Pathogenesis Of CTCL Research Articles

Monitoring malignant T-cell clones by direct TCR expression assay in patients with leukemic cutaneous T-cell lymphoma during extracorporeal photopheresis.

Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n=15) and during therapy at 3months and 6 months (n=12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vβ family members. At baseline, DTEA detected TCR-Vβ clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vβ clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vβ clones, DTEA also detected additional TCR-Vβ clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVβ usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vβ and TCRVα usages over therapy were consistent with patients' clinical responses. Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vβ usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

The polymorphisms of IL-6/STAT3 signaling pathway may contribute to cutaneous T-cell lymphomas susceptibility

IL-6/STAT3 signaling pathway has been suggested to play a role in CTCL pathogenesis. Polymorphisms in STAT3 signaling pathway-related genes might be a risk factor for CTCL. However, the exact role of inherited gene polymorphisms of IL-6 and STAT3 in the pathogenesis of CTCL is still not fully understood. The aim was to examine whether IL-6 cytokine and polymorphisms of IL-6 and STAT3 gene are associated with CTCL susceptibility, stage of disease and pruritus intensity. We compared the IL-6 serum level and the frequency of selected single nucleotide polymorphisms of IL-6 and STAT3 in 106 CTCL and 198 control group using polymerase chain reaction with sequence-specific primers method and ELISA. We have found that serum IL-6 level in CTCL patients was significantly higher than in healthy controls (p < 0.05). We also demonstrated that two genotypes, CC of IL-6 and GG of STAT3, were overexpressed in CTCL patients compared to healthy controls, and that they increase the risk of malignancy development (OR = 1.8, p = 0.04 for IL-6 and OR 2.53, p = 0.0064 for STAT3). Moreover, the GG genotype of STAT3 polymorphism seems to be associated with lack of pruritus or mild pruritus in CTCL patients. Our results indicate that IL-6 is involved in pathogenesis of CTCL but not pruritus. Moreover, CC of IL-6 and GG genotype of STAT3 genes might be considered as the risk factor for development of CTCL.

Bnz-1, a Selective γ-Chain Cytokine Inhibitor Has Clinical Activity in Patients with Cutaneous T Cell Lymphoma By Selectively Blocking IL-2, IL-15 and IL-9 Signaling: Results of a Multicenter, Open-Label Phase 1 Trial

Background: Cutaneous T cell lymphoma is incurable with current therapies and there is an urgent need for more effective therapies. BNZ-1 is a pegylated peptide antagonist that binds to the common γc signaling receptor for the cytokines IL-2, IL-9 and IL-15. These cytokines, particularly IL-2 and IL-15, have been implicated in the pathogenesis of CTCL through activation of JAK/Stat signaling pathways, Therefore, we hypothesized that inhibition of the IL-2 and IL-15 signaling pathways in CTCL will induce antitumor activity in patients with CTCL. Methods: A multicenter, open-label Phase 1 study is ongoing to characterize the safety and tolerability of BNZ-1 (NCT03239392). Patients with a diagnosis of mycosis fungoides (MF) of any stage or Sézary syndrome (SS) are eligible for this trial. Pts are enrolled in sequential dose cohorts of 0.5 mg/kg, 1mg/kg, 2 mg/kg, and 4 mg/kg to receive intravenous dose of BNZ-1 to characterize safety, pharmacokinetics, pharmacodynamics, and evidence of antitumor activity. Infusions are administered weekly for four doses to evaluate for safety. Thereafter, patients are enrolled on an extension phase for 3 months of weekly dosing of BNZ-1. If patient attain a response, they are eligible for a long-term extension arm, as approved by the FDA. Blood samples are collected to assess the impact of BNZ-1 on the anti-tumor response. Results: pts with MF/SS (11 M/5F, median age 61 years, range 32-89) have been enrolled. Clinical stages include IB (n=6), IIA (n=1), IIB (n=6), IVA1 (n=2), IVB (n=1). Patients were previously treated with a median of 2 ( 1-5) topical therapies and 3 (1-11) systemic therapies. Single and sequential doses of weekly 1 mg, 2 mg, or 4 mg BNZ-1 infusions have been well tolerated. The most frequently reported adverse events were pruritus (n=9), fatigue (n=5) and dry skin (n=3). All treatment-related AEs were Grade 1 or 2 in severity. No SAEs or dose limiting toxicity have been observed to date. Notably reductions in mSWATs and CAILs was noted even in patients with advanced stage disease and/or with features of large cell transformation and folliculotropic subtype. Flow cytometry of peripheral blood at baseline and during treatment indicated activation of anti-lymphoma immune responses associated with the downregulatio of PD1. Concommittantly, excess expression of cytotoxic granules (perforin &amp; Granzyme B) has been downregulated, suggesting the silencing of inflammatory T-cell responses. Conclusions: These preliminary Phase 1 results suggest that pegylated BNZ-1 is well-tolerated and inhibition of IL-2 and IL-15 leads to clinical improvement in patients with CTCL. Evidence for the rejuvenation of anti-lymphoma immunity and a decreasing inflammatory responses was seen in cases showing clinical response consistent with our hypothesis. An expansion cohort in CTCL is currently underway to validate these promising early results. Disclosures Querfeld: Trillium: Consultancy, Other: Investigator, Research Funding; Soligenix: Other: Investigator; Celgene: Other: Investigator, Research Funding; Medivir: Consultancy; Elorac: Other: Investigator, Research Funding; miRagen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Investigator; Bioniz: Membership on an entity's Board of Directors or advisory committees, Other: Investigator; Kyowa: Membership on an entity's Board of Directors or advisory committees, Other: Investigator; Eisai: Other: Investigator; Helsinn: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Investigator; City of Hope Cancer Center and Beckman Research Institute: Employment. William:Techspert: Consultancy; Celgene Corporation: Consultancy; Kyowa Kirin, Inc.: Consultancy; Guidepoint Global: Consultancy; Defined Health: Consultancy. Brammer:Celgene: Research Funding; Seatlle Genetics: Honoraria, Speakers Bureau. Sokol:EUSA: Consultancy. Tagaya:Bioniz: Research Funding; Bioniz: Membership on an entity's Board of Directors or advisory committees. Frohna:Bioniz: Employment. Azimi:Bioniz: Employment. Zain:Seattle Genetics: Honoraria, Speakers Bureau; spectrum: Honoraria.

Argx-110 for Treatment of CD70-Positive Advanced Cutaneous T-Cell Lymphoma in a Phase 1/2 Clinical Trial

Argx-110 for Treatment of CD70-Positive Advanced Cutaneous T-Cell Lymphoma in a Phase 1/2 Clinical Trial

Ph 1 study of MRG-106, an inhibitor of miR-155, in CTCL.

2511Background: MRG-106 is an inhibitor of miR-155, a microRNA with a strong link to CTCL pathogenesis. The goals of this FIH study are to evaluate the safety, tolerability, PK, and efficacy of MRG...

Skin Microbiota as Antigenic Triggers for Cutaneous T Cell Lymphoma

Abstract Background Cutaneous T Cell lymphoma (CTCL) is a debilitating malignancy of lymphocytes that home to the skin, thereby manifesting in a variety of chronic skin lesions. CTCL is thought to arise from a combination of genetic, epigenetic and environmental factors, but specific triggers remain unknown. We propose a model in which adaptive immune responses to particular skin commensals evolve into clonal proliferation of T cells in genetically predisposed patients. Methods and Results 10 CTCL patients with active skin lesions were sampled at 3 time points (blood, stool and lesional vs non-lesional skin). Skin swabs of SLE patients and healthy donors served as control. Dual-index V1–3 and V4 16S rRNA sequencing was performed on the MiSeq platform and revealed significantly more Firmicutes in patient skin compared to controls. In addition, single bacterial colonies were cultured from 3 subjects. Full-length 16S rRNA sequencing demonstrated unique bacterial strains in lesional compared to non-lesional cultures. Malignant cutaneous T cells from patient biopsies were extracted and cultured in vitro for antigenic challenge with patient-isolated skin commensal candidates. One patient’s T cells showed significant proliferation to lesionally enriched B. safensis, while not responding to non-lesional bacteria. Discussion Dissecting cancer cell-microbiota interactions revealed potential skin commensal candidates involved in the pathogenesis of CTCL. Pathobionts enriched in lesional skin might act as antigenic drivers for progression to clonality similar to processes involved in H. pylori-induced MALT lymphoma, a paradigm that may be applicable to skin lymphomagenesis.

PAK1 overexpression promotes cell proliferation in cutaneous T cell lymphoma via suppression of PUMA and p21

BackgroundCutaneous T cell lymphoma (CTCL) comprises a heterogeneous group of skin-homing T cell tumors. The small guanosine triphosphate effector p21-activated kinase 1 (PAK1) plays an important role in many fundamental cellular functions, including cell motility, proliferation, and apoptosis. The expression of PAK1 is up-regulated in several types of human cancers. However, little is known about the role of PAK1 in the pathogenesis of CTCL. ObjectiveThe aim of this study was to evaluate the expression pattern and underlying mechanism of PAK1 in CTCL. MethodsQuantitative real-time polymerase chain reaction(qRT-PCR) was used to detect PAK1 mRNA expression in the peripheral blood mononuclear cells (PBMCs) of patients with CTCL. The expression of PAK1 protein in CTCL tumor tissues was determined by immunohistochemistry. CTCL cell lines were treated with a small molecule inhibitor of PAK1, p21-activated kinase inhibitor III (IPA3), at concentrations of 2, 3.5 and 5 μM for 24 h. Hut 78 and HH CTCL cells were transfected with lentiviral-based PAK1 gene knockdown vectors. We determined the effects of PAK1 knockdown on cell proliferation and apoptosis in CTCL cells by MTS assay and flow cytometry. Animal experiments were performed to investigate the effects of PAK1 knockdown on the growth of tumors in vivo. Transcriptomic sequencing was performed to detect the direct downstream targets of PAK1 silencing. Reverse transcription polymerase chain reaction and western blot analysis were applied to verify the results of the transcriptomic analysis. ResultsWe detected PAK1 overexpression in PBMCs and skin lesions from patients with CTCL compared with benign inflammatory dermatoses (BID). Knockdown of PAK1 inhibited cell proliferation and promoted spontaneous apoptosis. In addition, the inhibitory effect of IPA3 was validated in the CTCL cell lines. Additionally, mice injected with PAK1-silenced cells presented with a decreased rate of tumor growth compared with the control groups. Moreover, the mRNA and protein expression of PUMA (BBC3) and p21 (CDKN1A) were increased in PAK1-silenced Hut 78 and HH cells. ConclusionsOur data indicated that PAK1 is upregulated in CTCL. PAK1 silencing induced apoptosis and inhibited cell growth by stimulating the expression of PUMA and p21. Thus, PAK1 may be a potential tumor marker and therapeutic target of CTCL.

Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of-function alteration in RLTPR (p.Q575E)

AbstractCutaneous T-cell lymphoma (CTCL) is an incurable non-Hodgkin lymphoma of the skin-homing T cell. In early-stage disease, lesions are limited to the skin, but in later-stage disease, the tumor cells can escape into the blood, the lymph nodes, and at times the visceral organs. To clarify the genomic basis of CTCL, we performed genomic analysis of 220 CTCLs. Our analyses identify 55 putative driver genes, including 17 genes not previously implicated in CTCL. These novel mutations are predicted to affect chromatin (BCOR, KDM6A, SMARCB1, TRRAP), immune surveillance (CD58, RFXAP), MAPK signaling (MAP2K1, NF1), NF-κB signaling (PRKCB, CSNK1A1), PI-3-kinase signaling (PIK3R1, VAV1), RHOA/cytoskeleton remodeling (ARHGEF3), RNA splicing (U2AF1), T-cell receptor signaling (PTPRN2, RLTPR), and T-cell differentiation (RARA). Our analyses identify recurrent mutations in 4 genes not previously identified in cancer. These include CK1α (encoded by CSNK1A1) (p.S27F; p.S27C), PTPRN2 (p.G526E), RARA (p.G303S), and RLTPR (p.Q575E). Last, we functionally validate CSNK1A1 and RLTPR as putative oncogenes. RLTPR encodes a recently described scaffolding protein in the T-cell receptor signaling pathway. We show that RLTPR (p.Q575E) increases binding of RLTPR to downstream components of the NF-κB signaling pathway, selectively upregulates the NF-κB pathway in activated T cells, and ultimately augments T-cell-receptor-dependent production of interleukin 2 by 34-fold. Collectively, our analysis provides novel insights into CTCL pathogenesis and elucidates the landscape of potentially targetable gene mutations.

Abstract 481: Diminished microRNA-29b results in overexpression of BRD4 and BRD4-regulated oncogenes in cutaneous T-cell lymphoma

Abstract Cutaneous T-cell lymphoma (CTCL) is a CD4+ T-cell malignancy that affects the skin but may disseminate systemically, and there are no long-term effective therapeutic strategies. Here, we characterized the therapeutic efficacy of manipulating the tumor suppressor microRNA-29b (miR-29b) and its target bromodomain-containing protein 4 (BRD4) in CTCL pathogenesis. Using primary CD4+ T-cells we determined that miR-29b expression is significantly decreased in patients compared to healthy controls (0.007±0.002, n=9 vs 1.008±0.052, n=6, p&amp;lt;0.0001). We utilized miR-29b-/- mice and bortezomib, a proteasome inhibitor known to increase miR-29b levels, to confirm the inverse relationship between miR-29b and BRD4 (Mishra, A. et al. Cancer Cell, 2012). Diminished miR-29b level resulted in increased BRD4 protein expression (1.87±0.29, p=0.014), while increase in miR-29b, as in bortezomib-treated CTCL cell lines, results in nearly undetectable BRD4 protein. We also observed increased genome-wide occupancy of BRD4 at regulatory regions in CTCL patients, an effect that is reversed with BRD4 inhibitor, JQ1, to similar levels as observed in healthy control CD4+ T-cells. Specifically, oncogene loci of NOTCH1 and RBPJ demonstrated enhanced BRD4 binding in CTCL patients, with resultant increases in mRNA in patient vs normal donors (4.16±0.98, p=0.024; 3.02±0.54, p=0.012). Further, in vivo treatment of interleukin-15 (IL-15) transgenic mice (Mishra, A. et al. Cancer Discovery, 2016) with JQ1 prevented CTCL development and decreased lesion severity (3.37±0.49, n=8 vs 6.0±0.45, n=5, p=0.004). Skin from JQ1-treated mice demonstrated decreased protein expression of BRD4, NOTCH1, and RBPJ, consistent with our findings in patients and CTCL cell lines. Bortezomib treatment of CTCL mice yielded similar results to JQ1 in vivo, while miR-29b levels were elevated in treated mice vs control animals. Since IL-15 signals through its receptor complex, we evaluated and observed increased BRD4 binding at IL-15 receptor αβγ gene loci in CTCL patients, which was reversed with both JQ1 and bortezomib treatment. Thus, we conclude that diminished miR-29b results in increased expression and subsequent binding of BRD4 at regulatory regions of known oncogenes in CTCL cells including NOTCH1, RBPJ, and the IL-15 receptor complex. We confirm the potential therapeutic utility of targeting this pathway in vivo, by direct displacement of BRD4 by JQ1, or by rescue of miR-29b expression by bortezomib. We therefore describe a novel targetable oncogenic pathway featuring IL-15, miR-29b, and BRD4 in CTCL. Citation Format: Rebecca Kohnken, Jing Wen, Bethany Mundy-Bosse, Max Yano, Leah Grinshpun, Kathleen McConnell, Ashleigh Keiter, Alex Hartlage, James Bradner, Michael Caligiuri, Pierluigi Porcu, Anjali Mishra. Diminished microRNA-29b results in overexpression of BRD4 and BRD4-regulated oncogenes in cutaneous T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2017-481

The Prevalence of Chlamydophila pneumoniae in the Blood Samples of Patients with Primary Cutaneous Lymphomas

Microbial infection and associated super antigens have been implicated in the pathogenesis of CTCL, and many patients die from complicating bacterial infections. It has been postulated that Chlamydophila pneumoniae infection may be involved in the pathogenesis of Mycosis fungoides (MF) but published data are limited and controversial. Aim of the study: The aim of the study was to analyze the frequency of C. pneumoniae DNA presence in blood samples of cutaneous T-cell and B-cell lymphomas (CTCL, CBCL) cases. Material and Methods: Using Q-PCR method we analyzed the presence of C. pneumoniae DNA in the blood samples obtained from 57 patients with CTCL (55-MF/Sezary Syndrome (SS), 1-primary cutaneous anaplastic large cell lymphoma (CD30+) and 1-NKT cell lymphoma) and 3 patients with cutaneous B-cell lymphomas and 120 individuals from control groups (40 patients with psoriasis, 40 patients with atopic dermatitis and 40 healthy controls). Results: C. pneumoniae DNA was identified in 13 of 55 cases in MF/SS group (23, 6%), in one patient with CD30+ large cell lymphoma and in 1 of 3 patients with B-cell lymphoma. The presence of C. pneumoniae was confirmed in 1 of 40 psoriatic patients (2, 5%), in 5 of 40 patients with atopic dermatitis (12, 5%) and in none of 40 healthy individuals. The frequency of C. pneumoniae DNA occurrence in MF patients group was strongly associated with the progression of the disease; rs = 0.756; p=0.0123 for groups IA→IVB, also for MF + SS patients divided by stages, the presence of C. pneumoniae was noted more frequently in advanced (III + IV) stages than in early (I-II) stages (p=0.0139). There are no differences in the main age of MF/SS patients with and without infection. Conclusion: Our results indicated that the presence of C. pneumoniae DNA in the blood cells is frequent event in late stages of MF/SS and may be explained by Th2 shift and suppression of immune system during the course of the disease.

Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here, we report that patients with CTCL show increased IL15 in a clinical stage-dependent manner. Mechanistically, we show that ZEB1 is a transcriptional repressor of IL15 in T cells and that hypermethylation of the ZEB1 binding region within the IL15 promoter, as seen in patients with CTCL, prevents ZEB1 binding and causes increased transcription of IL15 Using a transgenic mouse model of IL15, we provide evidence that overexpression of IL15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL15 in T cells inhibits an HDAC1-mediated negative autoregulatory loop, resulting in the upregulation of HDAC1 and HDAC6 and transcriptional induction of the onco-miR-21. Interruption of IL15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides preclinical evidence supporting a hierarchical model of oncogenic signaling in CTCL. To date, CTCL pathogenesis remains unknown, and there are no curative therapies. Our findings not only demonstrate a critical role for IL15-mediated inflammation in cutaneous T-cell lymphomagenesis, but also uncover a new oncogenic regulatory loop in CTCL involving IL15, HDAC1, HDAC6, and miR-21 that shows differential sensitivity to isotype-specific HDAC inhibitors. Cancer Discov; 6(9); 986-1005. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

Epigenetic Silencing of Mir-203 Contributes to IL2Rb Overexpression and Malignant Transformation in Cutaneous T-Cell Lymphoma

IntroductionCutaneous T-cell lymphomas (CTCL) are rare malignancies, which present in the skin and presumably arise from malignant transformation of T-cells normally destined to home to the cutaneous environment. MicroRNAs (miRs) regulate gene expression at the post transcriptional level. Many studies have shown that altered miR expression is a central event in lymphomagenesis, and that miRs have potential as both diagnostic and predictive tumor markers.In CTCL we have previously identified and validated a 3 miR classifier that distinguishes CTCL from BID with > 95% accuracy, based upon the up-regulation of miR-155 combined with the down-regulation of miR-203 and miR-205. In normal adult tissues, miR-203 is mainly associated with keratinocyte differentiation, acting to repress stemness, and to induce cell cycle arrest and differentiation. In cancer, miR-203 has been shown to hold tumor suppressor properties, and may be down-regulated by promoter hyper-methylation. The function and implications of miR-203 for CTCL has not previously been described. In this study we have investigated the regulation and function of miR-203 in primary CTCL biopsies and cell lines.Materials and MethodsTwenty-one fresh frozen primary CTCL biopsies, IL-2 independent CTCL cell lines (MyLa2059 and PDB2B), and the IL-2 dependent CTCL cell lines (SeAx and SeZ4) were analyzed in this study. Promoter methylation was analyzed by methylation specific melting curve analysis. Cell lines were transfected by electroporation of miR-203 mimic or non-template-control (mirVana, Ambion). Proliferation was measured by 3H-Thymidine and apoptosis by MMT assays. MiR-203 mimic and mock transfected cells were examined by Affymetrix RNA expression arrays (GeneChip Human Gene 2.0 ST). IL2Rβ mRNA expression was confirmed by qPCR and IL2Rβ protein levels by flow cytometry as measured by CD122 (IL2Rβ-chain), compared to CD25 (IL2Rα-chain) and CD132 (IL2Rγ-chain). Cloning was done according to the manufacturers’ recommendation (In-Fusion, Clontech) and luciferase reporter assays were performed using the Dual-Glo system (Promega).ResultsWe show that miR-203 is epigenetically silenced by DNA methylation in both CTCL cell lines and in 9 of 21 (43%) of primary CTCL samples, and that miR-203 can be up-regulated by the hypo-methylating agents 5-azacytidine and 5-aza-2-deoxycytidine in vitro. We also show, that forced miR-203 expression in CTCL cells targets known oncogenes such as p63, Survivin and CREB. Furthermore, it is shown that induction of miR-203 reduces cell viability and decreases proliferation.mRNA array analysis of miR-203 mimic and mock transfected cells lead to the identification of 19 significantly de-regulated genes (P<0.5/log fold change>2), including the as yet unrecognized miR-203 target molecule IL2Rb, which is essential for IL-2 induced JAK/STAT signaling. qPCR and FACS analysis confirmed this up-regulation both at the mRNA and protein level. The IL-2 dependent cell line SeAx showed significantly more profound down-regulation of IL2Rβ upon in miR203 transfected cell lines. Preliminary luciferase reporter assays confirm that IL2Rβ expression is regulated by miR-203, providing novel evidence that miR-203 may act in concert with IL-2/STAT in CTCL pathogenesis. These experiments are currently being validated.ConclusionWe provide the first evidence that miR-203 acts as a tumor suppressor in CTCL. Furthermore we show that down-regulation of miR-203 leads to increased expression of an as yet unidentified target gene, IL2Rβ, which is directly involved in JAK/STAT signaling, that plays an essential role in the regulation of T-cell proliferation. Thus, we suggest that epigenetic miR-203 down-regulation and IL2Rβ up-regulation are important early and driving events in CTCL pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

Role of IL-13 and its receptors in the pathogenesis of cutaneous T-cell lymphomas (TUM7P.961)

Abstract Cutaneous T-cell lymphomas (CTCL) affect primarily skin and are characterized by proliferation of mature CD4+ T-helper cells. Diagnose is difficult in the absence of specific markers for malignant lymphocytes. The pattern of cytokine production in the skin and blood is considered to be of major importance for the pathogenesis of CTCL. Abnormal cytokine expression in CTCL may be responsible for enhanced proliferation of the malignant cells and/or the depression of the anti-tumor immune response. Cytokine IL-13 promotes growth or survival of several tumors as well as suppresses immunosurveillance. Here we show that IL-13 and its receptors IL-13Rα1 and IL-13Rα2 are highly expressed in the clinically involved skin of CTCL patients. We also show that the tumor cells, identified by the expression of CD4 and TOX (thymus high-mobility group box), in the skin and blood of CTCL patients produce IL-13 and express both receptors. This finding is intriguing since CTCL is a T-cell origin tumor yet T cells do not express IL-13 receptors. Thus IL-13 is implicated as a possible autocrine factor for CTCL. Furthermore expression of the decoy IL-13Rα2 by CTCL cells may represent a novel marker of CTCL malignancy. Soluble human IL-13Rα2 and an anti-IL-13 monoclonal antibody are being tested for inhibition of tumor cell proliferation in the peripheral blood of patients. Altogether these results identify potential tumor markers for the early diagnosis of CTCL as well as novel therapeutic targets.

PLCG1 mutations in cutaneous T-cell lymphomas

AbstractCutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR–signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

Abstract 3857: Targeting HDAC1 in a novel model of cutaneous T-cell lymphoma.

Abstract Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma composed of a spectrum of diseases of malignant clonal helper CD4+ T lymphocytes. Previous studies have shown that IL-15 acts as viability factor for CTCL cells, inhibiting apoptosis, and may work in association with other factors to maintain the malignant infiltrate in the epidermis. IL-15 is highly expressed both at mRNA and protein levels in the skin of CTCL patients. Using an IL-15 transgenic (tg) mouse model for spontaneous CTCL that clinically and pathologically mimics the human malignancy, we described a system for studying lymphomagenesis in CTCL (97th Annual Meeting of the AACR; 2010. Abstract nr. 2858). Skin lesions in these mice revealed the presence of characteristic Pautrier's microabscesses and these mice had poor survival. Cutaneous CD4+ T-cells from these mice were capable of engrafting into the skin of secondary SCID recipients, producing histological lesions of similar pathology. Consistent with human CTCL, we found that these tg mice are predominantly CD62L- (P=0.0009, N=14) with overexpression of CCR4, CLA, and CD44 (P=0.0001, N=14). Interestingly, we also observed elevated histone deacetylase-1 (Hdac1) expression in IL-15 tg mice compared to the wild type (WT) cohort (P=0.0013, N=3). We hypothesized that IL-15 regulates epigenetic changes in normal CD4+ lymphocytes by modulating Hdac1 and Hdac6. We analyzed normal human- and WT mouse- CD4+ T-cells and found that IL-15 induces upregulation of Hdac1 protein and cytoplasmic translocalization of Hdac6. To elucidate the mechanism controlling Hdac1 induction, we tested whether IL-15 stimulation leads to autoregulation of Hdac1 in vitro. Chromatin immunoprecipitation (ChIP) PCR analysis of IL-15 stimulated CD4+ T-cells revealed that Hdac1 binds to its own promoter as early as 12 hours post incubation increasing its own transcription. Similar observations were made in CD4+ T-cells from CTCL patients when compared to normal donor CD4+ T-cells suggesting that Hdac1 could be upregulated in CD4+ T-cells as a result of autoregulation from endogenous IL-15. To test Hdac-inhibitor therapy in IL-15 tg CTCL mice, we performed a randomized, placebo-controlled trial of an oral HDAC inhibitor invented at The Ohio State University, AR-42 (Arno Therapeutics Inc., Flemington, NJ). Interestingly, 100% of mice treated with AR-42 showed clinical improvement and reduced dermal lymphocytic infiltration after the treatment when compared to the placebo arm. In summary, our study provides a murine model of spontaneous CTCL that mimics the human disease, and also, demonstrate for the first time IL-15 mediated autoregulation of a histone-modifying enzyme Hdac1 in CD4+ T-cells that may contribute towards pathogenesis of CTCL. Finally, we show that AR-42 has significant efficacy in this preclinical CTCL model, which has in part paved the way for its current evaluation in a Phase I human clinical trial. Citation Format: Anjali Mishra, Laura Sullivan, Gregory Sams, Jessica Johns, Douglas Curphey, Lauren Falkenberg, Heather Gibson, Shujun Liu, Laura Jaroncyk, Kathleen McConnell, Henry Wong, Krista La Perle, Ramiro Garzon, Guido Marcucci, Pierluigi Porcu, Michael Caligiuri. Targeting HDAC1 in a novel model of cutaneous T-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3857. doi:10.1158/1538-7445.AM2013-3857

Abstract 51: Activating PLCG1 mutations in cutaneous T-cell lymphomas.

Abstract Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of diseases characterized by clonal expansion of malignant T-cells in the skin. The two predominant clinical forms of CTCL are mycosis fungoides (MF) and Sezary syndrome (SS). Tumor-stage MF has an unfavorable prognosis with a 10-year survival of approximately 40%. Despite the molecular pathogenesis of CTCL is still basically unknown, some data including Gene expression profiling (GEP) studies have shown that increased signaling from the T-cell receptor (TCR) can be considered a driving force of CTCL and that the neoplastic T-cells can acquire Treg and Th17 phenotypes. However, the molecular mechanisms responsible for this activation have not been fully clarified. Based on the hypothesis that deregulated TCR activity may depend, at least in part, on somatic mutations, we have studied the coding exons of a selection of genes with known biological relevance in T-cells, including TCR and MAPK signaling, cytokine activity and survival/apoptosis regulators. By means of deep sequencing we analyzed paired (non tumoral vs. tumoral) genomic DNA from 11 CTCL patients. Under these premises, we first prepared enriched libraries for exons and regulatory regions for 524 genes belonging to the aforementioned pathways, and then proceeded to sequence with a Genome Analyzer GA2 (Illumina). The sequencing data was checked by FastQC and aligned to the human reference genome (GRCh37) and somatic variants were identified using GATK. Candidate variants were manually reviewed and validated by capillary sequencing. Our results showed multiple somatic mutations in essential genes implicated in the biology of T cells. Strikingly, we found recurrent mutations in PLCG1 affecting 3/11 patients (two of them sharing the same mutation) in our cohort. This protein can be considered a mayor effector of TRC signaling as well as to play a central role in the differentiation processes of T-cells towards the acquisition of a Treg/Th17 phenotype. Using a combination of PLCG1-targeted DNA sequencing and immune-staining in FFPE patient samples, we corroborated that up to 20% of a new cohort of 60 patients harbored the recurrent PLCG1 mutation. Moreover we found a correlation between the presence of this mutation and the nuclear accumulation of activated NFAT, a well-known PLCG1downstream effector, suggesting that these mutations may confer a gain of function activity to this enzyme. We corroborated this observation by comparing the activity of exogenous expression of WT and mutant PLCG1 proteins towards the activation of NFAT using a luciferase reporter gene assay. Moreover we also found that the resultant activity from these mutants was highly sensitive to the specific inhibition of CaN and PLC in our system which may suggest that the finding of these mutations in CTCL patients might be used as an indicative for targeted therapy. Citation Format: Jose P. Vaque, Gonzalo Gomez-Lopez, Veronica Monsalvez, Sagrario Gomez de Benito, Ignacio Varela, Nerea Martinez, Laura Cereceda, Soraya Curiel, Osvaldo Graña, Maria S. Rodriguez-Pinilla, Carmen M. Gonzalez-Vela, Jose L. Rodriguez-Peralto, Miriam Rubio-Camanillo, Esperanza Martin, Miriam Mendez, Jose A. Garcia-Marco, Mariano Provencio, Gaston Roustan, Mercedes Hospital, Dolores Suarez, Orlando Dominguez, Pablo Ortiz-Romero, Miguel A. Piris, Margarita Sanchez-Beato. Activating PLCG1 mutations in cutaneous T-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 51. doi:10.1158/1538-7445.AM2013-51

Role of STAT3 and Th17 Cells in Cutaneous T Cell Lymphoma

AbstractAbstract 66 BackgroundCutaneous T cell Lymphoma (CTCL) is a heterogeneous group of non-Hodgkin's lymphomas characterized by skin-homing malignant T cells. The etiology of CTCL remains enigmatic, but constitutive Signal Transducer and Activator of Transcription 3 (STAT3) activity is characteristic of this malignancy. CTCL cell lines undergo growth arrest and apoptosis following inhibition of STAT3 signaling, suggesting that STAT3 may be a pro-survival factor in these cells. STAT3 phosphorylation is also required for the initiation and maintenance of the Th17 differentiation program, a recently identified subset of CD4+ T helper cells, implicated in a number of chronic inflammatory conditions including rheumatoid arthritis and psoriasis. Furthermore, we know that Histone Deacetylase inhibitors (HDACi) namely Vorinostat and Romidepsin are very active agents for treatment of CTCL but their mechanism of action is still to be elucidated. Our goal is to probe the contribution of Th17 mediated inflammation in CTCL and to examine whether HDAC inhibitors work by modulating Th17 inflammation in the context of this malignancy. MethodsUsing conditional gene targeting approach we have generated a transgenic 100% penetrant mouse model of CTCL wherein expression of a hyper-active STAT3 mutant protein (STAT3C) selectively in T lymphocytes results in skin pathology highly reminiscent of this human malignancy. To have a better understanding of the contribution of STAT3 in CD4 T cell differentiation program and CTCL, we used lymphocytes and splenocytes from 8 weeks old STAT3Cstopfl/fl (without CD4Cre, thus no pathology) and control YFPstopfl/fl (Yellow Fluorescent Protein floxed) mice (Figure 1a). We isolated naïve CD4 cells using Dynabeads and treated these CD4 cells with a transducible Cre enzyme (Tat-Cre) to delete the upstream stop cassette ex-vivo. These cells were then plated with stimulating α CD3/CD28 antibodies in various cytokine cocktails as shown in Figure 1b. These cells were harvested after 84 hours and stimulated with PMA/Inomycin for 4 hours for flowcytometric analysis. We are now using this in-vitro approach as well as our mouse model and primary tissue samples from CTCL patients to explore the impact of HDAC inhibitors on Th17/Treg balance. ResultsUpon Tat-Cre treatment we were able to analyze GFP+ (and therefore STAT3C expressing) vs. GFP- (control) T cells in the same differentiation conditions. Hyperactive STAT3C mutant protein favored TH17 differentiation as GFP Positive cells had significantly augmented IL17A production (Figure 1b). On the other hand, STAT3C expressing GFP positive cells in Th0 and Th1 differentiation conditions had less IFN-γ production compared to the GFP negative cells and control YFP fractions. Even the GFP negative fraction of STAT3C had less IFN-γ and more IL17A production than the control YFP fractions due to the cytokine milieu created by STAT3C expressing cells present in the same well. Conclusions:These results highlight an intriguing possibility that STAT3 dependent Th17 cells play a role in CTCL pathogenesis. Using our newly developed mouse model and CTCL patients' blood and skin samples (IRB approved), we are trying to distinguish whether Th17 cells are instigators of chronic inflammation that contributes to the malignant transformation of T cells or that Th17 cells may actually be the cells of origin in this malignancy. Our ongoing experiments also include the effects of HDACi on this ex-vivo CD4 T cell differentiation. In addition, we are studying the hallmarks of Th17 differentiation namely IL-17A, IL-22 and pSTAT3 positivity in the skin biopsy and blood samples of CTCL patients before and after treatment with HDACi. These experiments will help us determine the impact of HDAC inhibition on STAT3 activity and Th17 differentiation and have a better understanding of their mechanism of action in CTCL. Our hope is that our study will guide us to comprehend the contribution of chronic inflammation in carcinogenesis and potentially identify novel treatment targets and strategies in the field of hematological malignancies. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Classification and molecular pathogenesis of CTCL

Classification and molecular pathogenesis of CTCL

Absence of Human T-Lymphotropic Virus Type I in Japanese Patients With Cutaneous T-Cell Lymphoma

AbstractCutaneous T-cell lymphoma (CTCL) is a disease entity characterized by a primary sporadic T-cell proliferation in the skin. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma. Recently, several authors have detected the HTLV-1 genome in genomic DNA from patients with CTCL and proposed a causal relation of HTLV-1 to CTCL. However, it remains controversial because these studies contain some problems in materials used to detect HTLV-1. We investigated both fresh and cultured T lymphocytes (128 specimens) derived from 50 Japanese patients with CTCL, where HTLV-1 is endemic, by using polymerase chain reaction with four sets of primers including gag, pol, env, and pX regions of HTLV-1 to elucidate the relationship between HTLV-1 and CTCL in Japan. However, none of the 128 DNA specimens revealed positive for HTLV-1 in contrast to the previous studies. We conclude that CTCL, which does not include HTLV-1, is present although the pathogenesis of CTCL may be different by areas or races. © 1997 by The American Society of Hematology.