Published OnlineFirst October 20, 2009; DOI: 10.1158/0008-5472.CAN-09-2367 Published Online First on October 20, 2009 as 10.1158/0008-5472.CAN-09-2367 Molecular Biology, Pathobiology, and Genetics Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays Elisabetta Caprini,1 Cristina Cristofoletti,1 Diego Arcelli,1 Paolo Fadda,1 Mauro Helmer Citterich,1 Francesca Sampogna,1 Armando Magrelli,2 Federica Censi,2 Paola Torreri,2 Marina Frontani,1 Enrico Scala,1 Maria Cristina Picchio,1 Paola Temperani,3 Alessandro Monopoli,1 Giuseppe Alfonso Lombardo,1 Domenica Taruscio,2 Maria Grazia Narducci,1 and Giandomenico Russo1 1Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico and 2National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy; and 3Università di Modena e Reggio Emilia, Unità di Ematologia, Dipartimento di Oncologia ed Ematologia, Modena, Italy Abstract Conventional cytogenetics, allelotyping, and comparative genomic In this study, we used single nucleotide polymorphism and hybridization (CGH) analyses have shown that these tumor cells comparative genomic hybridization array to study DNA copy exhibit chromosomal instability, manifested as allelic gain, loss, number changes and loss of heterozygosity for 28 patients af- or rearrangement (2). Although numerous efforts have been made fected by Sézary syndrome (SS), a rare form of cutaneous T- to clarify how the genomic aberrations might predispose to the cell lymphoma (CTCL). Our data identified, further confirm- development of this disease, the identification of pathogenically ing previous studies, recurrent losses of 17p13.2-p11.2 and relevant genes still remains a challenge. Recently, the application 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; of high-throughput technologies for genome-wide surveys of genet- common gains were detected for 17p11.2-q25.3 (64%) and ic or expression profiles in CTCL are giving new insights into this – chromosome 8/8q (50%). Moreover, we identified novel ge- malignancy (3 6). Furthermore, there is an increasing tendency to nomic lesions recurring in >30% of tumors: loss of 9q13- integrate mapping and expression data to study the relationship q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal between copy number changes and gene expression levels as a aberrations did not show a significant correlation with significant fraction of genes are altered in a manner consistent prognosis; however, when more than three recurrent chromo- with the underlying genomic alterations in a variety of tumors – somal alterations (gain or loss) were considered, a statistical (7 11) including CTCL (12, 13). The presence of recurrent chromo- association was observed using Kaplan-Meier survival analy- somal copy number alterations (CNA) that correlate with disease sis. Integrating mapping and transcriptional data, we were outcome suggests that changes in the expression of specific genes able to identify a total of 113 deregulated transcripts in aber- within these regions are critical to the disease process (14). rant chromosomal regions that included cancer-related genes Here, we describe a genome-wide analysis, at submegabase res- such as members of the NF-κB pathway (BAG4, BTRC, olution, of DNA copy number changes in 28 SS samples using the NKIRAS2, PSMD3, and TRAF2) that might explain its constitu- single nucleotide polymorphism (SNP) and array CGH (aCGH) tive activation in CTCL. Matching this list of genes with those technology. Our findings identified six regions of highly recurrent discriminating patients with different survival times, we copy number aberrations affecting chromosomes 8, 9, 10, and 17. identify several common candidates that might exert critical Additionally, the SNP technology allowed us to distinguish be- roles in SS, such as BUB3 and PIP5K1B. Altogether, our study tween loss of heterozygosity (LOH) associated with either copy confirms and maps more precisely the regions of gain and number changes, such as hemizygous deletions, or copy number loss and, combined to transcriptional profiles, suggests a neutral status, underlying the involvement of different genetic me- novel set of genes of potential interest in SS. [Cancer Res chanisms that lead to uniparental disomy (UPD) in SS (15). In the 2009;69(21):8438–46] attempt to correlate copy number data and clinical parameters, we find a relationship between complex pattern of chromosomal aber- rations, involving at least three recurrent CNAs, and shorter surviv- Introduction al. We then combined copy number results with gene expression Sézary syndrome (SS) is an aggressive leukemic variant of data from the same SS patients to generate a signature of genes cutaneous T-cell lymphoma (CTCL) that typically presents with differentially expressed and located within the chromosomal erythroderma, peripheral lymphadenopathy, severe pruritus, and regions of interest that include novel potential candidates of SS + malignant circulating CD4 T lymphocytes: the Sézary cells (1). tumor development. Note: Supplementary data for this article are available at Cancer Research Online Materials and Methods (http://cancerres.aacrjournals.org/). Requests for reprints: Giandomenico Russo and Maria Grazia Narducci, Istituto Patients. Peripheral blood samples from 28 SS-affected individuals were Dermopatico dell'Immacolata-Istituto Di Ricovero e Cura a Carattere Scientifico, Via analyzed. All the patients were enrolled in clinical protocols approved by dei Monti di Creta 104, 00167 Rome, Italy. Phone: 39-6-66464798; Fax: 39-6-66462430; the Ethical Committee of the Istituto Dermopatico dell'Immacolata, and E-mail: [email protected] and [email protected]. ©2009 American Association for Cancer Research. informed consent was obtained in accordance with the Declaration of doi:10.1158/0008-5472.CAN-09-2367 Helsinki. Diagnosis of SS was based on described criteria (16). The major Cancer Res 2009; 69: (21). November 1, 2009 8438 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst October 20, 2009; DOI: 10.1158/0008-5472.CAN-09-2367 SNP and Expression Profiling of Sézary Syndrome Table 1. Highly recurrent CNAs in SS Chr Copy number loss Mb* Genes in SS patients Copy Mb* Genes in SS patiens region† (%) number gain region† (%) Start Stop Size Start Stop Size 8 8p23.3-q24.3 1,13 142,1 140,94 1029 14/28 (50%) 9 9q13-q21.33 69,11 86,66 17,56 115 9/28 (32%) 10 10p12.1-q26.3 25,1 133,8 108,70 1016 19/28 (68%) 10p15.3-10p12.2 0,14 24,02 23,88 187 10/28 (36%) 17 17p13.2-p11.2 3,57 20,15 16,58 388 20/28 (71%) 17p11.2-q25.3 17,35 76,40 59,05 1171 18/28 (64%) *Numerical position based on human genome assembly hg 18 (National Center for Biotechnology Information Build 36.1). † Number of coding transcripts based on genomic sequence information available on Mar 24, 2008 (National Center for Biotechnology Information Build 36.3). clinical and immunologic characteristics, including T-cell receptor clonal hybridization were performed following the Agilent manufacturer's proto- analysis (17) of the samples, are listed in Supplementary Table S1. cols (version 5.0). Images were analyzed using Agilent Feature Extraction Tumor, normal cell isolation, and genomic DNA extraction. Periph- Software version 9.5.1 and data were imported into Agilent CGH analytics eral blood mononuclear cells were separated by Ficoll-Histopaque density software version 3.5 for a graphical overview and analysis. Analysis was per- gradient centrifugation (Sigma-Aldrich). Lymphomonocytes and granulo- formed using the ADM-2 algorithm (Agilent Technologies) with a threshold cytes were separately collected from the gradient fractions. Lymphomono- of 5.3. Experiments showing a derivative log ratio spread value of >0.3 were cytes were subsequently purified by positive selection using anti–human excluded from the analysis. CD3-conjugated dynabeads (Oxoid Ltd.). Granulocytes (normal matched RNA isolation, labeling, and gene expression analysis. Five micro- cells) were obtained collecting the upper phase overlaying the Ficoll density grams of total RNA extracted from sorted T lymphocytes, according to Af- gradient sediment; residual erythrocytes were then lysed by repetitive fymetrix procedure, were reverse transcribed, synthesized in cRNA, washes with a solution of 10 mmol/L Tris-HCl (pH 7.6), 5 mmol/L MgCl2, fragmented, labeled, and hybridized to Human Genome U133A arrays. and 10 mmol/L NaCl. Tumor genomic DNA was isolated from sorted CD3+/ The scanned data were analyzed with a customized script that uses Bio- CD4+ T lymphocytes of SS patients, whereas normal matched DNA was ex- conductor packages6 based on the R language,7 for quality control assess- tracted from the granulocytes of the same individuals according to pub- ment, data normalization, unsupervised and supervised clustering analysis, lished protocols (17). The calculated percentage of tumor cells after and identification of differentially expressed transcripts (19). This R-script purification is indicated for each sample in Supplementary Table S1 and provides utilization of different Bioconductor packages; gcrma package was ranged from 35% to 91%. used for normalization and background correction, and Genefilter package SNP genotyping, LOH, and DNA copy number change. The samples was used to filter genes with highly variance by interquartile range method; were analyzed