New Genetic Markers of Skin T-Cell Lymphoma Treatment.

AIM: Cutaneous T-cell lymphomas (CTCL) can be described as chronic skin inflammation lesions with the content of malignant T cells and they are considered to be T-cell-mediated skin diseases. CD147 is recognized as a 58-kDa cell surface glycoprotein of the immunoglobulin superfamily; it can induce the synthesis of MMPs (matrix metalloproteinases) on the surface of tumor cells where it was originally identified. It can also function in adjacent tumor fibroblasts using CD147-CD147 interactions. The polymorphism rs8259 T/A is situated in the untranslated region (3'UTR) of the CD147 gene. HLA DRB1*1501 takes part in the process of presentation and recognition of different antigens to T cells. It can be expressed by antigen-presenting cells-macrophages, dendritic cells, and B cells. The aim of the study is to test genotype-phenotype associations of both polymorphisms including therapy in a large cohort of CTCL patients. MATERIALS AND METHODS: A final total of 104 CTCL patients were enrolled in the study. For the first remission at the clinic department, they were treated by means of local skin-directed therapy, phototherapy, and systemic therapy. Genomic DNA was isolated from peripheral blood leukocytes. A standard technique using proteinase K was applied. The polymorphisms rs8259 T/A (CD147 gene) and rs3135388 (HLA DRB1*1501) were detected through standard PCR-restriction fragment length polymorphism methods. RESULTS: The severity of the disease (patients with parapsoriasis, stages IA and IB, vs patients with stages IIB, IIIA, and IIIB) was associated with the CD147 genotype: the AA variant was 3.38 times more frequent in more severe cases, which reflects the decision on systemic therapy (p = 0.02, specificity 0.965). The AA genotype in the CD147 polymorphism was 12 times more frequent in patients who underwent systemic therapy of CTCL compared to those not treated with this therapy (p = 0.009, specificity 0.976). The same genotype was also associated with radiotherapy-it was observed 14 times more frequently in patients treated with radiotherapy (p = 0.009, specificity 0.959). In patients treated with interferon α therapy, the AA genotype was observed to be 5.85 times more frequent compared to the patients not treated with interferon therapy (p = 0.03, specificity 0.963). The HLA DRB1*1501 polymorphism was associated with local skin-directed therapy of CTCL. The CC genotype of the polymorphism was observed to be 3.57 times more frequent in patients treated with local therapy (p = 0.008, specificity 0.948). When both polymorphisms had been calculated together, even better results were obtained: the AACC double genotype was 11 times more frequent in patients with severe CTCL (p = 0.009, specificity 0.977). The TACT double genotype was associated with local skin-directed therapy (0.09 times lower frequency, p = 0.007, sensitivity 0.982). The AACC genotype was 8.9 times more frequent in patients treated by means of systemic therapy (p = 0.02, specificity 0.976) and as many as 18.8 times more frequent in patients treated with radiotherapy (p = 0.005, specificity 0.969). Thus, the AACC double genotype of CD147 and DRB1*1501 polymorphisms seems to be a clinically highly specific marker of severity, systemic therapy and radiotherapy of patients with T-cell lymphoma. CONCLUSION: Although genotyping results were not known during the treatment decision and could not modify it, the clinical decision on severity and therapy reflected some aspects of the genetic background of this complicated T-cell-associated disease very well.

GATA-3-dependent Gene Transcription is Impaired upon HDAC Inhibition.

PURPOSE: Many peripheral and cutaneous T-cell lymphoma (CTCL) subtypes are poorly responsive to conventional chemotherapeutic agents and associated with dismal outcomes. The zinc finger transcription factor GATA-3 and the transcriptional program it instigates are oncogenic and highly expressed in various T-cell neoplasms. Posttranslational acetylation regulates GATA-3 DNA binding and target gene expression. Given the widespread use of histone deacetylase inhibitors (HDACi) in relapsed/refractory CTCL, we sought to examine the extent to which these agents attenuate the transcriptional landscape in these lymphomas. EXPERIMENTAL DESIGN: Integrated GATA-3 chromatin immunoprecipitation sequencing and RNA sequencing analyses were performed in complementary cell line models and primary CTCL specimens treated with clinically available HDACi. RESULTS: We observed that exposure to clinically available HDACi led to significant transcriptional reprogramming and increased GATA-3 acetylation. HDACi-dependent GATA-3 acetylation significantly impaired both its ability to bind DNA and transcriptionally regulate its target genes, thus leading to significant transcriptional reprogramming in HDACi-treated CTCL. CONCLUSIONS: Beyond shedding new light on the mechanism of action associated with HDACi in CTCL, these findings have significant implications for their use, both as single agents and in combination with other novel agents, in GATA-3-driven lymphoproliferative neoplasms.

Cutaneous T Cell Lymphoma: A Difficult Diagnosis Demystified.

Cutaneous T cell lymphoma (CTCL) represents a heterogeneous group of extranodal non-Hodgkin lymphomas in which monoclonal T lymphocytes infiltrate the skin. The mechanism of CTCL development is not fully understood, but likely involves dysregulation of various genes and signaling pathways. A variety of treatment modalities are available, and although they can induce remission in most patients, the disease may recur after treatment cessation. Owing to relatively low incidence and significant chronicity of disease, and the high morbidity of some therapeutic regimens, further clinical trials are warranted to better define the ideal treatment option for each subtype of CTCL.

NRAS mutations in cutaneous T cell lymphoma (CTCL) sensitize tumors towards treatment with the multikinase inhibitor Sorafenib.

Therapy of cutaneous T cell lymphoma (CTCL) is complicated by a distinct resistance of the malignant T cells towards apoptosis that can be caused by NRAS mutations in late-stage patients. These mutations correlate with decreased overall survival, but sensitize the respective CTCL cells towards MEK-inhibition-induced apoptosis which represents a promising novel therapeutic target in CTCL. Here, we show that the multi-kinase inhibitor Sorafenib induces apoptosis in NRAS-mutated CTCL cells. CTCL cell lines and to a minor extent primary T cells from Sézary patients without NRAS mutations are also affected by Sorafenib-induced apoptosis suggesting a sensitizing role of NRAS mutations for Sorafenib-induced apoptosis. When combining Sorafenib with the established CTCL medication Vorinostat we detected an increase in cell death sensitivity in CTCL cells. The combination treatment acted synergistically in apoptosis induction in both non-mutant and mutant CTCL cells. Mechanistically, this synergistic apoptosis induction by Sorafenib and Vorinostat is based on the downregulation of the anti-apoptotic protein Mcl-1, but not of other Bcl-2 family members. Taken together, these findings suggest that Sorafenib in combination with Vorinostat represents a novel therapeutic approach for the treatment of CTCL patients.

Psoralen with ultraviolet A-induced apoptosis of cutaneous lymphoma cell lines is augmented by type I interferons via the JAK1-STAT1 pathway.

BACKGROUND: Photochemotherapy with psoralen and ultraviolet A (PUVA), with or without adjuvant interferon-α (IFN-α), is a first-line therapy for early-stage mycosis fungoides and other forms of cutaneous T-cell lymphoma (CTCL). However, the mechanism by which PUVA with IFN-α work in CTCL is poorly understood. PURPOSE: To develop a model to investigate the mechanisms of PUVA and PUVA with IFN-α in CTCL cells. METHODS: An in vitro model to study the molecular mechanisms of PUVA was created using two different CTCL cell lines, MyLa, which has functional p53, and HuT-78, in which p53 is inactivated due to a homozygous nonsense mutation. RESULTS: PUVA caused G2/M cell cycle block and apoptosis of MyLa and HuT-78 accompanied by increase in the expression of the mitochondrial pro-apoptotic genes Bax, BAK, and PUMA and a downregulation in anti-apoptotic Bcl-2. p53 was induced and c-Myc was repressed by PUVA, but neither were essential for PUVA-induced apoptosis. IFN-α augmented PUVA-induced apoptosis via the JAK1 pathway, and this activity could be inhibited by ruxolitinib. CONCLUSION: PUVA induces p53-independent apoptosis in CTCL cell lines, and this process is augmented by type I interferons via the JAK1 pathway.

MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor.

To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

Cutaneous T cell lymphoma: translating immunobiology into therapeutic opportunities.

Cutaneous T cell lymphoma (CTCL) has always served as a proving ground where conceptual advances in immunology can be tested and the results translated into clinical practice. From the earliest studies that used sheep red blood cells to identify the malignant cell as a T lymphocyte to molecular demonstration of the clonalilty of the disease, basic science techniques have provided sign posts that allow us to understand the clinical features seen in the patients. We continue to apply this paradigm to develop new insights into the role of the immune system in CTCL with the goal of using this knowledge to enhance the therapeutic options available to the patient. This article will review the studies that have led to our current understanding of the immunobiology of CTCL and the new therapeutic approaches that are being tested in this disease.

Zinc in human health: effect of zinc on immune cells.

Although the essentiality of zinc for plants and animals has been known for many decades, the essentiality of zinc for humans was recognized only 40 years ago in the Middle East. The zinc-deficient patients had severe immune dysfunctions, inasmuch as they died of intercurrent infections by the time they were 25 years of age. In our studies in an experimental human model of zinc deficiency, we documented decreased serum testosterone level, oligospermia, severe immune dysfunctions mainly affecting T helper cells, hyperammonemia, neurosensory disorders, and decreased lean body mass. It appears that zinc deficiency is prevalent in the developing world and as many as two billion subjects may be growth retarded due to zinc deficiency. Besides growth retardation and immune dysfunctions, cognitive impairment due to zinc deficiency also has been reported recently. Our studies in the cell culture models showed that the activation of many zinc-dependent enzymes and transcription factors were adversely affected due to zinc deficiency. In HUT-78 (T helper 0 [Th(0)] cell line), we showed that a decrease in gene expression of interleukin-2 (IL-2) and IL-2 receptor alpha(IL-2Ralpha) were due to decreased activation of nuclear factor-kappaB (NF-kappaB) in zinc deficient cells. Decreased NF-kappaB activation in HUT-78 due to zinc deficiency was due to decreased binding of NF-kappaB to DNA, decreased level of NF-kappaB p105 (the precursor of NF-kappaB p50) mRNA, decreased kappaB inhibitory protein (IkappaB) phosphorylation, and decreased Ikappa kappa. These effects of zinc were cell specific. Zinc also is an antioxidant and has anti-inflammatory actions. The therapeutic roles of zinc in acute infantile diarrhea, acrodermatitis enteropathica, prevention of blindness in patients with age-related macular degeneration, and treatment of common cold with zinc have been reported. In HL-60 cells (promyelocytic leukemia cell line), zinc enhances the up-regulation of A20 mRNA, which, via TRAF pathway, decreases NF-kappaB activation, leading to decreased gene expression and generation of tumor necrosis factor-alpha (TNF-alpha), IL-1beta, and IL-8. We have reported recently that in both young adults and elderly subjects, zinc supplementation decreased oxidative stress markers and generation of inflammatory cytokines.

Tratamiento del linfoma cutáneo de células T con ligandos selectivos del receptor retinoide X: alteraciones endocrinológicas y metabólicas / Treatment of cutaneous T-cell lymphoma with retinoid receptor x- selective ligands: endocrine and metabolic disorders

Los ligandos selectivos del receptor retinoide X se utilizan actualmente en el tratamiento de estadios avanzados del linfoma cutáneo de células T resistentes a otros tratamientos sistémicos, siendo el bexaroteno el primer fármaco de este grupo aprobado en Europa. Se han descrito numerosos efectos adversos asociados a su utilización, entre los que destacan las alteraciones endocrino-metabólicas. Presentamos a 2 pacientes con linfoma cutáneo de células T, tratados con bexaroteno, que desarrollaron hipotiroidismo central y dislipemia de forma precoz tras su inicio. Se describen también la respuesta de estas alteraciones al tratamiento y su remisión completa tras suspender el bexaroteno (AU)

The retinoid X receptor-selective ligands has been used for advanced stages of cutaneous T-cell lymphoma refractory to previous systemic therapy, being bexarotene the first drug in this group approved in Europe. Multiple drug-related adverse events has been reported such as endocrine-metabolic disorders. We report 2 patients with cutaneous T-cell lymphoma, treated with bexarotene, that developed central hypothyroidism and dyslipidaemia immediately after the beginning of this treatment. We also showed the successfully treatment response of these alterations and the total clinical remission after discontinuing the drug (AU)

[Association of polymorphic variants in endothelin-1 (EDN1) genes with the therapy of patients with cutaneous T-cell lymphomas]. / Asociace variant polymorfismu v genu pro endotelin-1 (EDN1) s terapií u pacientu s kozními T lymfomy.

BACKGROUND: The cutaneous T-cell lymphomas (CTCL) are diseases characterised by cutaneous infiltrates of malignant, clonally expanded T-cells. Because individual genetic determination of angiogenetic and antioxidant properties of blood vessels could take part in responsibility to phototherapy in CTCL patients, the association of two frequent polymorphisms in endothelin-1 gene with phototherapy was tested. METHODS AND RESULTS: 77 patients with CTCL, diagnosed and treated at the First Dermatological Clinic of St. Ann's Faculty Hospital Bmo (46 men and 31 women, median age 62, range 28-82 years) were included in the study. Diagnosis of CTCL according to the clinical picture was verified histologically. The genotype distributions and allelic frequencies between CTCL with phototherapy and those without phototherapy were compared. The 4A4A variant of -3A/-4A EDN1 is more frequent in patients treated with phototherapy (8/30 vs. 1/38, OR=10.13; P=0.01). The GA and AA genotypes of G8002A EDN1 polymorphism are more frequent in CTCL patients treated with phototherapy compared to those without it (15/23 vs. 7/32, OR=2.98; P=0.03). CONCLUSIONS: Some polymorphic variants in EDN1 genes, a homozygote -4A-4A in -3A/-4A EDN1 and genotypes GA and GG in G8002A EDN1) seem to carry an advantage for phototherapy effectiveness in patients with CTCL.

Chromosomal abnormalities in relation to clinical disease in patients with cutaneous T-cell lymphoma: a 5-year follow-up study.

BACKGROUND: Patients with cutaneous T-cell lymphoma (CTCL) show chromosomal aberrations in skin and blood lymphocytes. OBJECTIVES: To evaluate the significance of peripheral blood clonal or non-clonal chromosomal abnormalities in comparison with the clinical course of cutaneous T-cell lymphoma patients. PATIENTS/METHODS: Five patients with large-plaque parapsoriasis (LPP) or with follicular mucinosis, eight with mycosis fungoides and two with Sézary syndrome were followed for an average of 54 months. G-banding and enzyme-detected in situ hybridization (EDISH) were used to identify aberrations in chromosomes 1, 6, 8, 9, 11, 13/21, 15 or 17, that had previously showed frequent aberrations. RESULTS: The aberration rates of all chromosomes studied differed between patients with active disease and healthy or photochemotherapy-treated controls by EDISH or G-banding (P < 0.01 to P < 0.05). Patients in complete remission differed from healthy controls for aberrations of chromosomes 1, 6 and 11, and from patients with active, progressing disease for chromosomes 1, 6, 8, 11 and 17 (P < 0.01 to P < 0.05, EDISH or G-banding). All 11 samples representing active, progressing disease showed elevated levels of chromosome 8 aberrations in EDISH. The change in chromosomal aberration rate and clinical condition between two consecutive samples agreed for chromosomes 1, 8, 9 and 15 (G-banding) and for chromosome 17 (G-banding and EDISH; kappa > 0.5-0.6). Six of seven patients (five CTCL, one LPP patient) with clonal chromosomal aberrations by G-banding showed continuously active disease and four of them, but none of the other patients, died within 30 months of the detection of the clone. CONCLUSIONS: The rate of chromosomal aberrations associates with the activity of CTCL, and has prognostic significance. Aberrations of chromosomes 1, 6 and 11, although increasing with activity of the disease, seem to be a hallmark of existing disease, detectable even in remission. Aberrations of chromosomes 8 and 17 especially associate with active or progressive disease.

Reversible resistance to apoptosis in cutaneous T cell lymphoma.

Mycosis fungoides and its leukemic variant, Sézary syndrome, represent the most common forms of cutaneous T cell lymphomas (CTCL). These disorders are clonal neoplasms characterized by the progressive accumulation of cells that resemble activated/memory CD4+ T cells. Unlike their normal counterparts, these malignant lymphocytes have prolonged life spans and are resistant to dying following treatment with most chemotherapeutic agents. This suggests that CTCL undergo abnormal programmed cell death; however, data regarding apoptotic defects in CTCL are limited. Regulation of apoptosis in lymphocytes that regularly undergo clonal expansion is necessarily complex and will be reviewed here. Clonally expanded lymphocytes rely primarily on Fas-mediated pathways to initiate apoptosis. Factors leading to the resistance of apoptosis in CTCL and new therapeutic approaches for reversing this resistance will be discussed, including the important role that the Fas death pathway may play in the pathogenesis and treatment of CTCL.

Etiopatogenia de los linfomas cutáneos de células T (micosis fungoide/síndrome de sézary) / Etiophatogenesis of cutaneous T-cell lymphomas (mycosis fungoides/Sézary syndrome)

La micosis fungoide y el síndrome de Sézary son los linfomas cutáneos de células T más frecuentes. Su etiopatogenia es poco conocida, como tampoco se conocen los mecanismos por los que las fases indolentes se hacen más agresivas desarrollan tumores y se afectan ganglios y órganos internos. Se revisan los factores implicados en su desarrollo y evolución: herencia, contaminantes ambientales, agentes infecciosos, antígenos del complejo mayor de histocompatibilidad, inestabilidad genética, citocinas y oncógenes. Aunque el desarrollo de avanzadas técnicas de laboratorio ha aumento el conocimiento de su etiopatogénesis, el signicado de muchos de los factores implicados es controvertido y objeto de debate. Serán necesarios nuevos estudios basados en la epidemiología y en la biología molecular para profundizar en estas cuestiones y en un mayor conocimiento de esta enfermedad (AU)

Densitometry of Pautrier's microabscess cells in cutaneous T cell lymphoma.

BACKGROUND: Epidermotropic lymphoid T cell infiltrates are part of a continuous spectrum of lesions ranging from "benign" parapsoriasis to frank cutaneous T cell lymphoma (CTCL, mycosis fungoides). Either the clinical or histologic differentiation between these entities prove often difficult and the prognosis may be difficult to assess. PATIENTS AND METHODS: We studied 15 patients, men aged 50 to 81 years, mean +/- SD 68 +/- 12 years, with patch-plague CTCL. Sections from punch biopsies from active lesions were stained with Feulgen reagent, coupled with a "twin" adjacent hematoxylin and eosin (H&E)-stained section and analyzed with a VIDAS Zeiss-Kontron Image Analyzer. At least 50 dermal infiltrating cells and 50 epidermotropic Pautrierian cells per specimen were counted and the biopsies were repeated periodically. RESULTS: Nine patients with aneuploid Pautrierian cell DNA patterns dis well after conventional phototherapy (dermal cell ploidy was irrelevant), whereas six patients with euploid Pautrierian cell DNA patterns had to be treated aggressively (IFN + retinoids, COP). Clinical and histopathologic aspects of the first group were comparable to those of the second group. CONCLUSIONS: The classical cytophotometric aphorism seems to be reversed in this sample: "The more abnormal the ploidy of epidermotropic Pautrierian cells, the better the prognosis." Euploid epidermotropic cell prove more efficient in invading the skin and other areas and this efficiency may be reflected in more aggressive trends in the evolution of the lymphoma. Thus, DSM analysis of epidermotropic cells could prove very useful as an inexpensive tool for routine CTCL grading.

Cytogenetic effects during extracorporeal photopheresis treatment of two patients with cutaneous T-cell lymphoma.

The effect of extracorporeal photopheresis (EP) on various cytogenetic parameters has been investigated. During EP the photoactivatable agent 8-methoxypsoralen (8-MOP) was administered orally. After 2 h a leukocyte-enriched blood fraction was collected by haemocentrifugation, irradiated with UVA extracorporeally, and reinfused to the patient. Two patients suffering from cutaneous T-cell lymphoma showed a marked clinical improvement in response to therapy. In order to investigate the cytogenetic effects and mutagenic risk of EP, the mitotic index (MI), the type and number of chromosomal aberrations and the rate of sister chromatid exchanges (SCE) were studied. Following EP treatment the patients' lymphocytes were cultured and stimulated with phytohaemagglutinin (PHA) for 48 or 72 h. The cultured lymphocytes showed a decreased MI after 48 h as an indicator of cytotoxic effects, but not after 72 h. In lymphocyte cultures not stimulated with PHA, the MI was decreased even after 72 h. The number of chromosomal aberrations and SCE were increased upon treatment, but only transiently, returning to basal levels between consecutive treatments. Our data provides no evidence for increased mutagenic risk as a consequence of effective EP treatment.