels and serum IgE levels was observed. Peripheral blood mononuclear cells (PBMC) isolated from SzS patients and stimulated with phytohemagglutinin (PHA) produced significantly higher levels of interleukin (IL)-4 and significantly lower levels of IL-2 and interferon (IFN)-gamma than did PBMC from normal controls (p less than 0.02, p less than 0.05, and p less than 0.02, respectively). PBMC from SzS patients in remission produced IL-4 and IL-2 levels similar to that of the normal controls. To determine if IFN-gamma could inhibit the increased IL-4 production by PHA-stimulated PBMC from SzS patients, IFN-gamma was added to culture at 0, 24, or 48 h prior to the addition of PHA. Significant decreases in IL-4 production were seen only in cultures incubated with IFN-gamma for 24 and 48 h prior to the addition of PHA. IL-2 levels were not affected by IFN-gamma incubation. The increased IL-4 and decreased IL-2 and IFN-gamma production by PBMC from SzS patients suggests that Sezary cells have a cytokine profile similar to murine Th2 cells. Moreover, this cytokine secretion pattern may play an integral role in the immunopathogenesis of advanced CTCL. The results also suggest that IFN-gamma be a useful component in the spectrum of therapeutic approaches to SzS.

Natural cell-mediated cytotoxicity in cutaneous T-cell lymphomas.

Natural cell-mediated cytotoxicity was studied in 24 patients with cutaneous T-cell lymphomas and in 18 age- and sex-matched controls studied concomitantly. Percent cytotoxicity was determined by 4-h 51Cr release assay using K562 targets at effector to target ratios of 100:1, 50:1, and 25:1. Mean percent cytotoxicity was significantly lower in patients than in controls at an effector to target cell ration of 100:1. Likewise, decreased cytotoxicity was found at effector to target ratios of 50:1 and 25:1, although this difference was not significant. When natural killer activity was analyzed separately for males and females, cytotoxicity was lower in both, although the decrease was significant only for male patients. Impairment of natural killer activity did not correlate with blood zinc levels, but appeared to correlate with stage of disease.

Clonal characteristics of cutaneous T cell lymphomas: cytogenetic evidence from blood, lymph nodes, and skin.

Chromosome studies were done on mitogen-stimulated lymphocytes from one or more tissues (blood, lymph nodes, skin lesions) in 15 patients with mycosis fungoides or Sézary syndrome. A cytogenetically abnormal clone was found in 10 individuals, including 6 with data from several tissues. In 4 cases the same aberrant clone was identified in a skin lesion as well as in blood and/or lymph node. The abnormal chromosome patterns ranged from hypodiploid to hypertetraploid, and there was no evidence of unrelated karyotypically-altered lines at different sites. A 6q- chromosome, previously reported in acute lymphocytic leukemia, was found in 2 patients. The results support the concept that cutaneous T cell lymphomas (CTCL) are clonal disorders, presumably unifocal in origin, with the skin lesions populated by cells from the same neoplastic clone that involves lymph nodes and blood.

Normal T-lymphocyte function in psoriatic patients undergoing methoxsalen photochemotherapy.

Peripheral blood lymphocytes from psoriatic patients undergoing photochemotherapy using oral 8-methoxypsoralen and high-intensity long-wave ultraviolet radiation, were isolated and their ability to respond to phytohemagglutinin and to form spontaneous rosettes with sheep red blood cells was measured in vitro. Seventeen patients were investigated who had received 200 to 3700 Joules/cm2 of ultraviolet radiation over a 6- to 33-month period. No significant defects in lymphocyte blastogenesis or E-rosette formation were detected in these patients.

The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis.

This study was undertaken to determine the clonality of lymphomatoid papulosis (LyP), its clonal relationship to lymphomas, which occur at high frequency in LyP patients, and to define the cell lineage of Reed-Sternberg-like cells in type A lesions of LyP. Punch biopsies of skin of 11 adult patients with LyP were analyzed for morphologic subtype of LyP, surface antigens, and clonal T-cell receptor (TCR) gene rearrangements. Clonal rearrangements were identified by semiquantitative polymerase chain reaction amplification and sequencing of TCR-beta chain genes in nine patients and TCR-gamma chain genes in two patients. A single dominant clone was detected in multiple separate LyP lesions, often of different histologies, in nine patients. The same clone was detected in LyP lesions and the anaplastic large cell lymphoma (ALCL) of 2 patients and the mycosis fungoides (MF) of 2 other patients. No dominant clone could be detected in one patient with LyP uncomplicated by lymphoma or in a second patient with LyP and MF. A T-cell lineage was evident for RS-like cells in cell culture and in type A lesions. These results show that multiple regressing skin lesions and associated T cell lymphomas (MF and ALCL) are clonally related in most LyP patients, which suggest that the disease in these patients was initiated by a non-random genetic event.

Biophysical characterization of normal T-lymphocytes and Sézary cells.

Blood lymphocytes from 18 patients with cutaneous T-cell lymphoma (Sézary syndrome and mycosis fungoides) were characterized using multiparameter laser flow microfluorimetry (FMF) and automated image analysis (AIA) and the results correlated with routine blood smears, cytogenetic studies and observations made on PHA-stimulated normal T-lymphocytes in vitro. Specimens from all 9 patients with Sézary syndrome and 5 of 9 patients with mycosis fungoides contained one or more discrete subpopulations of neoplastic (Sézary) lymphocytes that were detected by FMF. Studies with AIA demonstrated that neoplastic T-lymphocytes are distinguished from normal quiescent (G0) lymphocytes not only by alterations in DNA content (aneuploidy) but also by chromatin structuring (increased chromatin dispersion), which may be a more sensitive index of neoplastic transformation than ploidy levels. In several patients, small and large Sézary cells were present with DNA-chromatin properties quite similar to normal cycling G1 and G2 lymphocytes respectively, but their presence was not explained by an increase in proliferative activity in the blood. These findings indicate that Sézary syndrome consists of a heterogeneous group of related disorders differing in terms of the Sézary cell population. The response to treatment and prognosis may differ accordingly.

A clonal CD4-positive T-cell line established from the blood of a patient with Sézary syndrome.

The reported inability to establish long-term T-cell lines from the blood of cutaneous T-cell lymphoma patients with circulating neoplastic T cells has hindered the development of an in vitro system to investigate Sézary syndrome. We have established a rapidly proliferating T-cell line from the peripheral blood of a patient with Sézary syndrome, which expresses a mature helper T-cell phenotype and contains cytogenetic abnormalities and T-cell receptor gene rearrangements identical to those in the patient's blood. The method of establishment and characteristics of this line are described.

DNA-cytophotometry of lymph node touch imprints in cutaneous T-cell lymphoma.

Scanning DNA-cytophotometry was performed on touch imprints of 26 lymph nodes (LN) obtained from 25 patients with cutaneous T-cell lymphoma (CTCL), stained by the Feulgen technique, and interpreted without knowledge of histopathologic diagnosis. Four patterns of DNA distribution were identified, but only histograms that demonstrated cells containing nuclei with more than 4C DNA content (hypertetraploidy) reliably distinguished LN involved with CTCL from LN with reactive changes; for example, dermatopathic lymphadenitis. An abnormal DNA histogram with evidence of hypertetraploidy was demonstrated in 9 of 12 LN showing histopathologic evidence of involvement compared with no abnormal histograms in 14 LN without histopathologic involvement. One LN that was diffusely involved with CTCL had a DNA distribution characteristic of a relatively high level of cell proliferation, but without definite hypertetraploidy. Cytogenetic studies on the blood of this patient, who had Sézary syndrome, demonstrated a clone of lymphocytes with a pseudodiploid karyotype without a related polyploid subline. The remaining two histopathologically involved LN had normal DNA histograms; these LN were only focally involved with CTCL. These observations indicate that DNA-cytophotometry correlates well with the histopathologic findings in LN diffusely involved with CTCL, but may be normal in LN with focal involvement or in those that contain cytogenetically abnormal cells with a near-diploid DNA content.

Variable CD7 expression on T cells in the leukemic phase of cutaneous T cell lymphoma (Sézary syndrome).

CD7, a molecule normally expressed on 90% of normal CD4+ T cells, is often deficient on the malignant T cells of cutaneous T cell lymphoma. To investigate the clinical and biologic implications of CD7 expression, blood lymphocytes from 42 patients with the leukemic phase of cutaneous T cell lymphoma (CD4/CD8 ratio of 10 or more with evidence of a T cell clone in the blood) were analyzed for level of expression of CD7 by flow cytometry. CD7 expression by cells did not clearly segregate into two distinct subgroups that are either CD7 positive or CD7 negative as generally thought; however, nine of 17 patients with a predominantly CD4+CD7+ tumor population on early studies became CD4+CD7- over time whereas the converse situation was not observed. In addition, of three patients with evidence of large tumor cells in the blood coexisting with smaller cells, discordant CD7 expression was observed in one instance. In lymph node specimens, the percentage of cells expressing CD7 and other T cell markers did not correlate with histologic evidence of involvement. CD7 expression on blood lymphocytes also did not correlate with patients' survival nor to serum IgE levels or blood eosinophil counts, a finding suggesting that this marker does not identify functional cell subsets that produce serum interleukin-4 or -5, respectively. We speculate that the level of CD7 expression on malignant T cells may be the effect of sustained antigen stimulation in vivo analogous to what has been proposed to occur with normal T cells during aging.

The prognostic significance of delayed hypersensitivity to dinitrochlorobenzene and mechlorethamine hydrochloride in cutaneous T cell lymphoma.

Recent studies suggest that cells elaborating type 1 cytokines are important mediators of anti-tumor cell-mediated immunity in cutaneous T cell lymphoma. Type 1 cell-mediated immune responsiveness was assessed in 276 patients with cutaneous T cell lymphoma (mycosis fungoides and Sézary syndrome) using 2,4-dinitrochlorobenzene (DNCB) skin testing as part of the initial evaluation. The overall rate of sensitization after one and two DNCB challenges was 32% and 67%, respectively, which is much decreased compared with the expected rate of more than 95% for normal individuals. Moreover, the frequency of DNCB sensitization and allergic contact dermatitis to topically applied mechlorethamine decreased with advancing stage of disease. In addition to the expected strong correlation with stage, we observed that patients who were DNCB test positive were significantly less likely to experience disease progression and had a better overall prognosis compared with DNCB-negative patients. These results support the concept that cell-mediated responses are important in cutaneous T cell lymphoma, and that augmentation of these responses would be therapeutically beneficial.

Combined moderate dose electron beam radiotherapy and topical chemotherapy for cutaneous T-Cell lymphoma.

Between October, 1978 and June, 1979, nine patients with biopsy-proven cutaneous T-Cell lymphoma were treated with combined total-skin electron beam radiation (TSEB) and topical chemotherapy. TSEB was administered using 3.8 MeV electron and dual exposure technique. All patients received skin dose of 400 rad once weekly to a total dose of 2000 to 2400 rad followed by topical chemotherapy with mechlorethamine hydrochloride (HN2) two to four weeks after completion of radiation. A complete response followed TSEB in seven of nine patients, but a relapse of disease activity has subsequently occurred within the first year for all the patients despite adjunct therapy, except for one patient who remains disease free for more than 21 months. Generalized severe erythema developed during or shortly after completion of radiation in six of nine patients, with blistering at the overlapping treatment fields and body folds in four patients. In addition four patients developed diffuse permanent telangiectasia of skin and one patient developed linear sclerosis, telangiectasis and painful ischemic ulceration on the fingertips two years after completion of electron beam therapy. Most patients had evidence of mild depression of lymphocyte responsiveness to Phytohemagglutinin after TSEB. Our conclusion is that the short-term benefits and convenience of this particular technique do not justify the acute and chronic toxicity encountered.

Total skin electron beam and total nodal irradiation for treatment of patients with cutaneous T-cell lymphoma.

Sixteen patients with advanced cutaneous T-cell lymphoma (CTCL) with or without lymph node involvement, but without evidence of extranodal manifestations, were treated with a combination of total skin electron beam therapy (TSEB) and total nodal irradiation (TNI). Fourteen (87%) patients achieved a complete response (CR) lasting from 1 to 84+ months (median, 8+ months) from the completion of treatment. The best results occurred in 6 patients with pretumorous intracutaneous CTCL (Stages IB and IIA) where the CR has lasted in all patients from 8 to 84+ months (median about 27+ months). Conversely, a long-term CR occurred in only one of five patients with tumor-phase intracutaneous CTCL (Stage IIB) and in none of the 5 patients with histopathologically proven nodal involvement (Stage IVA). Radiotherapy was well tolerated with the major toxicity being bone marrow suppression. We conclude that combined TSEB and TNI is a relatively safe and effective treatment for patients with CTCL prior to the development of lymph node involvement. Long-term follow-up is needed to assess the curative potential of this treatment.

The radiation therapy of early stage cutaneous T-cell lymphoma.

Results of different radiotherapy schedules used for early stage (T1-2, N0-1, M0) cutaneous T-cell lymphoma (CTCL) are compared in a series of 45 patients (22 patients treated with high dose total skin electron beam therapy (TSEB) with curative intent, 18 patients treated with palliative radiotherapy, and 5 patients treated with high dose local electron beam). At 3, 5, and 10 years after diagnosis the high dose TSEB treatment group had a probability of overall survival of 91%, 86%, and 75%, respectively, compared with 94%, 88%, and 88% for the palliative treatment group. The complete response (CR) rate for the high dose TSEB treatment group was 82% (18/22), compared with a 57% (4/7) complete response rate for seven patients in the palliative group who received low dose TSEB (less than 25 Gy in 6-7 weeks) followed by daily application of topical mechlorethamine hydrochloride (HN2). However, the probability of continued remission at 3, 5, and 10 years was 44%, 44%, and 33%, respectively, for the high dose TSEB group and 25%, 25%, and 0%, respectively, for the low dose TSEB + HN2 group. The median disease-free survival was 17.5 months for the high dose TSEB group versus 5.5 months for the low dose TSEB + HN2 group. The five patients who were treated with high doses of local electrons to a single local field had an overall survival rate of 80%, a median survival rate of 64 months, and a median length of continued remission of 31 months. These results indicate that high-dose electron beam can result in long-term disease-free survival in patients with localized and limited extent skin involvement with cutaneous T-cell lymphoma.

Total skin electron beam and total nodal irradiation of cutaneous T-cell lymphoma.

Nineteen patients with cutaneous T-cell lymphoma (CTCL) limited to the skin and/or lymph nodes were treated at Hahnemann University with a combination of total skin electron beam and total nodal irradiation (TSEB + TNI). The patients were classified as Stage Ib (1 patient), Stage IIa (8 patients), Stage IIb (5 patients), and Stage IVa (5 patients). Treatment resulted in a complete response in 100% (14/14) of patients with Stage Ib, IIa, and IIb disease, and a CR in 60% (3/5) of patients with Stage IVa disease. The Stage Ib and IIa patients had an overall survival of 100% and a disease-free survival of 44% at 6 years. Four of the five patients with Stage IIb CTCL relapsed within 3 months after completing TSEB + TNI with an overall survival in the group of 40% at 5 years. The Stage IVa patients all relapsed within 7 months and died of their disease within 50 months of completing treatment. The acute effects of TSEB + TNI were well tolerated, but three patients developed second malignancy (lung, kidney and skin) and one patient developed myelodysplasia, possibly the result of radiotherapy.

Radiotherapy for unilesional mycosis fungoides.

PURPOSE: To evaluate the treatment outcome and natural history of patients with the diagnosis of unilesional mycosis fungoides, treated according to a prospective radiotherapy protocol in our institution since July 1975. METHODS AND MATERIALS: A total of 325 patients with the diagnosis of mycosis fungoides have been referred to the Department of Radiation Oncology at Allegheny University of Health Sciences from July 1975 through September 1996. Of these, 18 patients (5%) were classified as having unilesional mycosis fungoides and were irradiated with a curative intent using local electron fields. One patient received 22 Gy; 1 patient received 40 Gy, and the rest of the patients 30.6 Gy. Daily fractions ranged from 1.8 to 2.0 Gy. Treatments prior to radiation consisted of topical steroids and/or antifungal creams in the majority of patients, with temporary partial responses. One patient had received 2 years of topical mechlorethamine (HN2) and another patient had received topical carmustine solution (BCNU) without response prior to irradiation. RESULTS: The responses were measured clinically; posttreatment skin biopsy was not performed routinely unless there was clinical evidence of disease persistence. Complete response rate was 100%; all treated lesions cleared completely within 4 to 8 weeks after the completion of radiation. With a median follow-up of 43 months (range 12 to 240 months), 2 relapses have occurred, 2 and 71 months after the completion of radiation. Both relapses were confined to the skin and were remote from the original site. Both relapses responded to topical application of HN2. There have been no recurrences in the irradiated field nor systemic dissemination. No long-term side effects were found related to treatment, and all the patients are currently alive and without evidence of disease. Actuarial relapse-free and overall survival at 10 years are, respectively, 86.2% and 100%. CONCLUSION: Unilesional mycosis fungoides has a long natural history, is possibly the earliest manifestation of a malignant process, and local treatments, including local radiotherapy, result in long-term disease-free intervals and, possibly, cure. Total skin electron beam radiotherapy is not indicated for this disease entity.