Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151 Clinical Cancer Human Cancer Biology Research Integrative Genomics Analyses Reveal Molecularly Distinct Subgroups of B-Cell Chronic Lymphocytic Leukemia Patients with 13q14 Deletion Laura Mosca1, Sonia Fabris1, Marta Lionetti1, Katia Todoerti1, Luca Agnelli1, Fortunato Morabito2, Giovanna Cutrona3, Adrian Andronache1, Serena Matis3, Francesco Ferrari4, Massimo Gentile2, Mauro Spriano5, Vincenzo Callea6, Gianluca Festini7, Stefano Molica8, Giorgio Lambertenghi Deliliers1, Silvio Bicciato4, Manlio Ferrarini3,9, and Antonino Neri1 Abstract Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL. Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease. Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups. Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. Ó2010 AACR. B-cell chronic lymphocytic leukemia (CLL) is a lympho- advanced stages whereas others survive for a long time proliferative disorder characterized by a variable clinical without disease progression (1). Recently, considerable course: some patients progress rapidly toward more efforts have been addressed to the identification of geno- mic aberrations that could explain the pathogenetic mechanisms and the clinical heterogeneity of the disease (2–5). Deletions of 13q14, 11q22.3, and 17p13, and 1 Authors' Affiliations: Dipartimento di Scienze Mediche, Universitadi trisomy 12 are common in CLL and may play a role in Milano, U.O. Ematologia 1, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2U.O.C. di Ematologia, Azienda Ospe- pathogenesis and disease progression (4). 13q14 deletion daliera di Cosenza, Italy; 3Divisione di Oncologia Medica C, Istituto occurs in approximately 50% of CLL and is associated with 4 Nazionale per la Ricerca sul Cancro, IST, Genoa, Italy; Dipartimento di a favorable prognosis when present as the sole abnormality Scienze Biomediche, Universita di Modena e Reggio Emilia, Modena, Italy; 5Dipartimento di Ematologia, Azienda Ospedaliera S. Martino, Genoa, (6). Notably, 2 microRNA genes, miR-15a and miR-16-1, Italy; 6Divisione di Ematologia, Azienda Ospedaliera, Reggio Calabria, located at 13q14, have been reported to be downregulated Italy; 7Centro di Riferimento Ematologico-Seconda Medicina-Azienda Ospedaliero-Universitaria, Ospedali Riuniti, Trieste, Italy; 8U.O.C. di in del(13)(q14) patients (7) and strongly suggest their Oncologia, Azienda Ospedaliera "Pugliese-Ciaccio", Catanzaro, Italy; potential role in the disease. However, the 13q14 deletion and 9Dipartimento di Oncologia, Biologia e Genetica, Universita degli is not always accompanied by defects in miR-15a/16-1 Studi di Genova, Genoa, Italy expression, suggesting a more complex heterogeneity of Note: Supplementary data for this article are available at Clinical Cancer the deletion itself (8, 9). Research Online (http://clincancerres.aacrjournals.org/). The recent introduction of microarray technology has L. Mosca and S. Fabris contributed equally to this work. improved the possibility of combining genome-wide Corresponding Author: Prof. Antonino Neri, Department of Medical DNA analyses with transcriptomic profiles, thus allowing Sciences, University of Milan, Via F. Sforza 35, 20122 Milano, Italy. Phone. þ39.02.55033328; Fax: þ39.02.50320403. E-mail: [email protected] the identification of potential candidate tumor genes related doi: 10.1158/1078-0432.CCR-10-0151 to underlying chromosomal alterations. To further elucidate the genomic complexity of the 13q14 deletion Ó2010 American Association for Cancer Research. www.aacrjournals.org 5641 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151 Mosca et al. Translational Relevance CD38 and ZAP-70 expression were determined by flow cytometry (11). IgVH gene usage and mutational status The 13q14 deletion represents the most common were established as previously described (12), with a 2% genomic aberration in CLL (50%). Although it is asso- cutoff value being used to define mutated and unmutated ciated with a favorable prognosis when present as the patients. For the microarray analyses, the CLL cells were sole abnormality, the imprecise knowledge of the genes enriched by negative selection when less than 90% (11). implicated and its genetic heterogeneity have limited in situ our understanding on the pathogenetic mechanisms Fluorescence hybridization contributing to the disease. Microarray technology The most common genomic aberrations, del(17)(p13), has improved the possibility of combining genome- del(11)(q23), del(6)(q23), del(13)(q14), and trisomy 12, wide DNA with transcriptomic profiles to identify were investigated by interphase FISH hybridization. All of potential candidate tumor genes related to underlying the probes are commercially available (Vysis; ref. 13). chromosomal aberrations. Based on SNP array, our study shows the presence of 2 distinct molecular groups High-density SNP arrays and data analysis of patients with del(13)(q14) based on the deletion size Total genomic DNA (250 ng) were processed in accor- and the presence of biallelic deletions. Notably, global dance with the manufacturer’s protocol (Affymetrix), gene expression profiling identified a significant tran- hybridized using Affymetrix GeneChip Human Mapping scriptional deregulation specifically associated with the 250K NspI microarrays, and subsequently scanned using a 2 groups. Our data highlight the presence of 2 distinct GeneChip Scanner 3000 7G. The images were acquired molecular types of 13q14 deletion that may be of using the Affymetrix GeneChip Operating System (GCOS clinical relevance for the biology and the prognostic 1.4). The accuracy of the SNP array data were confirmed by stratification of the disease. the mean and median call rates of 95.75% and 96.12% (the quality control specification for 250K arrays is a call rate greater than 93%), respectively. The entire procedure for the copy number (CN) estima- in CLL, we applied single-nucleotide polymorphism (SNP)- tion has been fully described previously (14). Briefly, the array technology to a panel of 100 untreated patients with raw data relating to the individual SNPs were extracted Binet stage A disease including 44 patients with 13q deletion from CEL files and converted into signal intensities using as assessed by fluorescence in situ hybridization (FISH). We GTYPE 4.1 and Affymetrix Copy Number Analysis Tool then investigated the expression levels of the miR-15a/16 (CNAT 4.0.1) software. Each sample was compared with a cluster by quantitative real-time PCR (qRT-PCR) and global set of 48 normal Caucasian HapMap references available gene expression profiling in a representative panel of cases on the Affymetrix web site (http://www.affymetrix.com/ (see flow chart in Supplementary Figure S1). The integration support/technical/sample_data/500k_data.affx) and the between genomic and expression data allowed the identi- genomic smoothing window of the Hidden Markov Model fication of 2 distinct molecular groups of patients with algorithm was set at 0. After preprocessing, piecewise con- 13q14 deletions. Our data may have implications for the stant estimates of the underlying local DNA CN alterations biology and the prognostic stratification of CLL. were calculated using the DNAcopy Bioconductor package, which looks for optimal breakpoints on the basis of circular Materials and Methods binary segmentation (15), and the median values of the estimated profiles were scaled back to a nominal multi- Patients plicity of 2. After scaling, we determined all the clusters The study included samples from 100 untreated CLL appearing on the frequency distribution (histogram) of the patients in Binet stage A: 60 from a retrospective collabora- SNP values for all the SNP probes using the k-means tive Italian study (10) and 40 enrolled in an Italian pro- algorithm