Copyright @ 2012 John Wiley & SonsThe 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.This work was funded by grants from
Netherlands Organization for Scientific Research (NWO) (MHV) and the Fondation Rene´ Touraine (MvK), and grants from the Leukaemia and Lymphoma Research (EB) and the Julian Starmer-Smith Memorial Fund (CHL)

Ballabio, E

Benner, MF

Lawrie, CH

Saunders, NJ

Tensen, CP

van Kester, MS

Vermeer, MH

Willemze, R

Experimental Dermatology

English

Benner, M.F.

Ballabio, E.

Kester, M.S. van

Saunders, N.J.

Vermeer, M.H.

Willemze, R.

Lawrie, C.H.

Tensen, C.P.

Leiden University Scholary Publications

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

Brunel University Research Archive

DOI: 10.1111/j.1600-0625.2012.01548.xwww.blackwellpublishing.com/EXDLetter to the EditorPrimary cutaneous anaplastic large cell lymphoma shows a distinctmiRNA expression profile and reveals differences from tumor-stagemycosis fungoidesMarchina F. Benner1, Erica Ballabio2, Marloes S. van Kester1, Nigel J. Saunders3, Maarten H. Vermeer1,Rein Willemze1, Charles H. Lawrie2,4,5,† and Cornelis P. Tensen1,†1Department of Dermatology, LUMC, Leiden, The Netherlands; 2Lymphoid Malignancy Research Group, Nuffield Department of ClinicalLaboratory Sciences, John Radcliffe Hospitals, University of Oxford, Oxford, UK; 3Sir William Dunn School of Pathology, University of Oxford,Oxford, UK; 4Biodonostia Research Institute, Paseo Doctor Begiristain s/n, San Sebastia´n, Spain; 5IKERBASQUE, Basque Foundation for Science,Bilbao, SpainCorrespondence: Dr. C. P. Tensen, Department of Dermatology, T02-03, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden,The Netherlands, Tel.: +31 (0)715269369, Fax: +31 (0)715248106, e-mail: c.p.tensen@lumc.nl†These authors share senior authorship.Abstract: The miRNA expression profiles of skin biopsies from 14primary cutaneous anaplastic large cell lymphoma (C-ALCL)patients were analysed with miRNA microarrays using the samecontrol group of 12 benign inflammatory dermatoses (BID) aspreviously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentiallyexpressed miRNAs between C-ALCL and BID. The up-regulation ofmiR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validatedby miRNA-Q-PCR on independent study groups. Additionally, themiRNA expression profiles of C-ALCL were compared with those oftumor-stage MF. Although miRNA microarray analysis did notidentify statistically significant differentially expressed miRNAs,miRNA-Q-PCR demonstrated statistically significantly differentialexpression of miR-155, miR-27b, miR-93, miR-29b and miR-92abetween tumor-stage MF and C-ALCL. This study, the firstdescribing the miRNA expression profile of C-ALCL, revealsdifferences with tumor-stage MF, suggesting a differentcontribution to the pathogenesis of these lymphomas.Key words: miR-155 – miRNA microarray – mycosis fungoides –primary cutaneous anaplastic large cell lymphoma – Q-PCRAccepted for publication 4 June 2012BackgroundPrimary cutaneous anaplastic large cell lymphoma (C-ALCL) is char-acterized by large cells with an anaplastic, pleomorphic or immunob-lastic cytomorphology and by expression of the CD30 antigen bymore than 75% of the tumor cells (1). Although its histology indi-cates an aggressive lymphoma, C-ALCL often shows an indolent clin-ical behaviour with a 10-year disease-specific survival (DSS) ofapproximately 90% (2–4). The molecular mechanisms involved inthe development of this disorder are largely unknown (5).MicroRNAs (miRNAs) are small non-coding RNA moleculesthat regulate gene expression posttranscriptionally. They may haveoncogenic or tumor suppressing properties depending on theirtarget genes (6,7). Several studies have demonstrated specificmiRNA expression profiles in different types of CTCL suggesting arole in the pathogenesis of these disorders (8–12). For instance,recently, van Kester et al. (8) have elaborated on the miRNAexpression profile of tumor-stage mycosis fungoides (MF), aCTCL with a 10-year DSS of 42%. So far, only one study focusingon the miRNA expression profiling of CTCL in general hasincluded C-ALCL biopsies (10). Hence, the miRNA expressionprofile of C-ALCL is as yet unknown.Questions addressedThe aim of this study was to determine the miRNA expressionprofile of C-ALCL compared with benign inflammatory dermato-ses (BID) and with tumor-stage MF to identify miRNAs whichmay contribute to the pathogenesis of these malignancies.Experimental designFor detailed material and methods, please see Supporting informa-tion (Data S1). Formalin fixed paraffin embedded (FFPE) skinbiopsies containing more than 75% of CD30+ large cells wereselected from 21 C-ALCL patients. In total, 14 biopsieswere selected for array analysis, and seven additional biopsies wereselected as a test group for validation by miRNA-Q-PCR. As acontrol group, FFPE biopsies of 12 benign inflammatorydermatoses (BID) containing T-cell-rich infiltrates were selected:five eczema and seven lichen planus cases for array analysis andfive additional eczema and six lichen planus cases for miRNA-Q-PCR.The complete miRNA profile of samples was elucidated usinglRNA microarrays (8,14). The arrays were used within 3 monthsafter printing. ANOVA analysis was performed to identifymiRNAs differentially expressed between sample types, and multi-ple testing correction was done using the Benjamini–Hochbergmethod.Results of miRNA microarray analysis of C-ALCL were com-pared with those recently published on 19 tumor stage MF (8),which were generated using the same platform (9).MiRNA cDNA synthesis was performed as described before (8).MiRNA-Q-PCR was performed using Taqman miRNA assays and29 Universal PCR mastermix (Applied Biosystems-Life Technolo-gies, Carlsbad, CA, USA). All reactions were run on the Light-Cycler480 (Roche, Almere, the Netherlands), according to632ª 2012 John Wiley & Sons A/SExperimental Dermatology, 2012, 21, 630–642manufacturer’s protocol (Applied Biosystems). MiRNA expressionlevels were analysed using the ΔCt method expressed relative toU6.ResultsMiRNA microarray analysis showed twelve miRNAs higherexpressed and one miRNA lower expressed in C-ALCL comparedwith BID (adjusted P < 0.05; Table S1). To validate these results,miRNA-Q-PCR was performed for miR-155, miR-29b, miR-30cand miR-27b in an additional group of C-ALCL (n = 7) and BID(eczema n = 5 and lichen planus n = 6). All miRNA-Q-PCRresults are consistent with the microarray data, showing significantdifferential expression between C-ALCL and BID (P < 0.05;Fig. 1a).Comparison of miRNA microarray results from C-ALCL withthose from tumor-stage MF showed differential expression of sev-eral miRNAs, but none remained statistically significant afterp < 0.01 p < 0.01p = 0.01 p = 0.02p < 0.01 p < 0.01p < 0.01 p = 0.01p = 0.56(a)(b)Figure 1. Expression levels of selected miRNAs as measured by miRNA-Q-PCR inan additional test group. Horizontal bars represent the mean. (a) Confirmation offour miRNAs selected for validation. (b) Four of five miRNAs additionally tested aresignificant differentially expressed between C-ALCL and benign controls (P-valuesmeasured by Mann–Whitney U-test).p = 0.02p < 0.01p = 0.04 p = 0.05p = 0.05Figure 2. Expression levels of miR-155, miR-27b, miR-93, miR-29b and miR-92a asmeasured by miRNA-Q-PCR in C-ALCL and tumor-stage MF. Horizontal barsrepresent the mean (P-values measured by Mann–Whitney U-test).ª 2012 John Wiley & Sons A/SExperimental Dermatology, 2012, 21, 630–642 633Letter to the Editormultiple testing correction. However, we noticed that the set ofmiRNAs discriminating C-ALCL from benign controls (this study)is different from the set discerning tumor-stage MF from the samecontrols (8). We therefore extended the analysis of C-ALCL andused miRNA-Q-PCR to determine the expression of miR-93,miR-92a, miR-30b, miR-16 and miR-383, which were differentiallyexpressed between tumor-stage MF and BID. Except for miR-383,all miRNAs showed significant up-regulation in C-ALCL vs. BID(Fig. 1b). In analogy, we also measured miR-29b and miR27b intumor-stage MF, using the validation set as described by van Kest-er et al. (8), resulting in the up-regulation of miR-29b in tumor-stage MF vs. BID (P < 0.01), while miR-27b is not differentiallyexpressed (data not shown). Finally, we determined whether anyof the miRNAs assayed by miRNA-Q-PCR is differentiallyexpressed between C-ALCL and MF. As depicted in Fig. 2, thisanalysis identified differential expression of miR-155, miR-27b,miR-93, miR-92a and miR-29b.ConclusionsUsing miRNA microarrays, we found thirteen miRNAs differen-tially expressed between C-ALCL and BID, twelve up-regulatedand one down-regulated. We validated expression of miRNA-155,miR-27b, miR-30c and miR-29b with miRNA-Q-PCR in twoindependent groups of C-ALCL and BID. Because these array-based analyses of miRNA profiles of C-ALCL were performedusing the same platform as the recently published experiments onmiRNA profiling of tumor-stage MF, a proper comparison(including normalization and statistical analysis) was feasible(8,9). Under stringent statistical conditions, no differences betweenC-ALCL and MF were identified. However, the miRNA-Q-PCR-based analysis of an additional selection of individual miRNAs(chosen as being differentially expressed between tumor-stage MFor C-ALCL and BID) revealed not only further differencesbetween the two disease entities (miR-155, miR-27b, miR-93,miR-92a and miR-29b) but also additional differences betweenC-ALCL and BID (miR-93, miR-92a, miR-27b, miR-30b and miR-16). Therefore, miRNA arrays are well suited for initial genome-wide screening, but apparently are less sensitive for detection ofmore subtle changes in miRNA expression. A similar observationwas recently made by Ralfkiaer et al. (10) in the course of theirmiRNA profiling studies for diagnostic markers of CTCL whospeculated that this might relate to cross-hybridization problemsor a restricted dynamic range of miRNA microarrays. Moreover,miRNA microarrays are only capable of measuring the relativeexpression levels of known (annotated) miRNAs, and hithertounknown miRNAs might be differentially expressed between thedifferent disease entities as well. These restrictions might be over-come in the future using techniques like next generation deepsequencing, though the limited availability of suitable (frozen)material with a high tumor cell content will remain a challenge.The current miRNA expression profile of C-ALCL and the differ-ences compared with tumor-stage MF provide a framework forfurther (functional) studies and may help to reveal the molecularpathogenesis of these lymphomas.AcknowledgmentsMvK, EB and MFB performed the research; MvK, EB, MFB, CPT and CHLanalysed the data; MFB, CPT, CHL, MHV and RW wrote the paper. NJScontributed essential reagents and tools; MFB, MHV and RW collectedpatient samples; CHL and CPT designed the research study. The authorswould like to thank WH Zoutman, JJ Out-Luiting (Department of Derma-tology, LUMC), PA van der Velden (Department of Ophthalmology,LUMC) and RHAM Vossen (Center of Human and Clinical Genetics,LUMC) for their excellent assistance. This work was funded by grants fromNetherlands Organization for Scientific Research (NWO) (MHV) and theFondation Rene´ Touraine (MvK), and grants from the Leukaemia and Lym-phoma Research (EB) and the Julian Starmer-Smith Memorial Fund (CHL).Conflict of interestsThe authors have declared no conflicting interests.References1 Willemze R, Jaffe E S, Burg G et al. Blood 2005:105: 3768–3785.2 Bekkenk M W, Geelen F A, van Voorst Vader PC et al. Blood 2000: 15: 3653–3661.3 Benner M F, Willemze R. Arch Dermatol 2009:145: 1399–1404.4 Liu H L, Hoppe R T, Kohler S et al. J Am AcadDermatol 2003: 49: 1049–1056.5 Greisser J, Doebbeling U, Roos M et al. Exp Der-matol 2005: 14: 380–385.6 Lawrie C H. J Cell Moll Med 2008: 12: 1432–1444.7 Fabbri M, Croce C M. Curr Opin Haematol2011: 18: 266–272.8 Van Kester M S, Ballabio E, Benner M F et al.Mol Oncol 2011: 5: 273–280.9 Ballabio E, Mitchell T, van Kester M S et al.Blood 2010: 16: 1105–1113.10 Ralfkiaer U, Hagedorn P H, Bangsgaard N et al.Blood 2011: 24: 5891–5900.11 Narducci M G, Arcelli D, Picchio M C et al. CellDeath and Disease 2011: 28: 2.12 Maj J, Jankowska-Konsur A, Sadakiewska-ChudyA et al. Br J Dermatol 2012: 166: 331–336.13 Van Doorn R, Van Haselen C W, van Voorst VaderP C et al. Arch Dermatol 2000: 136: 504–510.14 Lawrie C H, Saunders N J, Soneji S et al. Leuke-mia 2008: 22: 1440–1446.Supporting InformationAdditional Supporting Information may be found inthe online version of this article:Table S1. Differentially expressed miRNAs with chro-mosomal location.Data S1. Material and methods.Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than miss-ing material) should be directed to the correspondingauthor for the article.634ª 2012 John Wiley & Sons A/SExperimental Dermatology, 2012, 21, 630–642Letter to the Editor

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Leukemia 2008: 22: 1440–1446. Supporting Information Additional Supporting Information may be found in the online version of this article:

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Sadakiewska-Chudy A et al.

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

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