Current status of histone deacetylase inhibitors in cutaneous T-cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin's lymphoma with a heterogenous presentation and highly variable disease course. The most common subtypes of CTCL are mycosis fungoides (MF) and Sézary Syndrome (SS). Treatment varies based on the stage of the disease with skin directed therapies typically utilized for early stage disease, and systemic therapies employed for more advanced disease. There are few highly effective treatments available, and systemic therapies have limited response rates. Histone deacetylase inhibitors have emerged as mainstream treatments for MF/SS over the past several years. Here, we discuss the mechanism of action of histone deacetylase inhibitors in relation to the pathogenesis of MF/SS, evaluate the clinical trials that led to Food and Drug Administration approval of two of the histone deacetylase inhibitors for MF/SS and describe the results for those still under investigation. Additionally, we discuss the potential for combination therapies in order to optimize outcomes of treatment with histone deacetylase inhibitors.

Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.

Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

Thymopentin down-regulates both activity and expression of iNOS in blood cells of Sézary syndrome patients.

While thymopentin has been used for many years in the experimental treatment of Sézary syndrome (SS), a rare and very aggressive lymphoma, its mechanism of action is still not known. Herein we show that this peptide acts as an inhibitor of isolated iNOS and nNOS isoforms, and reduces iNOS protein/mRNA levels and iNOS activity in blood cells obtained from both healthy donors and SS patients. Similar results were obtained with TPN-2, the N(ω)-nitro analogue of the Arg-Lys motif present in thymopentin. Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of iNOS down-regulation in the therapeutic action of these peptides against SS.

High expression of Dicer reveals a negative prognostic influence in certain subtypes of primary cutaneous T cell lymphomas.

BACKGROUND: Aberrant expression of microRNAs (miRNAs) has been implicated in oncogenesis of various tumors and primary cutaneous T cell lymphomas. Dicer, a ribonuclease III-like enzyme is essential for miRNA processing. OBJECTIVE: We initiated a retrospective study to characterize the alterations in the expression profile of Dicer in patients with primary cutaneous T cell lymphomas (CTCL). METHODS: A total of 50 consecutive patients with primary CTCL were studied, with the majority having mycosis fungoides (n=34). Five patients had primary cutaneous CD 30+ anaplastic large cell lymphoma, four patients each had lymphomatoid papulosis and primary cutaneous CD4-positive small/medium T-cell lymphoma, one primary cutaneous γδ T cell lymphoma, one Sézary syndrome and another subcutaneous panniculitis-like T cell lymphoma of αß-phenotype. Immunohistochemistry was performed on paraffin sections using a commercially available antibody against Dicer. Intensity of expression was correlated with clinical parameters including disease specific survival (DSS) and time to progression (TTP). RESULTS: After a median follow-up of 74 months (range: 1-271), 12/50 patients (24%) have died. Univariate and multivariate analysis for disease-specific survival showed Dicer expression and stage as a negative predictive factor in the sole group of MF patients (n=34) as well as in the heterogeneous group of patients (n=50), but not gender, histological subtype, primary localization of disease, age and recurrence of lymphoma (p>0.05). CONCLUSION: Our data suggest Dicer expression as a possible molecular marker in patients with MF and apparently indicate that miRNA(s) might be of clinical relevance in CTCL.

Programmed cell death-10 enhances proliferation and protects malignant T cells from apoptosis.

The programmed cell death-10 (PDCD10; also known as cerebral cavernous malformation-3 or CCM3) gene encodes an evolutionarily conserved protein associated with cell apoptosis. Mutations in PDCD10 result in cerebral cavernous malformations, an important cause of cerebral hemorrhage. PDCD10 is associated with serine/threonine kinases and phosphatases and modulates the extracellular signal-regulated kinase pathway suggesting a role in the regulation of cellular growth. Here we provide evidence of a constitutive expression of PDCD10 in malignant T cells and cell lines from peripheral blood of cutaneous T-cell lymphoma (Sezary syndrome) patients. PDCD10 is associated with protein phosphatase-2A, a regulator of mitogenesis and apoptosis in malignant T cells. Inhibition of oncogenic signal pathways [Jak3, Notch1, and nuclear factor-κB (NF-κB)] partly inhibits the constitutive PDCD10 expression, whereas an activator of Jak3 and NF-κB, interleukin-2 (IL-2), enhances PDCD10 expression. Functional data show that PDCD10 depletion by small interfering RNA induces apoptosis and decreases proliferation of the sensitive cells. To our knowledge, these data provide the first functional link between PDCD10 and cancer.

Inhibition of IkappaB kinase subunit 2 in cutaneous T-cell lymphoma down-regulates nuclear factor-kappaB constitutive activation, induces cell death, and potentiates the apoptotic response to antineoplastic chemotherapeutic agents.

PURPOSE: A key molecular feature of cutaneous T-cell lymphomas (CTCL) is the constitutive activation of the nuclear factor-kappaB (NF-kappaB) transcription factor. We investigated in vitro the effects on CTCL survival and chemoresistance of a specific inhibition of IkappaB kinase subunit 2 (IKK2). EXPERIMENTAL DESIGN: Selective IKK2 inhibition was carried out by transfection of SeAx and MyLa CTCL lines with an inactive form of IKK2 and by exposing these lines and tumor cells from 10 patients with Sézary syndrome (SS) to AS602868, a new IKK2 inhibitor. The constitutive nuclear translocation of NF-kappaB was analyzed by electrophoretic mobility shift assay and confocal microscopy. Apoptosis was determined by Annexin V/propidium iodide-positive staining and mitochondrial transmembrane potential alterations as well as poly(ADP-ribose)polymerase cleavage. The expression of Bcl-2 family oncoproteins and survivin was studied by immunoblotting. RESULTS: Specific IKK2 inhibition resulting from transfection or from incubation with AS602868 allowed a down-regulation of NF-kappaB transcriptional activity. As shown by electrophoretic mobility shift assay and apoptosis assays, AS602868 down-regulated the nuclear translocation of NF-kappaB and induced a potent apoptotic response in CTCL lines and in tumor cells from patients with SS while preserving the viability of both peripheral blood lymphocytes from healthy donors and of nonmalignant T cells from SS patients. Moreover, CTCL death induction by conventional antineoplastic agents etoposide and vincristine was potentiated by AS602868. Finally, AS602868-induced apoptosis of CTCL cells was associated with an up-regulation of Bax dimers and a decrease of survivin. CONCLUSION: These results indicate that IKK2 inhibition represents a promising strategy for the treatment of advanced stages of CTCL.

Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis.

Sézary syndrome (Sz) is a malignancy of CD4+ memory skin-homing T cells and presents with erythroderma, lymphadenopathy, and peripheral blood involvement. To gain more insight into the molecular features of Sz, oligonucleotide array analysis was performed comparing gene expression patterns of CD4+ T cells from peripheral blood of patients with Sz with those of patients with erythroderma secondary to dermatitis and healthy controls. Using unsupervised hierarchical clustering gene, expression patterns of T cells from patients with Sz were classified separately from those of benign T cells. One hundred twenty-three genes were identified as significantly differentially expressed and had an average fold change exceeding 2. T cells from patients with Sz demonstrated decreased expression of the following hematopoietic malignancy-linked tumor suppressor genes: TGF-beta receptor II, Mxi1, Riz1, CREB-binding protein, BCL11a, STAT4, and Forkhead Box O1A. Moreover, the tyrosine kinase receptor EphA4 and the potentially oncogenic transcription factor Twist were highly and selectively expressed in T cells of patients with Sz. High expression of EphA4 and Twist was also observed in lesional skin biopsy specimens of a subset of patients with cutaneous T cell lymphomas related to Sz, whereas their expression was nearly undetectable in benign T cells or in skin lesions of patients with inflammatory dermatoses. Detection of EphA4 and Twist may be used in the molecular diagnosis of Sz and related cutaneous T-cell lymphomas. Furthermore, the membrane-bound EphA4 receptor may serve as a target for directed therapeutic intervention.

The serine protease granzyme M is preferentially expressed in NK-cell, gamma delta T-cell, and intestinal T-cell lymphomas: evidence of origin from lymphocytes involved in innate immunity.

Granzyme M (GM) is a novel serine protease whose expression is highly restricted to natural killer (NK) cells, CD3(+)CD56(+) T cells, and gamma delta T cells. Using a GM-specific monoclonal antibody, we analyzed the expression of GM in 214 mature T-cell and NK-cell lymphomas. GM was preferentially expressed in nasal NK/T-cell lymphomas (100%), gamma delta T-cell lymphomas (100%), and intestinal T-cell lymphomas (85%). In contrast, GM expression was present at low prevalence in mycosis fungoides/Sézary syndrome (3%), anaplastic large-cell lymphoma (6%), panniculitis-like T-cell lymphoma (11%), and angioimmunoblastic T-cell lymphoma (0%) cases. Peripheral T-cell lymphomas of unspecified subtype showed an intermediate frequency (37%) of GM expression, consistent with their heterogeneous origin. We conclude that GM expression is a distinctive feature of the nasal NK/T-cell, gamma delta T-cell, and intestinal T-cell lymphomas, and suggest that these tumors develop from lymphocytes involved in innate immunity.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Isoform patterns of lysosomal glycosidases in phenotypically different leukemic lymphoid cells.

Isoform patterns of six lysosomal glycosidases were studied in leukemic lymphoid cells phenotypically related to B and T cells at distinct stages of differentiation. In all types of cells, the activity of glycosidases under study was expressed in two major isoforms. No correlation was observed between isoform patterns and cell phenotypes. The beta-hexosaminidase isoform ratios for phenotypically related leukemic lymphoid cells but isolated from different sources (blood and spleen) differed. It was suggested that cell localization affects isoform expression. An anomalous alpha-mannosidase was detected in lymphoid cells from lymph nodes, while it was lacking in the phenotypically related blood lymphoid cells from the same patients. Isoform I of beta-hexosaminidase was recorded in lymphoid cells of patients with anemias.

[Protein kinase activity in lymphoid cells in various forms of lymphoproliferative disorders]. / Aktivnost' proteinaz v limfoidnykh kletkakh pri raznykh formakh limfoproliferativnykh zabolevanii.

Activities of dipeptidyl-aminopeptidase IV, urokinase-like plasminogen activator, cathepsins B and L were studied in lymphoid cells of patients with various forms of lymphoproliferative disorders. Activity of the enzymes studied was found in all the T- and B-cell, although rate and ratio of the enzymatic activity were dissimilar in various cell types. The highest rate of activity exhibited cells at early stages of maturation obtained from patients with acute lymphoblastic leukemia, while low level of the proteinase activity was detected in cells of patients with chronic lymphoid leukemia, non-Hodgkin lymphoma, hairy cell leukemia and Sezary disease, corresponding to mature T- and B-subpopulations. As shown by analysis of the cells immunological phenotype and their proteolytic activity, the rate of lymphoid cells differentiation correlated with level of proteinases activity. Series of proteinases were firstly studied in human malignant lymphoid cells with known phenotype. The enzyme assay may be used in diagnosis and treatment of patients with lymphoproliferative disorders.

[Dipeptidyl aminopeptidase-IV in lymphocytes of patients with lymphoproliferative diseases]. / Dipeptidilaminopeptidaza-IV v limfotsitakh bol'nykh limfoproliferativnymi zabolevaniiami.

DAP-IV activity (Gly-Pro-MCA hydrolysis, pH 7.8) was found in lysates of peripheral blood lymphocytes of patients with T- and B-cell forms of malignant lymphoproliferative diseases. The highest DAP-IV activity was seen in the cells of patients with a rare variant of T-cell lymphocytic leukemia (T-CLL); these cells expressed simultaneously the antigens of T helpers and T suppressors (Th and Ts) (OKT4+ and OKT8+). The DAP-IV activity about ten times less was found in the pathological cells with a phenotype of mature Th (Sezary disease), as well as in the cells expressing antigens of both Ts and natural killers (a rare variant of T-CLL). The same activity was also found in Ts (T gamma-lymphocytosis). The data obtained show that the differences in DAP-IV expression are connected with the differentiation step rather than with the belonging to a particular subpopulation of T-cells. DAP-IV activity, which was somewhat lower than that of T-cells, was found in B-lymphocytes of patients with B-CLL, hair-cellular leukemia, and non-Hodgkin's lymphoma. No correlation of DAP-IV activity with the level of E-cellular differentiation was observed.

Cytochemical method for demonstrating magnesium-activated adenosine triphosphatase in haemic cells.

A modified cytochemical method was developed for demonstrating magnesium-activated adenosine triphosphatase in haemic cells. The enzyme was shown in a variety of normal and leukaemic, unfixed cells. The high sensitivity of this enzyme to all fixatives tried in this study hampered further exploration and characterization of this enzyme.

Dipeptidylaminopeptidase IV (DAP IV) in B- and T-cell leukaemias.

A cytochemical study in samples from 100 lymphoid leukaemias, 84 of B-cell type and 16 of T-cell type, was carried out with three acid hydrolases: DAP IV, acid phosphatase (AP) and alpha-naphthyl acetate esterase (ANAE). DAP IV was studied in leukaemic T-cells both by cytochemistry and by a monoclonal antibody with the immunoperoxidase technique. Both methods showed similar results. AP and ANAE gave weak reactions in immature B-cell leukaemias (common-ALL and B-CLL) and were strongly expressed in plasma cell disorders. DAP IV showed no activity in any of the types of B-cell leukaemia studied and was strongly positive in some T-cell leukaemias but with a more restricted distribution than ANAE and AP. T-lymphoblasts (T-ALL) and mature (T8+) leukaemias were DAP IV negative. Within the T4+ malignancies DAP IV was positive in four T-prolymphocytic leukaemias, one of two T-CLL and one of three Sezary syndrome cases. Although DAP IV is strictly T-cell specific it does not appear to aid the differentiation between B- and T-cell disorders or the identification of T-cell subsets as determined by monoclonal antibodies. It remains to be established whether this enzyme will define a functionally distinct T-cell subset.

Phenotypic heterogeneity of leukemic Sézary cells.

Leukemic blood cells from eight patients with Sézary's syndrome were analyzed for enzyme cytochemical features, Fc receptors, and surface phenotype. Enzyme cytochemically the cases were heterogeneous in their activity of acid esterase, acid phosphatase and dipeptidylaminopeptidase IV (DAP IV). Only one case showed positive staining for DAP IV. The expression of Fc receptors for IgG and IgM also varied. The DAP IV-positive case exhibited Fc mu receptors. In contrast, four other cases showed only Fc gamma receptors. Analysis of the surface antigen pattern of Sézary cells with monoclonal antibodies revealed the phenotype of helper T lymphocytes (Leu-3 a/OKT4+) in all but one case. The Leu-3 a/OKT4-negative case showed a phenotypic feature of natural killer cells (Leu-7+). The results obtained with the antibodies TU14 and Anti-human Lyt-1 were more heterogeneous. The heterogeneity of Sézary cells may be interpreted as a sign of differences in functional differentiation or of proliferation of different T-cell subclones.

Dipeptidylaminopeptidase IV (DAP IV) activity in normal and malignant T-cell subsets as defined by monoclonal antibodies.

The activity of dipeptidylaminopeptidase IV (DAP IV) was investigated by a cytochemical method in isolated fractions of normal peripheral blood mononuclear cells and malignant cells from 9 patients with chronic T-cell leukaemia. A positive DAP IV reaction was observed in 52% of normal T cells, a consistent negative reaction in normal B cells and monocytes. 2 distinct T-cell subsets, helper/inducer cells (T4+/T8-) and cytotoxic/suppressor cells (T4-/T8+), were negatively selected by complement-mediated cytolysis utilizing the monoclonal antibodies OKT4 and OKT8. On examination of these T-cell subsets a positive DAP IV reaction was restricted to the majority of normal T4+/T8- cells. The malignant cells from 3 patients with T-chronic lymphocytic leukaemia, expressing the cytotoxic/suppressor phenotype (T4-/T8+) lacked DAP IV activities. In contrast, almost all leukaemic cells from 3 other cases, expressing the helper/inducer phenotype (T4+/T8-), were strongly positive. Despite their T4+/T8- phenotype, the malignant cells from 3 patients with Sézary syndrome were completely DAP IV negative. These findings suggest that the DAP IV reaction may be helpful in the further characterization of normal and malignant T-cell subsets.

Comparison of purine degradative enzymes and terminal deoxynucleotidyl transferase in T cell leukaemias and in normal thymic and post-thymic T cells.

Adenosine deaminase (ADA), ecto 5' nucleotidase (5'NT), purine nucleoside phosphorylase (PNP) and terminal deoxynucleotidyl transferase (TdT) were measured in the cells of patients with acute or chronic T cell leukaemia and compared with normal putative prothymocytes (large, blast-like cortical thymocytes), cortical and medullary thymocytes and peripheral blood T lymphocytes. Distinct patterns of enzyme activities were found in the individual types of T cell leukaemia. Mean ADA, TdT and 5'NT activities in thymic acute lymphoblastic leukaemia (Thy-ALL) were 41.9 u/10(8) cells, 31.1 u/10(8) cells and 4.7 u/10(6) cells respectively; in chronic T cell leukaemia they were 7.1 u/10(8) cells, 0.6 u/10(8) cells and 18.1 u/10(6) cells respectively. Mean PNP activity was similar between these two groups of leukaemia (68.6 u/10(6)cells in Thy-ALL and 77.9 u/10(6) cells in chronic T cell leukaemia). The activities of these four enzymes in OKT4+ chronic T cell leukaemia did not differ significantly from those in the OKT8+ chronic T cell leukaemia cases. The activities of TdT, ADA, PNP and 5'NT in Thy-ALL closely resembled those in normal immature thymocytes, and in the chronic T cell leukaemias showed a similar pattern of enzyme activities to that of mature T lymphocytes. These findings are consistent with surface phenotypic studies of T cell malignancies which suggest that different T cell leukaemias represent malignant proliferation of T cell clones arrested at different stages of T cell differentiation. They also demonstrate the value of biochemical markers in defining the different subtypes of acute and chronic leukaemia.

Purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) activities examined cytochemically in unfixed lymphocytes of patients with lymphoproliferative disorders.

New techniques have been devised for the cytochemical demonstration of purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) activities in unfixed human lymphocytes. A suspension of living lymphocytes is mixed with agarose sol containing the reagents for the detection of PNP or ADA activity on a glass slide. The mixture solidifies, is incubated, and then dried for lightmicroscopic observation. Reactive cells are recognized by the diffusely deposited granules of formazan, the end-product of the cytochemical reaction, and are divided into three groups of the cell with the low, middle, and high enzyme activity by the number of the granule. In healthy adults, the mean percentages of PNP- and ADA-positive cells were more than 90% in unfractionated lymphocytes, T-cell fractions, and complement-receptor cell fractions and cells with middle PNP and ADA activities were predominant. The PNP and ADA staining was observed in lymphoid cells of patients with lymphoproliferative disorders. A decrease in the percentage of PNP-positive cells concomitant with a relative increase of cells with the low enzyme activity was observed in the lymphocytes of nine patients with chronic lymphocytic leukemia (CLL). Similar findings were obtained in the ADA staining of the lymphocytes of five patients with B-cell CLL.