Marloes S. van Kester

Oncogenomic analysis of mycosis fungoides reveals major differences with Sézary syndrome

Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is a malignancy of mature, skin-homing T cells. Sézary syndrome (Sz) is often considered to represent a leukemic phase of MF. In this study, the pattern of numerical chromosomal alterations in MF tumor samples was defined using array-based comparative genomic hybridization (CGH); simultaneously, gene expression was analyzed using microarrays. Highly recurrent chromosomal alterations in MF include gain of 7q36, 7q21-7q22 and loss of 5q13 and 9p21. The pattern characteristic of MF differs markedly from chromosomal alterations observed in Sz. Integration of data from array-based CGH and gene-expression analysis yielded several candidate genes with potential relevance in the pathogenesis of MF. We confirmed that the FASTK and SKAP1 genes, residing in loci with recurrent gain, demonstrated increased expression. The RB1 and DLEU1 tumor suppressor genes showed diminished expression associated with loss. In addition, it was found that the presence of chromosomal alterations on 9p21, 8q24, and 1q21-1q22 was associated with poor prognosis in patients with MF. This study provides novel insight into genetic alterations underlying MF. Furthermore, our analysis uncovered genomic differences between MF and Sz, which suggest that the molecular pathogenesis and therefore therapeutic requirements of these cutaneous T-cell lymphomas may be distinct.

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

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.

miRNA expression profiling of mycosis fungoides

MicroRNAs (miRNAs) are small RNA species that regulate gene expression post‐transcriptionally and are aberrantly expressed in many malignancies including lymphoma. However, the role of miRNAs in the pathogenesis of T‐cell lymphoid malignancies is poorly understood. Previously we examined the miRNA profile of Sézary syndrome (Sz), a leukemia of skin‐homing memory T cells. In this study we determined the complete miRNome of mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma. The miRNA profile of skin biopsies from 19 patients with tumor stage MF and 12 patients with benign inflammatory dermatoses (eczema and lichen planus) were compared by microarray analysis. We identified 49 miRNAs that are differentially expressed in tumor stage MF compared to benign inflammatory dermatoses using ANOVA analysis (P < 0.05, Benjamini–Hochberg corrected). The majority of the differentially expressed miRNAs (30/49) were up‐regulated in tumor stage MF. The most significant differentially expressed were miR‐155 and miR‐92a (both up‐regulated in tumor stage MF), while miR‐93 showed the highest up‐regulation in tumor stage MF with a fold difference of 5.8. Differential expression of a selection of these miRNAs was validated by miRNA‐Q‐PCR on additional test groups (tumors and controls). None of the miRNAs up‐regulated in tumor stage MF was previously shown to be up‐regulated in Sz, and only 2 of the 19 miRNAs down‐regulated in tumor stage MF were also down‐regulated in Sz. Taken together this report is the first describing the miRNA signature of tumor stage MF.

Cucurbitacin I Inhibits Stat3 and Induces Apoptosis in Sézary Cells

Sézary syndrome (Sz) is an aggressive cutaneous CD4(+) T-cell lymphoma with tumor cells (Sz cells) localized in the skin, lymph nodes, and peripheral blood. Using western blotting, we demonstrate the expression of phosphorylated (P)-Stat3 in the Sz-derived cell line Seax, and in freshly isolated tumor cells from Sz patients (n=6). In Vitro overnight culture without exogenous cytokines results in decreased expression of P-Stat3 (n=3), indicating that Stat3 is not constitutively activated. Incubation of the Seax cell line with the Jak/Stat3 inhibitor Cucurbitacin I resulted in a time- and concentration-dependent decrease of P-Stat3 and Stat3. In freshly isolated Sz cells (n=3), Cucurbitacin I induced a concentration-dependent decrease in Stat3 expression whereas P-Stat3 was undetectable. Finally, incubation of freshly isolated Sz cells (n=4) with 30 microM Cucurbitacin I for 6 hours induced apoptosis in the large majority (73-91%) of tumor cells. These data strengthen the notion that activation of Stat3 plays an essential part in the malignant transformation of Sz and provide further rationale for the therapeutical targeting of Stat3 in Sz.

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.

Cutaneous Anaplastic Large Cell Lymphoma and Peripheral T-Cell Lymphoma NOS Show Distinct Chromosomal Alterations and Differential Expression of Chemokine Receptors and Apoptosis Regulators

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) has an indolent clinical course and favorable prognosis. On the contrary, primary cutaneous peripheral T-cell lymphoma not otherwise specified (PTL-NOS) shows aggressive clinical behavior. To identify genomic events relevant in the pathogenesis of these cutaneous T-cell lymphomas (CTCLs), we carried out array-based comparative genomic hybridization (CGH) analysis. Simultaneously, gene expression profiling was conducted to gain insight into gene expression programs associated with the different clinical behavior of these CTCLs. C-ALCL was characterized by gains on chromosome 7q and 17q and losses on 6q and 13q. PTL-NOS similarly showed gains on 7q and 17q, but was distinguished by gains on chromosome 8 and loss of a focal overlapping region on 9p21. We identified minimal common regions harboring candidate oncogenes and tumor suppressor genes in C-ALCL and PTL-NOS. Genes with a role in lymphocyte chemotaxis, apoptosis, and proliferation were overrepresented among genes differentially expressed between these lymphomas. C-ALCL showed higher expression of the skin-homing chemokine receptor genes CCR10 and CCR8, which may explain the lower tendency to disseminate to extracutaneous sites. Furthermore, C-ALCL and PTL-NOS showed aberrant expression of distinct genes implicated in apoptosis and proliferation, such as IRF4/MUM1 and PRKCQ, which may account for differences in clinical aggressiveness.

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C‐ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor‐stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C‐ALCL and BID. The up‐regulation of miR‐155, miR‐27b, miR‐30c and miR‐29b in C‐ALCL was validated by miRNA‐Q‐PCR on independent study groups. Additionally, the miRNA expression profiles of C‐ALCL were compared with those of tumor‐stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA‐Q‐PCR demonstrated statistically significantly differential expression of miR‐155, miR‐27b, miR‐93, miR‐29b and miR‐92a between tumor‐stage MF and C‐ALCL. This study, the first describing the miRNA expression profile of C‐ALCL, reveals differences with tumor‐stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.

The absence of multiple atypical nevi in germline CDKN2A mutations: Comment on “Hereditary melanoma: Update on syndromes and management: Genetics of familial atypical multiple mole melanoma syndrome”

Fig 1. CDKN2A carrier with little nevi. Band-Aids mark the location of the most recent superficial spreading melanoma, Breslow thickness 0.4 mm. To the Editor: With great interest we have read the article by Soura et al and would like to compliment the authors with their elaborate overview of hereditary melanoma. In the article, the authors urge that familial melanoma in the presence of multiple nevi should raise suspicion for a germline CDKN2A mutation. We would like to add caution regarding the diagnostic pitfall of disregarding family members with few nevi as potential carriers of a germline CDKN2A mutation. At our Leiden Pigmented Lesions Clinic, we see more than 1000 patients per year with a germline CDKN2A mutation. Many patients do have atypical nevi but a substantial number do not. Illustrative is the case of a 35-year old CDKN2A carrier with 1 melanoma in situ, 1 nodular melanoma, and 2 superficial spreading melanomas with little nevi (Fig 1). Others showed a poor correlation between having the so-called atypical mole syndrome and carrying the mutant gene in families with germline CDKN2A mutations. This heterogeneity in phenotypes is one of the reasons identifying the CDKN2A mutation was difficult at that time. Familial melanoma without the presence of atypical nevi should also raise suspicion for a germline CDKN2Amutation. All family members, regardless of the number of atypical nevi, need to be considered as potential carriers. Therefore, clinicians should not use absence of atypical nevi to rule out a germline CDKN2A mutation.

Lymphangitis carcinomatosa of a cutaneous squamous cell carcinoma

An 83-year-old woman presented with erythema, papules and nodules on the left upper arm (figure 1A). She was treated with antibiotics by her general practitioner, with no disease response. Eight months earlier a 2.5 cm poorly differentiated primary cutaneous squamous cell carcinoma of the left preauricular area, with metastases to the regional lymph nodes, was staged T2N2M0 (tumour, node, metastases). She was treated with radical tumour resection, parotidectomy, and levels 2, 3 and 5 lymph node dissection, followed by postoperative radiotherapy. A total body skin examination was performed. On the left upper arm and adjacent to the axillary sulcus, a poor demarcated area of erythema with papules and nodules was noted. …