Willem H. Zoutman

MicroRNA-21 Expression in CD4+ T Cells Is Regulated by STAT3 and Is Pathologically Involved in Sézary Syndrome

MicroRNAs (miRNAs) are small RNAs that control gene expression, and are involved in the regulation of fundamental biological processes including development, cell differentiation, proliferation, and apoptosis. miRNAs regulate gene expression in normal hematopoiesis, and aberrant miRNA expression might contribute to leukomogenesis. Specifically, miR-21 is abundantly expressed in various tumors including leukemia and lymphoma, and is functionally involved in oncogenic processes. We investigated a role for miR-21 in Sézary Syndrome (SS), a cutaneous T-cell lymphoma (CTCL) with CD4+ tumor cells (Sézary cells) present in the skin, lymph nodes, and peripheral blood. It was shown previously that SS is characterized by constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling. In this study we show by chromatin immunoprecipitation that miR-21 is a direct STAT3 target in Sézary cells. Stimulation of Sézary cells or healthy CD4+ T cells with the common-γ chain cytokine IL-21 results in a strong activation of STAT3, and subsequent upregulation of miR-21 expression. Both pri- and mature miR-21 expression are increased in Sézary cells when compared with CD4+ T cells from healthy donors. Silencing of miR-21 in Sézary cells results in increased apoptosis, suggesting a functional role for miR-21 in the leukomogenic process. Consequently, miR-21 might represent a therapeutic target for the treatment of SS.

A Meta-Analysis of Gene Expression Data Identifies a Molecular Signature Characteristic for Tumor-Stage Mycosis Fungoides

Mycosis fungoides (MF) is the most common type of primary cutaneous T-cell lymphoma (CTCL). To identify a molecular signature characteristic of MF tumor stage, we used a bioinformatic approach involving meta-analysis of publicly available gene expression data sets combined with previously generated gene expression data. Results for a selection of genes were further refined and validated by quantitative PCR and inclusion of additional controls. With this approach, we identified a profile specific for MF tumor stage, consisting of 989 aberrantly expressed genes, the majority of which (718 genes) are statistically significantly more expressed in MF compared with normal skin, inflamed skin, and normal T cells. As expected, the signature contains genes reflecting the highly proliferative characteristic of this T-cell malignancy, including altered expression of cell cycle and kinetochore regulators. We uncovered details of the immunophenotype, suggesting that MF originates from IL-32-producing cells and identified previously unreported therapeutic targets and/or diagnostic markers, for example, GTSF1 and TRIP13. Loss of expression of the NF-κB inhibitor, NFKBIZ, may partly explain the enhanced activity of NF-κB, which is a hallmark of MF and other CTCLs.

Fine-Mapping Chromosomal Loss at 9p21: Correlation with Prognosis in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT) is the most aggressive type of primary cutaneous B-cell lymphoma. In a recent study on 12 patients it was found that inactivation of CDKN2A by either deletion of 9p21.3 or promoter hypermethylation is correlated with a worse prognosis. In the present EORTC multicenter study, skin biopsies of 64 PCLBCL, LT patients were analyzed by multiplex ligation-dependent probe amplification to validate these previous results and to fine-map the losses in this region. Although no minimal common region of loss could be identified, most homozygous loss was observed in the CDKN2A gene (43 of 64; 67%) encoding p16 and p14ARF. Promoter hypermethylation of p16 and p14ARF was found in six and zero cases, respectively. Survival was markedly different between patients with versus without aberrations in the CDKN2A gene (5-year disease-specific survival 43 versus 70%; P=0.06). In conclusion, our results confirm that deletion of chromosome 9p21.3 is found in a considerable proportion of PCLBCL, LT patients and that inactivation of the CDKN2A gene is associated with an unfavorable prognosis. In most patients the deletion involves a large area of at least several kilobase pairs instead of a small minimal common region.

Exploring the IL-21-STAT3 Axis as Therapeutic Target for Sezary Syndrome

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.

Diagnostic and prognostic significance of CDKN2A/CDKN2B deletions in patients with transformed mycosis fungoides and primary cutaneous CD30-positive lymphoproliferative disease

DEAR EDITOR, Large-cell transformation in mycosis fungoides (MF) has been associated with an aggressive clinical course and poor survival. However, not all patients with transformed MF run an aggressive clinical course. In a recent study, multivariate analysis revealed four risk factors that were independently associated with reduced survival. These included transformation at extracutaneous sites, the extent of skin lesions showing transformation, folliculotropic MF and negative staining for CD30. Histologically, differentiation between CD30+ transformed MF and primary cutaneous CD30+ lymphoproliferative disease (LPD), including primary cutaneous anaplastic large-cell lymphoma (C-ALCL) or lymphomatoid papulosis (LyP), may be very difficult, in particular when the infiltrates contain a diffuse proliferation of CD30+ blast cells. Differentiation is important, as both C-ALCL and LyP have an excellent prognosis, with 10-year survival rates of 90% and almost 100%, respectively. Furthermore, they require a different therapeutic approach from that in patients with transformed MF. However, reliable (bio)markers allowing differentiation between these types of cutaneous T-cell lymphomas are not yet available. Several studies have reported frequent loss of chromosomal region 9p21.3 in patients with MF. This region contains the CDKN2A and CDKN2B tumour suppressor genes, which encode cell-cycle proteins P16 and P14ARF, and P15, respectively. It has been shown that loss of CDKN2A/CDKN2B in MF is associated with reduced survival. However, it is unknown whether its prognostic value is independent of other known risk factors, such as CD30 expression. Studies investigating 9p21.3 loss in C-ALCL show conflicting results, and the CDKN2A/CDKN2B status in rare cases of C-ALCL or LyP developing systemic disease is unknown. In the present study genetic or epigenetic inactivation of CDKN2A/CDKN2B was investigated by Multiplex Ligation-dependent Probe Amplification (MLPA) in 54 patients with transformed MF and 25 patients with a CD30+ LPD. The aims of this study were to find out whether loss of CDKN2A/CDKN2B is an independent prognostic parameter in transformed MF, to assess the frequency and prognostic significance of this loss in CD30+ LPD, and in particular to find out whether there are differences in CDKN2A/CDKN2B loss between CD30+ transformed MF and CD30+ LPD, which may be used as an additional differential diagnostic marker. Fifty-four patients with transformed MF were selected from a recent study, in which large-cell transformation was defined by the presence of large T-cells exceeding 25% of the total lymphoid infiltrate or forming microscopic nodules. For the present study only those cases in which the skin biopsy contained at least 70% neoplastic T cells were selected. This group included 36 men and 18 women (male-to-female ratio 2 : 1), with a median age at the time of transformation of 66 years (range 33–87). Other relevant parameters are presented in Table 1. At the end of follow-up, 11 of 54 patients were still alive; 34 had died of lymphoma and nine of unrelated disease. The median survival after transformation was 33 months (range 2–291) and the 5-year disease-specific survival (DSS) and overall survival (OS) were 38% and 34%, respectively. For the detection of both copy number and methylation changes in the 9p21.3 CDKN2A/CDKN2B region, a commercially available MLPA Kit (SALSA MLPA Kit ME024; MRC-Holland, Amsterdam, the Netherlands) was used according to manufacturer’s recommendations (Fig. 1). In 30 of 54 cases (56%) of transformed MF a hemizygous (n = 15) or homozygous deletion (n = 15) of CDKN2A/CDKN2B was found, and in most cases both genes, encoding P16, P14ARF and P15, were affected. The median survival times were 17 months (range 2–69) for patients with a homozygous deletion, 21 months (range 5–119) for patients with a hemizygous deletion and 55 months (range 5–261) for patients without 9p21.3 loss. The 5-year DSS rates for these three groups were 10%, 29% and 61%, respectively (P = 0 003). These results are consistent with previous studies, which reported loss at 9p21.3 in 40–70% of cases of transformed MF. Methylation of the P16 promoter (two cases) or P15 promoter (four cases) was detected in only six of 54 cases. No significant difference in CDKN2A/CDKN2B deletions was found between CD30+ (nine of 19, 47%) and CD30 cases (21 of 35, 60%). To find out whether CDKN2A/CDKN2B deletion is an independent prognostic factor, both CDKN2A/CDKN2B deletion (homozygous and hemizygous deletion combined) and the same risk factors as analysed previously (Table 1) were included in univariate and multivariate analyses. Loss at 9p21.3 appeared to be the strongest independent parameter for reduced DSS and OS (Table 1). There are several

A restricted clonal T-cell receptor αβ repertoire in Sézary syndrome is indicative of superantigenic stimulation

Background  Sézary syndrome (SS) is a cutaneous T‐cell lymphoma characterized by erythroderma, lymphadenopathy and malignant clonal T cells in the skin, lymph nodes and peripheral blood. A role for superantigens in the pathogenesis of SS has been postulated before.

Genetic rearrangements result in altered gene expression and novel fusion transcripts in Sézary syndrome

Sézary syndrome (SS) is an aggressive, leukemic cutaneous T-cell lymphoma variant. Molecular pathogenesis of SS is still unclear despite many studies on genetic alterations, gene expression and epigenetic regulations. Through whole genome and transcriptome next generation sequencing nine Sézary syndrome patients were analyzed in terms of copy number variations and rearrangements affecting gene expression. Recurrent copy number variations were detected within 8q (MYC, TOX), 17p (TP53, NCOR1), 10q (PTEN, FAS), 2p (DNMT3A), 11q (USP28), 9p (CAAP1), but no recurrent rearrangements were identified. However, expression of five genes involved in rearrangements (TMEM244, EHD1, MTMR2, RNF123 and TOX) was altered in all patients. Fifteen rearrangements detected in Sézary syndrome patients and SeAx resulted in an expression of new fusion transcripts, nine of them were in frame (EHD1-CAPN12, TMEM66-BAIAP2, MBD4-PTPRC, PTPRC-CPN2, MYB-MBNL1, TFG-GPR128, MAP4K3-FIGLA, DCP1A-CCL27, MBNL1-KIAA2018) and five resulted in ectopic expression of fragments of genes not expressed in normal T-cells (BAIAP2, CPN2, GPR128, CAPN12, FIGLA). Our results not only underscored the genomic complexity of the Sézary cancer cell genome but also showed an unpreceded large variety of novel gene rearrangements resulting in fusions transcripts and ectopically expressed genes.

Prognostic Significance of Promoter Hypermethylation and Diminished Gene Expression of SYNPO2 in Melanoma

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RNA-seq analysis of Lgr6(+) stem cells and identification of an Lgr6 isoform.

We studied Lgr6+ stem cells in experimental UV carcinogenesis in hairless mice. For further characterization through RNA‐seq, these stem cells were isolated by FACS from transgenic hairless mice bearing an EGFP‐Ires‐CreERT2 reporter cassette inserted into exon 1 of the Lgr6 gene (purity confirmed by human ERT2 expression). Between Lgr6/EGFP+ and Lgr6/EGFP− basal cells (Tg/wt), 682 RNAs were differentially expressed, indicating stemness and expression of cancer‐related pathways in Lgr6/EGFP+ cells. We discovered that suspected “Lgr6 null” mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta‐Lgr6, lacking 74 N‐terminal aa) which could be functional and explain the lack of a phenotype.

Increased Expression of PLS3 Correlates with Better Outcome in Sézary Syndrome.

Figure 1. Survival curves. (a) Disease-specific survival and (b) overall survival curve according to the groups with and without up-regulation of PLS3. TO THE EDITOR Patients with Sézary syndrome (SS), a rare erythrodermic and leukemic form of cutaneous T-cell lymphoma, have a poor prognosis with a 5-year overall survival (OS) of 20e42% and a median OS between 2.5 and 5.0 years (Agar et al., 2010; Bernengo et al., 1998; Diamandidou et al., 1999; Kim et al., 2003; Kubica et al., 2012; Talpur et al., 2012). Prognostic factors associated with a worse survival reported in SS include advanced age (Agar et al., 2010; Diamandidou et al., 1999; Foulc et al., 2003; Kim et al., 2003; Kubica et al., 2012; Talpur et al., 2012), short duration of skin lesions before diagnosis of SS (Foulc et al., 2003), previous history of mycosis fungoides (Bernengo et al., 1998; Kubica et al., 2012), elevated serum lactate dehydrogenase levels (Agar et al., 2010; Bernengo et al., 1998; Diamandidou et al., 1999; Foulc et al., 2003; Kubica et al., 2012; Talpur et al., 2012), degree of nodal involvement (Diamandidou et al., 1999; Kim et al., 2003), and factors reflecting blood tumor burden, such as increased leukocyte counts (Bernengo et al., 1998; Talpur et al., 2012; Vidulich et al., 2009) or high Sézary cell counts (Bernengo et al., 1998). However, the results of these studies are not consistent, which may be due to different diagnostic criteria for SS, such as inclusion of patients without a T-cell clone in the peripheral blood, and analysis of mixed populations of patients with SS and erythrodermic mycosis fungoides. Recently, we investigated the diagnostic significance of a large number of immunophenotypic and molecular biomarkers for SS in a group of patients with SS (Boonk et al., 2016) that fulfilled the