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.

Differentiation of tumour-stage mycosis fungoides, psoriasis vulgaris and normal controls in a pilot study using serum proteomic analysis.

BACKGROUND: Serum proteomic analysis is an analytical technique utilizing high-throughput mass spectrometry (MS) in order to assay thousands of serum proteins simultaneously. The resultant 'proteomic signature' has been used to differentiate benign and malignant diseases, enable disease prognosis, and monitor response to therapy. OBJECTIVES: This pilot study was designed to determine if serum protein patterns could be used to distinguish patients with tumour-stage mycosis fungoides (MF) from patients with a benign inflammatory skin condition (psoriasis) and/or subjects with healthy skin. METHODS: Serum was analysed from 45 patients with tumour-stage MF, 56 patients with psoriasis, and 47 controls using two MS platforms of differing resolution. An artificial intelligence-based classification model was constructed to predict the presence of the disease state based on the serum proteomic signature. RESULTS: Based on data from an independent testing set (14-16 subjects in each group), MF was distinguished from psoriasis with 78.6% (or 78.6%) sensitivity and 86.7% (or 93.8%) specificity, while sera from patients with psoriasis were distinguished from those of nonaffected controls with 86.7% (or 93.8%) sensitivity and 75.0% (or 76.9%) specificity (depending on the MS platform used). MF was distinguished from unaffected controls with 61.5% (or 71.4%) sensitivity and 91.7% (or 92.9%) specificity. In addition, a secondary survival analysis using 11 MS peaks identified significant survival differences between two MF groups (all P-values <0.05). CONCLUSIONS: Serum proteomics should be further investigated for its potential to identify patients with neoplastic skin disease and its ability to determine disease prognosis.

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.

BCL2 and JUNB abnormalities in primary cutaneous lymphomas.

BACKGROUND: BCL2 is upregulated in nodal and extranodal B-cell non-Hodgkin's lymphomas, with a consequent antiapoptotic effect. However, loss of BCL2 has also been noted in some malignancies, suggesting a different molecular pathogenesis. OBJECTIVES: To investigate genomic and protein expression status of BCL2 and to compare the results with that of JUNB in primary cutaneous lymphomas (PCLs). METHODS: We analysed gene copy number of BCL2 and JUNB in 88 DNA samples from 80 patients with PCL consisting of Sézary syndrome/mycosis fungoides (SS/MF), primary cutaneous B-cell lymphoma (PCBCL) and primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) by the use of real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). Real-time PCR and IHC findings were subsequently compared with the results of additional fluorescent in situ hybridization (FISH) analysis of 23 cases of SS and Affymetrix cDNA expression microarray study of two primary cutaneous T-cell lymphoma (CTCL) cell lines. RESULTS: Real-time PCR analysis showed loss of BCL2 gene copy number in 22 of 80 PCL cases (28%), including 17 of 42 SS/MF, three of 13 C-ALCL and two of 33 PCBCL samples, and gain of BCL2 in four PCBCL samples. Gain of JUNB was identified in 18 of 71 PCL cases (25%), including nine of 35 SS/MF, seven of 13 C-ALCL and two of 31 PCBCL samples. IHC analysis revealed absent nuclear expression of BCL2 protein in 47 of 73 PCL cases, comprising 28 of 36 SS/MF, eight of eight C-ALCL and 11 of 29 PCBCL cases. In contrast, BCL2 protein expression was detected in 26 of 73 PCL cases, consisting of 18 of 29 PCBCL and eight of 36 SS/MF cases. JUNB protein expression was present in tumour cells from 30 of 33 of SS/MF and eight of eight C-ALCL, and was absent in tumour cells from 18 of 27 PCBCL cases. A comparison between BCL2 and JUNB revealed loss of BCL2 and gain of JUNB in five of 35 SS/MF samples, and expression of JUNB protein and absent BCL2 expression in 25 SS/MF and eight of eight C-ALCL cases. In contrast, expression of BCL2 and absent JUNB expression were detected in 67% of PCBCL cases. Additional FISH analysis revealed deletion of BCL2 in 19 of 23 SS cases (83%), including eight cases with BCL2 loss shown by real-time PCR. Furthermore, Affymetrix expression microarray demonstrated decreased expression of proapoptotic and antiapoptotic genes involved in BCL2 signalling pathways such as BOK, BIM, HRK, RASA1 and STAT2 in two CTCL cell lines with BCL2 loss and absent BCL2 expression. Increased expression of JUNB was also identified in the MF cell line. CONCLUSIONS: These findings provide a comprehensive assessment of BCL2 and JUNB status in PCL, and suggest that there is a selection pressure in a subset of CTCL cases for tumour cells showing BCL2 loss and upregulation of JUNB primarily through chromosomal deletion and amplification, respectively.

The role for interleukin-12 therapy of cutaneous T cell lymphoma.

Recent phase I and phase II trials using recombinant human interleukin-12 (rhIL-12) for cutaneous T cell lymphoma (CTCL) have been completed. Observations on 32 evaluable patients revealed an overall response rate approaching 50 percent. Biopsy of regressing lesions revealed an increase in numbers of CD8+ and/or TIA-1+ T cells. These results suggest that rhIL-12 may induce lesion regression by augmenting antitumor cytotoxic T cell responses. Future trials will be focused on strategies for further immune enhancement by the concomitant use of additional immune augmenting cytokines with rhIL-12.

Association between Sézary T cell-activating factor, Chlamydia pneumoniae, and cutaneous T cell lymphoma.

Sézary T cell-activating factor (SAF) was originally defined as an inducer of functional interleukin-2 (IL-2) receptors on normal and malignant T cells in patients suffering from Sézary syndrome. In fact, a combination of SAF and IL-2 stimulated the propagation of T cell lines from the peripheral blood mononuclear cells (PBMC) of those patients, with approximately one third of those cell lines containing the predominant malignant clone as determined via cytogenetic and/or T cell receptor gene rearrangement analysis. Although the primary source of SAF was mitogen-stimulated PBMC of a patient with Sézary syndrome, we were unable to isolate the gene encoding SAF from eukaryotic libraries. However, we observed SAF activity in the cytoplasm of one of the malignant cell lines in a complex containing RNA and DNA. This observation led us to consider the possibility that SAF is not of eukaryotic origin. Intracellular pathogens replicate in the cytoplasm of host cells and contain proteins, DNA, and RNA. Using a panel of antichlamydial antibodies with confirmation from polymerase chain reaction primers, we found that most patients with mycosis fungoides were positive for these determinants. Immunoelectron microscopy and protein blotting further confirmed antibody reactivity. We showed that Chlamydia pneumoniae were capable of infecting normal human keratinocytes in culture. We also demonstrated that C. pneumoniae antigen expression was associated with active disease because these determinants were not expressed after psoralen and ultraviolet A therapy. We hypothesize that chronic infection by C. pneumoniae leads to expansion of C. pneumoniae-specific T cells, thereby potentiating the development of cutaneous T cell lymphoma.

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.

A phase III, randomized, double-blind, placebo-controlled study of peldesine (BCX-34) cream as topical therapy for cutaneous T-cell lymphoma.

BACKGROUND: The purine nucleoside phosphorylase inhibitor peldesine is a new agent being evaluated as a T-cell inhibitor. OBJECTIVE: We attempted to determine the efficacy of peldesine (BCX-34) in a 1% dermal cream formulation as a treatment for cutaneous T-cell lymphoma (CTCL). METHODS: Ninety patients with patch and plaque phase CTCL, histologically confirmed by a referee dermatopathologist, were enrolled in a randomized, double-blind, placebo-controlled study. BCX-34 dermal cream 1% or the vehicle cream (as a placebo control) was applied twice daily to the entire skin surface for up to 24 weeks. Efficacy of the topical therapy was assessed in terms of complete or partial (> or = 50%) clearing of patches and plaques. RESULTS: Of the 89 patients able to be examined, 43 received BCX-34 and 46 received the placebo vehicle cream. One patient withdrew early and was not analyzed. The two groups were well balanced for potential prognostic factors. A total of 28% (12/43) of the patients treated with BCX-34 showed a response, but 24% (11/46) of patients who received vehicle also responded (P =.677). CONCLUSION: Although BCX-34 dermal cream 1% was not significantly better than the control as therapy for patch and plaque-phase CTCL, this appears to be the first published placebo-controlled trial evaluating treatment for CTCL. Whether the vehicle cream has more than a placebo therapeutic effect is unclear. The relatively high (24%) placebo response rate should be kept in mind in assessing other treatments for early-stage CTCL.

Increased interleukin 5 production in eosinophilic Sézary syndrome: regulation by interferon alfa and interleukin 12.

BACKGROUND: Peripheral eosinophilia occurs in a small subpopulation of patients with cutaneous T-cell lymphoma (CTCL) and denotes a poor prognosis. Clinical studies have suggested that the Sézary cell is a T(H)2 type helper T cell that produces cytokines that enhance the differentiation and activation of eosinophils. Interferon alfa (IFN-alpha) and interleukin 12 are effective therapeutic agents for CTCL and other hematologic disorders. OBJECTIVE: Our purpose was to determine the inhibitory activity of IFN-alpha and IL-12 on IL-5 production in vitro by peripheral blood mononuclear cells (PBMCs) obtained from patients with CTCL and eosinophilia. METHODS: Suppression of IL-5 production by IFN-alpha and IL-12 was assessed by comparing IL-5 production by PBMCs from patients with Sézary syndrome and eosinophilia when cultured alone or in the presence of either IFN-alpha or IL-12. RESULTS: A marked increase in IL-5 production by PBMCs from patients with Sézary syndrome and eosinophilia was observed. IL-5 production was markedly reduced when PBMCs were exposed to IFN-alpha or IL-12. CONCLUSION: These results suggest that IFN-alpha and perhaps IL-12 may produce a therapeutic response in patients with CTCL and eosinophilia through direct suppression of IL-5 production by malignant Sézary cells.

Relative frequency of various forms of primary cutaneous lymphomas.

BACKGROUND: Data on the relative frequency of the various forms of primary cutaneous lymphomas (PCLs) are largely limited to European institutions. OBJECTIVE: Our purpose was to document the relative frequencies of various PCLs seen at 3 US institutions with active cutaneous lymphoma programs and to compare those with the European data. METHODS: Included in this study are newly registered patients seen at MCP Hahnemann University, New York University, and the University of California, San Francisco from July 1, 1995 to June 30, 1998. RESULTS: A total of 755 patients were seen. The frequency distribution of the major diagnostic groups was as follows: mycosis fungoides/Sézary syndrome, 82.3%; lymphomatoid papulosis, 12.6% (including patients with associated mycosis fungoides/Sézary syndrome); CD30(+) anaplastic large-cell lymphoma, 0.9%; peripheral T-cell lymphomas, 2.9%; B-cell lymphoma, 4.5%. CONCLUSION: The most striking finding is the much lower relative frequency of primary cutaneous B-cell lymphomas at US institutions (4.5%) versus the approximately 20% reported by European groups. The reason for this difference requires further study.

Sarcoidal tissue reaction in Sézary syndrome.

Sézary syndrome (SS) is an erythrodermic and leukemic variant of cutaneous T-cell lymphoma (CTCL). Occasionally, the histology of CTCL exhibits evidence of a granulomatous infiltrate in the skin. A case of SS that showed epithelioid granulomas resembling sarcoidosis in the skin and lymph nodes is presented. The clinical course of this patient has been relatively indolent.

Fine-needle aspiration biopsy in the evaluation of lymphadenopathy associated with cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome).

We studied the role of fine-needle aspiration (FNA) in the evaluation of lymphadenopathy associated with cutaneous T-cell lymphoma (CTCL) in 11 patients with lymphadenopathy and compared findings with corresponding histologic material. Molecular genetic analysis for T-cell clonality by polymerase chain reaction (PCR) was performed on all aspirates. Immunophenotyping was successful in 4 of 7 cases in which flow cytometry was attempted from the aspirated material. Cytologic evaluation of FNA samples correlated strongly with histologic rating of involvement based on numbers of atypical cerebriform lymphocytes in the nodal specimen. Of 7 nodal specimens with scattered or small groups of atypical cells in the background of dermatopathic lymphadenopathy (LN1-2), the cytologic diagnosis was interpreted as reactive in all instances. Of 4 specimens with highly suspect (LN3) or definite histologic involvement (LN4), the cytologic diagnosis was likewise suspect or malignant. The correlation between molecular genetic studies on FNA samples and studies on tissue was not significant; in 2 cases, a T-cell clone was detected in the nodal tissue sample but not in the FNA sample, suggesting undersampling. A T-cell clone was detected by PCR in 5 of 7 nodal specimens judged reactive by FNA biopsy or histologic assessment. FNA for cytologic and molecular genetic analysis is a useful method to evaluate lymphadenopathy associated with CTCL and may obviate the need for surgical biopsy.

Mycosis fungoides with CD30-positive cells in the epidermis.

Large numbers of CD30-positive tumor cells are not typically observed in mycosis fungoides (MF), but CD30 expression may occur on large cells of MF that have transformed into high-grade large cell lymphoma. Of 202 patch/plaque phase MF cases studied by immunohistochemistry, we encountered 4 patients with patch-phase MF at stage Ia or Ib, whose lesions contained a high proportion (greater than 50%) of CD30-positive tumor cells within the epidermis. The morphologic and immunohistochemical features of these four cases were otherwise similar to those of other patch-phase MF cases, and were different from those of pagetoid reticulosis. More importantly, the clinical behavior of these cases did not differ from that of other cases of early MF without CD30 expression. The mechanism underlying the high levels of CD30 expression by epidermotropic tumor cells is unknown. With increased use of the CD30 immunohistochemical stain in the diagnosis of cutaneous lymphomas, similar cases are likely to be encountered, and perhaps will be evaluated for their clinical behavior.

Sézary T-cell activating factor is a Chlamydia pneumoniae-associated protein.

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it's role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated with Chlamydia pneumoniae bacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein of C. pneumoniae or against the lipopolysaccharide of Chlamydiae spp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence of C. pneumoniae DNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of the C. pneumoniae antigens and SAF appears to be associated with active disease in that C. pneumoniae antigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is a Chlamydia-associated protein and that further investigation is warranted to determine whether SAF and C. pneumoniae play a role in the pathogenesis of CTCL.

p53 tumor suppressor gene mutations in transformed cutaneous T-cell lymphoma: a study of 12 cases.

The transformation of cutaneous T-cell lymphoma (t-CTCL) is an uncommon phenomenon that is associated with histopathologic changes and follows an aggressive course. The factors contributing to this transformation are poorly understood. The aim of this study was to analyze the p53 status in t-CTCL and to correlate it with disease outcome. The p53 status was investigated by immunohistochemistry, single-strand conformation polymorphism (SSCP) and DNA sequencing in 12 patients with t-CTCL. Eight mutations were detected; including four in exon 5, one in exon 6 and three in exon 7. Five were point mutations and three were deletions. Paired samples from nontransformed patch and plaque lesions showed no p53 over-expression. Eight disease-related deaths were reported, six to 23 months after transformation, all of which had p53 mutations. Three other patients with wild phenotype (WT-p53) were last reported alive with the disease 19-33 months after transformation (p < 0.0002). One other case had a p53 mutation but a short period of follow-up. Our results suggest that phenotypic changes of t-CTCL are frequently associated with genotype alterations in the p53 gene. Because 70% of the mutations detected were either G to C transversions or deletions, nucleotide-pairing mismatch and not DNA damage by UVB represents a likely mechanism for mutagenesis. Furthermore, the data may help in the design of gene transfer therapies that target the p53 molecule.

Interleukin-12 therapy of cutaneous T-cell lymphoma induces lesion regression and cytotoxic T-cell responses.

Progression of cutaneous T-cell lymphoma (CTCL) is associated with profound defects in cell-mediated immunity and depressed production of cytokines, which support cell-mediated immunity. Because we have observed marked defects in interleukin-12 (IL-12) production in CTCL and because IL-12 is critical for antitumor cytotoxic T-cell responses, we initiated a phase I dose escalation trial with recombinant human IL-12 (rhIL-12) where patients received either 50, 100, or 300 ng/kg rhIL-12 twice weekly subcutaneously or intralesionally for up to 24 weeks. Ten patients were entered: 5 with extensive plaque, 3 with Sezary syndrome, and 2 with extensive tumors with large cell transformation. One patient with Sezary syndrome dropped out after 1 week for personal reasons. Subcutaneous dosing resulted in complete responses (CR) in 2 of 5 plaque and partial responses (PR) in 2 of 5 plaque, and 1 of 2 Sezary syndrome (overall response rate CR+PR 5 of 9, 56%). A minor response also occurred in 1 of 5 plaque patients. Intralesional dosing resulted in individual tumor regression in 2 of 2 patients. Biopsy of regressing lesions showed a significant decrease in the density of the infiltrate in all cases and complete resolution of the infiltrate among those with clinical lesion resolution. An increase in numbers of CD8-positive and/or TIA-1-positive T cells were observed on immunohistochemical analysis of skin biopsy specimens obtained from regressing skin lesions. Adverse effects of rhIL-12 on this regimen were minor and limited and included low-grade fever and headache. One patient discontinued rhIL-12 at week 6 because of depression. These results suggest that rhIL-12 may augment antitumor cytotoxic T-cell responses and may represent a potent and well-tolerated therapeutic agent for CTCL.