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Human Cancer Biology
Integrative Genomics Analyses Reveal Molecularly Distinct Subgroupsof 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
AbstractPurpose: 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. �2010 AACR.
B-cell chronic lymphocytic leukemia (CLL) is a lympho-proliferative disorder characterized by a variable clinicalcourse: some patients progress rapidly toward more
advanced stages whereas others survive for a long timewithout disease progression (1). Recently, considerableefforts have been addressed to the identification of geno-mic aberrations that could explain the pathogeneticmechanisms and the clinical heterogeneity of the disease(2–5). Deletions of 13q14, 11q22.3, and 17p13, andtrisomy 12 are common in CLL and may play a role inpathogenesis and disease progression (4). 13q14 deletionoccurs in approximately 50% of CLL and is associated witha favorable prognosis when present as the sole abnormality(6). Notably, 2 microRNA genes, miR-15a and miR-16-1,located at 13q14, have been reported to be downregulatedin del(13)(q14) patients (7) and strongly suggest theirpotential role in the disease. However, the 13q14 deletionis not always accompanied by defects in miR-15a/16-1expression, suggesting a more complex heterogeneity ofthe deletion itself (8, 9).
The recent introduction of microarray technology hasimproved the possibility of combining genome-wideDNA analyses with transcriptomic profiles, thus allowingthe identificationof potential candidate tumorgenes relatedto underlying chromosomal alterations. To furtherelucidate the genomic complexity of the 13q14 deletion
Authors' Affiliations: 1Dipartimento di Scienze Mediche, Universit�a diMilano, U.O. Ematologia 1, Fondazione IRCCS Ca’ Granda OspedaleMaggiore Policlinico, Milan, Italy; 2U.O.C. di Ematologia, Azienda Ospe-daliera di Cosenza, Italy; 3Divisione di Oncologia Medica C, IstitutoNazionale per la Ricerca sul Cancro, IST, Genoa, Italy; 4Dipartimento diScienze Biomediche, Universit�a di Modena e Reggio Emilia, Modena, Italy;5Dipartimento di Ematologia, Azienda Ospedaliera S. Martino, Genoa,Italy; 6Divisione di Ematologia, Azienda Ospedaliera, Reggio Calabria,Italy; 7Centro di Riferimento Ematologico-Seconda Medicina-AziendaOspedaliero-Universitaria, Ospedali Riuniti, Trieste, Italy; 8U.O.C. diOncologia, Azienda Ospedaliera "Pugliese-Ciaccio", Catanzaro, Italy;and 9Dipartimento di Oncologia, Biologia e Genetica, Universit�a degliStudi di Genova, Genoa, Italy
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
L. Mosca and S. Fabris contributed equally to this work.
Corresponding Author: Prof. Antonino Neri, Department of MedicalSciences, University of Milan, Via F. Sforza 35, 20122 Milano, Italy. Phone.þ39.02.55033328; Fax: þ39.02.50320403. E-mail: antonino.neri@unimi.it
doi: 10.1158/1078-0432.CCR-10-0151
�2010 American Association for Cancer Research.
ClinicalCancer
Research
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in CLL, we applied single-nucleotide polymorphism (SNP)-array technology to a panel of 100 untreated patients withBinet stageAdisease including44patientswith13qdeletionas assessed by fluorescence in situ hybridization (FISH). Wethen investigated the expression levels of the miR-15a/16cluster by quantitative real-time PCR (qRT-PCR) and globalgene expression profiling in a representative panel of cases(see flow chart in Supplementary Figure S1). The integrationbetween genomic and expression data allowed the identi-fication of 2 distinct molecular groups of patients with13q14 deletions. Our data may have implications for thebiology and the prognostic stratification of CLL.
Materials and Methods
PatientsThe study included samples from 100 untreated CLL
patients in Binet stage A: 60 from a retrospective collabora-tive Italian study (10) and 40 enrolled in an Italian pro-spective multicenter study (GISL O-CLL1). Eligibilityrequired a diagnosis of a typical CLL phenotype: CD5/CD19þ and CD23þ, weak surface immunoglobulin (sIg),
and the monotypical expression of k or l light chains byneoplastic cells. Patients were selected to provide a goodnumerical representation of cytogenetic lesions, as assessedby FISH, and IgVH mutation status. All the patients gavetheir informed consent in accordance with our institutionalguidelines.
Sample preparation, immunophenotype, andprognostic markers
Peripheral blood mononuclear cells were isolated byFicoll–Hypaque gradient centrifugation (Seromed), and
CD38 and ZAP-70 expression were determined by flowcytometry (11). IgVH gene usage and mutational statuswere established as previously described (12), with a 2%cutoff value being used to define mutated and unmutatedpatients. For the microarray analyses, the CLL cells wereenriched by negative selection when less than 90% (11).
Fluorescence in situ hybridizationThe most common genomic aberrations, del(17)(p13),
del(11)(q23), del(6)(q23), del(13)(q14), and trisomy 12,were investigated by interphase FISH hybridization. All ofthe probes are commercially available (Vysis; ref. 13).
High-density SNP arrays and data analysisTotal genomic DNA (250 ng) were processed in accor-
dance with the manufacturer’s protocol (Affymetrix),hybridized using Affymetrix GeneChip Human Mapping250K NspI microarrays, and subsequently scanned using aGeneChip Scanner 3000 7G. The images were acquiredusing the Affymetrix GeneChip Operating System (GCOS1.4). The accuracy of the SNP array data were confirmed bythemean andmedian call rates of 95.75% and 96.12% (thequality control specification for 250K arrays is a call rategreater than 93%), respectively.
The entire procedure for the copy number (CN) estima-tion has been fully described previously (14). Briefly, theraw data relating to the individual SNPs were extractedfrom CEL files and converted into signal intensities usingGTYPE 4.1 and Affymetrix Copy Number Analysis Tool(CNAT 4.0.1) software. Each sample was compared with aset of 48 normal Caucasian HapMap references availableon the Affymetrix web site (http://www.affymetrix.com/support/technical/sample_data/500k_data.affx) and thegenomic smoothing window of the Hidden Markov Modelalgorithm was set at 0. After preprocessing, piecewise con-stant estimates of the underlying local DNA CN alterationswere calculated using the DNAcopy Bioconductor package,which looks for optimal breakpoints on the basis of circularbinary segmentation (15), and the median values of theestimated profiles were scaled back to a nominal multi-plicity of 2. After scaling, we determined all the clustersappearing on the frequency distribution (histogram) of theSNP values for all the SNP probes using the k-meansalgorithm on the cumulative profile of all of the arraydata. The transition from one cluster to the other, namelythe threshold from one CN to another, was then estimatedas the meeting point of 2 Gaussian curves and approximat-ing the distributions of mean m and variance s2 of the 2neighboring clusters of SNPs identified in the final cluster-ing. Thus, the thresholds result as follows: inferred CNs ofmore than 2.1 and 2.5 corresponded to gain and amplifi-cation, whereas CNs of less than 1.9 and 1.34 correspondedto loss and biallelic deletion. After segmentation, the SNPdataset was compressed to 1346 probes by eliminatingredundant probes to better balance less represented regionswith those showing a large number of probes, as previouslydescribed (14). A probe was defined as redundant whenit showed CN values identical to those of the most
Translational Relevance
The 13q14 deletion represents the most commongenomic aberration in CLL (50%). Although it is asso-ciated with a favorable prognosis when present as thesole abnormality, the imprecise knowledge of the genesimplicated and its genetic heterogeneity have limitedour understanding on the pathogenetic mechanismscontributing to the disease. Microarray technologyhas improved the possibility of combining genome-wide DNA with transcriptomic profiles to identifypotential candidate tumor genes related to underlyingchromosomal aberrations. Based on SNP array, ourstudy shows the presence of 2 distinct molecular groupsof patients with del(13)(q14) based on the deletion sizeand the presence of biallelic deletions. Notably, globalgene expression profiling identified a significant tran-scriptional deregulation specifically associated with the2 groups. Our data highlight the presence of 2 distinctmolecular types of 13q14 deletion that may be ofclinical relevance for the biology and the prognosticstratification of the disease.
Mosca et al.
Clin Cancer Res; 16(23) December 1, 2010 Clinical Cancer Research5642
Research. on October 18, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151
contiguous upstream probe in all samples. Then a non-negative matrix factorization (NMF) procedure adapted toR from the original MATLAB package (16) was used toevaluate the meaningful clusters across the whole dataset.For each factor level from 2 to10, NMF was repeated 100times to build a consensus matrix, and the samples wereassigned to clusters on the basis of the consensus results.
Sequence copy number determination by quantitativereal-time PCRReal-time PCR was performed according to a published
protocol (17) using the ABI Prism 7900 sequence detec-tion system. Singleplex amplification reactions were car-ried out in triplicate using 40 ng of template DNA, 1XTaqMan Universal Master Mix, no AmpErase UNG, and a1X Primer-Probe Mix (Applied Biosystems) containingsequence-specific primers and a fluorogenic probe.The TaqMan RNase P Detection Reagents kit and aCustom TaqMan Gene Expression Assay (forward primer:50-GCAATGTCAGCAGTGCCTTAG-30; reverse primer:50-CAGCAGCACAGTTAATACTGGAGAT-30; probe: 50-FAM-CAGCACGTAAATATTG-30) were used to amplifythe ribonuclease P RNA component H1 gene mappedwithin 14q11.2 (present in 2 copies in all of the subjectsand thus used as an internal standard) and the miR-15a/16-1 cluster. The comparative DCt method was used forquantification purposes. DNA samples from 10 controlindividuals with 2 copies of the commonly deleted regionwere selected for PCR calibration. The estimated haploidgene CN was given by the formula 2�DDCt, and the pre-dicted CN was calculated as the closest integer number tothe estimated CN (determining Gene Copy Number usingTaqMan Real-Time PCR Assays on the 7900 HT-QuickReference Card; Applied Biosystems; ref. 18).
Quantification of specific gene expressionby Q-RT-PCRQ-RT-PCR of specific genes (TPT1, WBP4, PEA15,
and LGALS1) and mature miRNAs (hsa-miR-15a andhsa-miR-16) was performed using commercial TaqManassays (Applied Biosystems) as previously described(19). The relative gene and miRNA expression levels werecalculated using the 2�DCt method (Applied BiosystemsUser Bullettin No. 2) as previously described (19).Pearson’s correlation coefficient was calculated to testcorrelation between gene expression and Q-RT-PCR data.
Gene expression profilingTwenty-two CLL samples with 13q deletion included in
the retrospective database underwent gene expressionprofiling (GEP) analysis. Total RNA was extracted usingthe TRIZOL reagent (Invitrogen) and purified using theRNeasy total RNA Isolation Kit (Qiagen). The biotin-labeled complementary RNA was prepared and hybri-dized with GeneChip Human Genome U133A Arrays(Affymetrix Inc.), which were scanned (GeneChip Scan-ner 3000 7G; Affymetrix Inc.) in accordance with themanufacturer’s protocols. The probe data were converted
to expression values using the Bioconductor function forthe robust multiarray average procedure, as describedpreviously (20). Supervised analysis was made usingSignificant Analysis of Microarrays software, version3.0.2 (http://www-stat.stanford.edu/�tibs/SAM/index.html/; ref. 21). The cutoff point for significance at amedian false discovery rate (FDR) less than 5% (i.e., q< 0.05) was determined by tuning the D parameter on theFDR and controlling the q-value of the selected probes.dChip software (22) was used to represent the selectedprobe lists; NetAffx (https://www.affymetrix.com/analy-sis/netaffx/) was used for the functional annotation studyof the list. The genotyping and gene expression data areavailable at NCBI’s Gene Expression Omnibus throughGEO Series Accession no. GSE16746.
Statistical analysisThe data were statistically analyzed using conventional
procedures in R software (Kruskal–Wallis tests, Kendall’s tcorrelations, Fisher’s exact tests, and q-value calculations).
Results
Molecular characteristics of the CLL patientsThemain characteristics of the 100CLL patients included
in the study are reported in Supplementary Tables S1 andS2. Based on FISH analyses, 44 cases carried the 13q14deletion: 34 as the sole abnormality, and the remainingshowed 11q22.3 (6 patients), 17p13.1 (2 patients),11q22.3 and 17p13.1 deletions (1 patient), or 6q23 dele-tion (1 patient). Biallelic 13q14 deletions were identified in11 cases, 5 of which (CS3, CZ42, GE110, CS0100 andLD0062) showed the presence of subclones characterizedby the biallelic deletion (ranging from 68% to 83.5%).Trisomy 12 was present in 21 patients as a sole abnorm-ality. Overall, the 11q22.3, 17p13.1, and 6q23 deletionswere present in 15, 7, and 2 patients, respectively. Fifty-fivecases had unmutated IgVH genes; ZAP-70 and CD38 wereexpressed in 42 and 46 cases, respectively. 13q deletion waspresent in 25 of 45 (55.5%) patients with mutated IgVHgenes, 15 of 42 (35.7%) of the ZAP-70 positive patients,and 15 of 46 (32.6%) of the CD38 positive patients.
SNP array data were concordant with FISH results(Supplementary Tables S2 and S3). In addition, SNP arraysdetected short deletions involving the second 13q allele in3 del(13)(q14) patients (TS12, VB0013, and PS0044);deletions were, respectively, 467, 468, and 291-kb longand located centromerically to the LSI D13S25 FISH probe(Fig. 1A and B). Furthermore, SNP arrays revealed a 6qdeletion of approximately 39 Mb in length and locatedupstream of the LSI MYB FISH probe in a single patients(CZ47; data not shown). These aberrations were confirmedby specific FISH probes in all cases (data not shown).
Characterization of the 13q deletion by SNP arraysThe SNP arrays showed that the 13q deletions varied
considerably in size, ranging from a minimum of 291 kb(PS0044) to a maximum of 56 Mb (CZ36; Fig. 1A and B).
Integrative Genomics Analysis of 13q14-Deleted CLL
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C
A
B
Fig. 1. Chromosome 13 deletion pattern in 44 CLLs. A, monoallelic (gray lines) and biallelic losses (black lines) in the 44 deleted patients. B, theenlarged subregion spanning 13q14.2–q14.3 between physical positions 44.50 and 52.00 Mb, including the minimal monoallelic deletion. C, thelocalization of SNPs and the FISH probe encompassing the minimally altered region (gray bar). Gene locations and transcriptional orientation areindicated by the horizontal arrows at the bottom.
Mosca et al.
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The minimal monoallelic deletion was 635-kb longspanning from SNP_A-2003314 to SNP_A-2003318(physical position 49,635,024 bp to 50,270,550 bp;Fig. 1C); notably, the miR-15a/16-1 cluster is locatedupstream (�87 kb) of the centromeric SNP_A-2003314. With regard to the miR-15a/16-1 cluster, SNParray analysis depicted the following scenario: (i) reten-tion of 2 copies of the cluster in 56 patients with a normal
13q and in 2 cases (CD0018 and CS95) showing a 13qmonoallelic deletion telomeric to the cluster; (ii) reten-tion of 1 copy of the cluster in 29 patients with a 13qmonoallelic deletion and in 2 cases (CZ42 and PS0044)showing a deletion telomeric to the cluster in the secondallele; (iii) loss of both copies of the cluster in theremaining 11 patients showing biallelic deletions(Fig. 1A and B; Supplementary Table S4).
C
A
B
Fig. 2. Sequence copy number and expression quantification of miR-15a/16 as assessed by Q-RT-PCR. A, vertical axis: 2�DDCt values for the 32 investigatedpatients. The inferred CN values obtained on the basis of the criteria described in Material and Methods are shown for each patientsas white bars (CN ¼ 0), striped bars (CN ¼ 1), and black bars (CN ¼ 2); horizontal axis: patient distribution into 3 classes on the basis of the numberof alleles (0, 1, and 2) detected by SNP array. B and C, expression of miR-15a and miR-16 as calculated using the 2�DCt method and ranked accordingto the corresponding inferred CN value (CN0 ¼ 11 cases; CN1 ¼ 28 cases, including PS0044 patient; CN2 ¼ 39 cases, including CD0018 and CS95patients). P values of the comparison of between the biallelic and monoallelic groups.
Integrative Genomics Analysis of 13q14-Deleted CLL
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The status of the miR-15a/16-1 cluster was investigatedfurther using a custom Q-RT-PCR assay on DNA from 32patients: 10 with biallelic deletions, 10 with monoallelicdeletions, and 12 normal at 13q14 based on SNP arraydata. As shown in Figure 2A and B and SupplementaryTable S4, the estimated CN obtained with this approachwere concordant with the SNP array data (P < 0.0001)with 3 exceptions: 2 patients (CS3 and GE110) classifiedas CN ¼ 1 by RT-PCR and CN ¼ 0 by SNP array, whoshowed 2 distinct cell populations by FISH characterizedby either mono- or biallelic losses, and 1 patient (RC21)classified as normal by RT-PCR and CN ¼ 1 by SNP arraywho showed a monoallelic loss in a fraction of interphasenuclei (61%; Fig. 2A). These findings suggest that clonalheterogeneity may account for the discrepancies betweenthe real-time PCR and SNP array data.
Furthermore, we compared miR-15a and miR-16expressions in 78 of 100 cases including 11 with biallelic
deletions, 28 with monoallelic deletions and 39 normalcases based on SNP array CN values. Cases with biallelicdeletions had a miRNA expression significantly lowerthan cases retaining 1 or 2 copies (P < 0.0001 for miR-15a, P ¼ 0.0001 for miR-16) whereas no statisticallysignificant differences in miRNA expression levels couldbe observed between normal cases and those retaining asingle copy of the cluster (Fig. 2B and C; SupplementaryTable S5).
Finally, SNP array documented a loss of the RB1 gene in28 of 44 cases with 13q14 deletion (63.6%). Two samples(CS0100 and TS12) showed a biallelic loss of the RB1locus, which was associated with a homozygous deletion ofthe 13q14 FISH probe (case CS0100) or was the result of asmall deletion involving the RB1 locus on the second allele(case TS12; Fig. 1B). All these findings were validated byFISH using the RB1 specific clone RP11-305D15 (data notshown).
Fig. 3. Heat map of significantly altered DNA regions in 100 CLLs, as assessed by GeneChip Human Mapping 250K Nsp. The genomic profiles of the CLLsamples (horizontal lines) are clustered into 4 groups in accordance with the NMF method. Horizontal axis: chromosome localization. The dashed linesrepresent the centromeres. Light-green, biallelic deletion; dark-green, loss; white, normal copy number; red, gain.
Mosca et al.
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Identification of 2 genetically distinct patientsubgroups with del(13)(q14)To identify the most significant natural grouping of
genome profiles in our panel, we used the NMF algorithm.This analysis led to the identification of 4 major groups(correlation coefficient ¼ 0.95) characterized by 13q dele-tion (groups I and II, 27 and 13 cases, respectively) and bytrisomy 12 (group III, 21 cases), whereas no specific altera-tion could be associated with group IV (39 cases; Fig. 3;Table 1). Patients from groups I and II differed in thedeletion size and for the occurrence of mono- or biallelicdeletions: group I included samples with relatively smallerlosses, 10 of which showed biallelic deletions, whereasgroup II included samples with larger losses, all but one(GE110) showing monoallelic deletions. The presence of 4samples with 13q14 deletions in group IV was in all like-lihood related to the fact that the inferred CN values (ran-ging from 1.74 to 1.85) were very close to the threshold(1.9) between monoallelic deletion and the retention of 2copies. These "marginal" CNdata are consistent with clonalheterogeneity and are in accordance with the small percen-tage of interphase nuclei detected by FISH showing 13q14deletion (no more than 21% in all cases), which may affectthe clustering analysis. RB1 deletion occurred in 14 of 27cases in group I (52%), 12of 13 in group II (92%), and2of 4in group IV. Only 10 of the 44 patients with del(13)(q14)carried additional known genetic lesions, namely 11q22.3(6 patients), 17p13.1 (2 patients), 11q22.3 plus 17p13.1(1 patient), 6q23 deletions (1 patient); however, no corre-lation was found with the 2 specific subgroups I and II.Finally, there was no significant association between caseswithinNMF groups I and II andCD38or ZAP-70 expressionor IgVH mutational status (data not shown).
Gene expression patterns associated with distinctgroups of patients with 13q deletionFifteen cases from group I and 7 from group II, for whom
RNA material was available, were profiled on GeneChipHG-U133A arrays. To verify whether the 2 groups could bedivided on the basis of the differences of their expressionprofiles, we made a conventional unsupervised analysisusing hierarchical agglomerative clustering at differentlevels on (i) all of the probes in the array; (ii) all of theprobes mapped to the chromosome 13; or (iii) all ofthe probes within the 13q14 region. Notably, a significantgrouping (i.e., group I and group II patients in 2 separatebranches) was only detected when the 89 probes (65 genes)mapped on the 13q14 region were used (P < 0.0001;
Fig. 4A). This finding indicates that, albeit differences existbetween the 2 groups, these are not sufficient to drive theclustering when the global transcriptional profiling wasconsidered (i.e., the whole matrix). Thus, we carried out asupervised analysis to characterize the specific transcrip-tional profiles distinguishing NMF groups I and II. Weidentified 76 differentially expressed probe sets, specific to63 well-characterized genes (Fig. 4B), 10 of which weredownregulated and 53 upregulated in group II comparedwith group I. Six of the 10 downregulated genes mapped to13q13-q14 and exhibited amonoallelic deletion inmost orall cases within group II [3 of 7 (43%) for FOXO1; 4 of 7(57%) for EXOSC1 and WBP4; 6 of 7 (86%) for TPT1 andNUFIP1; 7 of 7 (100%) for ESD) compared with cases ingroup I (1 of 15 (6.7%) for EXOSC1, TPT1, ESD, FOXO1,and NUFIP1; 2 of 15 (13.3%) forWBP4]. The whole list ofdifferentially expressed genes is reported in Table 2.
We selected 2 downregulated (TPT1 and WBP4) and 2upregulated (PEA15 and LGALS1) genes in group II versusgroup I for Q-RT-PCR validation of the microarray data.The Q-RT-PCR analyses were made in a subset of 20 of 22patients for whom RNA material was available (14 belong-ing to group I, 6 to group II). The correspondence betweenthe microarray and Q-RT-PCR data were evaluated byassessing the correlation coefficients of the expressionlevels determined by the 2 analyses: the coefficients were0.71 for TPT1, 0.79 forWBP4, 0.60 for PEA15, and 0.78 forLGALS1 probe, thus indicating a very good concordance forall of the tested genes (Supplementary Fig. S2).
Discussion
This study focused on the molecular characterization ofthe 13q14 deletion based on SNP arrays and gene expres-sion profiling analyses. Several aspects distinguish thisfrom previous reports (9, 23) including a homogeneouscohort of untreated early-stage patients (Binet A), the use ofhighly purified CLL samples and a higher-resolution SNParray (250K NspI), the application of stringent statistics todefine the genetic groups, and the integration of genomicand gene expression data in a significant number of cases.The main findings relate to the description of the topo-graphy of 13q14 deletions, the reassessment of the fate ofmiR-15a/16-1 cluster in relation to the genomic losses, and,perhaps more importantly, the definition of 2 major geno-mic groups of patients with 13q14 deletions also charac-terized by distinct transcriptional patterns.
Table 1. Molecular characteristics of the 4 genomic groups
NMF cluster No. ofcases
del(11q22.3) Monoallelicdel(13q14)
Biallelicdel(13q14)
del(17p13.1) 12 CD38þ ZAP70þ UnmutatedIgVH
I 27 3 (11.1%) 17 (63.0%) 10 (37.0%) 1 (3.7%) 0 (0%) 8 (29.6%) 10 (37.0%) 11 (40.7%)II 13 4 (30.8%) 12 (92.3%) 1 (7.7%) 2 (15.4%) 0 (0%) 4 (30.8%) 3 (23.1%) 6 (46.1%)III 21 0 (0%) 0 (0%) 0 (0%) 0 (0%) 21 (100%) 17 (81.0%) 12 (57.1%) 15 (71.4%)IV 39 8 (20.5%) 4 (10.3%) 0 (0%) 4 (10.3%) 0 (0%) 17 (43.6%) 17 (43.6%) 23 (59.0%)
Integrative Genomics Analysis of 13q14-Deleted CLL
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Tab
le2.
Func
tiona
lann
otations
ofthe63
gene
siden
tifiedas
differen
tially
expressed
amon
gthe2NMFgrou
psbySAM
2-clas
san
alysis
Probese
tID
Gen
esy
mbol
Gen
etitle
Sco
re(d)
Fold
chan
ge
q(%
)Cytoban
d
2128
69_x
_at
TPT1
Tumor
protein,
tran
slationa
llyco
ntrolled1
�6.330
80.79
013
q12
–q1
4
2090
09_a
tESD
Esteras
eD/formylglutathion
ehy
drolase
�5.013
00.56
013
q14
.1–q14
.2
2151
36_s
_at
EXOSC8
Exo
someco
mpon
ent8
�4.588
80.61
2.19
13q13
.122
2182
_s_a
tCNOT2
CCR4-NOTtran
scrip
tion
complex,
subun
it2
�4.491
10.69
2.19
12q15
2027
23_s
_at
FOXO1
Forkhe
adbox
O1
�4.390
60.67
3.26
13q14
.120
5134
_s_a
tNUFIP1
Nuc
lear
frag
ileXmen
tal
retardationprotein
interactingprotein
1
�4.325
70.66
3.26
13q14
2103
46_s
_at
CLK
4CDC-likekina
se4
�4.314
60.65
3.26
5q35
2132
56_a
tMARCH3
Mem
brane
-ass
ociated
ringfin
ger(C3H
C4)
3�4
.169
60.51
4.20
5q23
.2
2035
97_s
_at
WBP4
WW
dom
ainbinding
protein
4(fo
rmin
binding
protein21
)
�4.125
80.64
4.20
13q14
.11
2041
55_s
_at
QSK
Serine/threon
ine-protein
kina
seQSK
�4.066
20.72
4.72
11q23
.3
2130
11_s
_at
TPI1
Triose
pho
spha
teisom
eras
e1
3.93
551.60
4.72
12p13
2010
13_s
_at
PAICS
Pho
spho
ribos
ylam
inoimidaz
ole
carbox
ylas
e,pho
spho
ribos
ylam
inoimidaz
ole
succ
inoc
arbox
amidesynthe
tase
3.94
071.73
4.72
4q12
2040
47_s
_at
PHACTR
2Pho
spha
tase
andac
tinregu
lator2
3.94
281.33
4.72
6q24
.2
2018
50_a
tCAPG
Cap
pingprotein(actin
filam
ent),
gelsolin-like
3.94
392.24
4.72
2p11
.2
2015
77_a
tNME1
Non
metas
tatic
cells
1,protein
(NM23
A)ex
pres
sedin
3.96
021.62
4.72
17q21
.3
2178
65_a
tRNF1
30Ringfin
gerprotein13
03.96
172.02
4.72
5q35
.320
3857
_s_a
tPDIA5
Protein
disulfid
eisom
eras
efamily
A,mem
ber
53.99
101.25
4.01
3q21
.1
2011
05_a
tLG
ALS
1Le
ctin,ga
lactos
ide-binding,
soluble,1
3.99
673.27
4.01
22q13
.1
4048
9_at
ATN
1Atrop
hin1
4.03
881.23
4.01
12p13
.31
(Con
tinue
don
thefollo
wingpag
e)
Mosca et al.
Clin Cancer Res; 16(23) December 1, 2010 Clinical Cancer Research5648
Research. on October 18, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151
Tab
le2.
Func
tiona
lan
notatio
nsof
the
63ge
nes
iden
tified
asdifferen
tially
expressed
amon
gthe
2NMF
grou
ps
by
SAM
2-clas
san
alysis
(Con
t'd)
Probese
tID
Gen
esy
mbol
Gen
etitle
Sco
re(d)
Fold
chan
ge
q(%
)Cytoban
d
4412
0_at
ADCK2
aarF
domainco
ntaining
kina
se2
4.06
161.18
4.01
7q32
–q34
2093
29_x
_at
HIG
D2A
HIG
1hy
pox
iaindu
cible
dom
ainfamily,mem
ber
2A4.06
861.27
4.01
5q35
.2
4085
0_at
FKBP8
FK50
6bindingprotein
8,38
kDa
4.07
681.35
4.01
19p12
2035
07_a
tCD68
CD68
molec
ule
4.08
091.22
4.01
17p13
2016
05_x
_at
CNN2
Calpon
in2
4.10
171.59
4.01
19p13
.321
1793
_s_a
tABI2
Abl-interactor
24.10
521.26
4.01
2q33
2178
06_s
_at
POLD
IP2
Polym
eras
e(DNA-dire
cted
),delta
interactingprotein
24.13
881.26
3.26
17q11
.2
2212
69_s
_at
SH3B
GRL3
SH3dom
ainbind
ingglutam
icac
id-richprotein
like3
4.19
761.58
3.26
1p35
–p34
.3
2116
22_s
_at
ARF3
ADP-ribos
ylationfactor
34.20
201.25
3.26
12q13
2174
57_s
_at
RAP1G
DS1
RAP1,
GTP
-GDPdisso
ciation
stim
ulator
14.22
391.26
1.95
4q23
–q25
2048
75_s
_at
GMDS
GDP-m
anno
se4,6-deh
ydratase
4.24
551.63
1.95
6p25
2177
36_s
_at
EIF2A
K1
Euk
aryo
tictran
slation
initiationfactor
2-alph
akina
se1
4.27
281.57
1.95
7p22
2057
70_a
tGSR
Glutathione
reduc
tase
4.28
001.35
1.95
8p21
.120
7722
_s_a
tBTB
D2
BTB
(POZ)do
mainco
ntaining
24.30
071.50
1.95
19p13
.320
6200
_s_a
tANXA11
Ann
exin
A11
4.30
521.62
1.95
10q23
2088
77_a
tPAK2
p21
protein(Cdc4
2/Rac
)-ac
tivated
kina
se2
4.34
491.84
1.95
3q29
2125
66_a
tMAP4
Microtubule-as
sociated
protein
44.36
081.50
1.95
3p21
2007
88_s
_at
PEA15
Pho
spho
protein
enric
hed
inas
troc
ytes
154.38
381.57
1.95
1q21
.1
2191
62_s
_at
MRPL1
1Mito
chon
dria
lribos
omal
protein
L11
4.42
121.29
1.95
11q13
.3
2172
11_a
t—
—4.44
141.41
1.95
—
3220
9_at
FAM89
BFa
mily
with
seque
nce
similarity89
,mem
ber
B4.46
151.70
1.95
11q23
2123
30_a
tTF
DP1
Tran
scrip
tionfactor
Dp-1
4.50
851.95
1.95
13q34
2020
41_s
_at
FIBP
Fibroblast
grow
thfactor
(acidic)
intrac
ellularbindingprotein
4.51
751.71
1.95
11q13
.1
2086
77_s
_at
BSG
Bas
igin
(Okblood
grou
p)
4.55
871.78
1.95
19p13
.3
(Con
tinue
don
thefollo
wingpag
e)
Integrative Genomics Analysis of 13q14-Deleted CLL
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Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151
Tab
le2.
Func
tiona
lan
notatio
nsof
the
63ge
nes
iden
tified
asdifferen
tially
expressed
amon
gthe
2NMF
grou
ps
by
SAM
2-clas
san
alysis
(Con
t'd)
Probese
tID
Gen
esy
mbol
Gen
etitle
Sco
re(d)
Fold
chan
ge
q(%
)Cytoban
d
2000
01_a
tCAPNS1
Calpain,
smalls
ubun
it1
4.61
761.71
019
q13
.12
2180
37_a
tFA
M13
4AFa
mily
with
seque
ncesimilarity
134,
mem
ber
A4.70
821.48
02q
35
3796
5_at
PARVB
Parvin,
beta
4.78
961.48
022
q13
.2–q13
.33
2137
39_a
t—
—4.81
741.28
0—
2210
59_s_a
tCOTL
1Coa
ctos
in-like1(Dictyos
telium)
4.85
242.20
016
q24
.121
1672
_s_a
tARPC4///TT
LL3
Actin-related
protein2/3
complex,
subun
it4,
20kD
a///
tubulin
tyrosine
ligas
e-likefamily,mem
ber
3
4.90
821.68
03p
25.3
2008
38_a
tCTS
BCathe
psinB
5.09
361.58
08p
2220
1426
_s_a
tVIM
Vim
entin
5.12
721.82
010
p13
2134
53_x
_at
GAPDH
Glyce
raldeh
yde-3-ph
ospha
tedeh
ydroge
nase
5.29
861.70
012
p13
2141
19_s_a
tFK
BP1A
FK50
6binding
protein1A
,12
kDa
5.32
261.78
020
p13
2090
41_s_a
tUBE2G
2Ubiquitin
-con
juga
tingen
zyme
E2G
2(UBC7ho
molog
,ye
ast)
5.38
331.65
021
q22
.3
2012
51_a
tPKM2
Pyruv
atekina
se,mus
cle
5.41
681.92
015
q22
2088
29_a
tTA
PBP
TAPbindingprotein
(tapas
in)
5.52
711.55
06p
21.3
2010
82_s_a
tDCTN
1Dyn
actin
1(p15
0,glue
dho
molog
,Droso
phila)
5.53
972.09
02p
13
2183
88_a
tPGLS
6-Pho
spho
gluc
onolac
tona
se5.88
841.89
019
p13
.220
1516
_at
SRM
Spermidinesy
ntha
se5.90
121.57
01p
36–p22
2011
36_a
tPLP
2Proteolipid
protein
2(colon
icep
ithelium-enriche
d)
6.04
221.62
0Xp11
.23
2009
66_x
_at
ALD
OA
Aldolas
eA,fruc
tose
-bispho
spha
te6.10
411.72
016
p11
.220
3683
_s_a
tVEGFB
Vas
cularen
dothe
lialg
rowth
factor
B6.33
301.51
011
q13
2083
08_s_a
tGPI
Gluco
sepho
spha
teisom
eras
e7.12
611.94
019
q13
.1
Mosca et al.
Clin Cancer Res; 16(23) December 1, 2010 Clinical Cancer Research5650
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The natural grouping of genome profiles by SNP arrayanalysis showed that the complex scenario of CN altera-tions affecting CLL is mainly driven by the presence of the13q14 deletion and trisomy 12. Our SNP array approachextends previous limited evidence showing that del(13)(q14) patients are characterized by differently sized dele-tions. Ouillette et al. (9) documented a significant correla-tion between a wider deletion encompassing the RB1 geneand a higher Rai clinical stage at diagnosis or in previouslytreated patients. In preliminary observations on our cohortof Binet stage A patients, we did not find any significantdifferences in the time to treatment between the patientswith shorter/biallelic (group I) or wider/monoallelic losses(group II; data not shown), although this finding awaitsconfirmation by ongoing prospective studies.
Our data support the notion that loss of the miR-15a/16-1 locus occurs in virtually all the CLL cases with del(13)(q14); in fact, among 44 cases with 13q deletion in ourstudy, both copies of miRNA cluster were retained in only 2patients with a monoallelic 13q14 deletion. Furthermore,we found that reduced miR-15a and miR-16 expressionlevels in patients with del(13)(q14) significantly correlatedonly with the presence of a biallelic loss, which is inagreement with some recent data (8, 9) but not with theolder findings (7). Overall, these findings suggest a need toredefine the pathogenetic role of miR-15a and miR-16-1 inthe context of the molecular subtypes of 13q14-deletedpatients.
The availability and integration of GEP data allowed amore comprehensive overview of the genetic complexity ofthe 13q14 deletion. Indeed, this approach led to thedetermination of distinct transcriptional signatures asso-ciated with different groups of 13q14-deleted patients(short/biallelic versus long/monoallelic lesion) identifiedby the NMF algorithm. In particular, a significant genedosage effect has been observed involving the downregula-tion of genes in group II (long/monoallelic deletion), 6 ofwhich localized within the 13q14 region. Among thedownregulated genes, we should note TPT1/TCTP (trans-lationally controlled tumor protein) encoding for a highlyconserved multifunctional protein acting as a prosurvivaland growth stimulating factor (24), which inhibits BAX-induced apoptosis (25).
Most of the upregulated genes in group II are involved incell motility and adhesion, regulation of cell proliferation,tumor cell migration, metastasis, angiogenesis, and apop-tosis, and some of these may contribute to lymphomagen-esis. This is the case of the autocrine motility factor (AMF)/glucose phosphate isomerase (GPI) gene, which is upre-gulated in several human cancers and encodes for a house-keeping cytosolic enzyme involved in both glycolysis andgluconeogenesis (26). The basigin gene (BSG) encodes acell surface glycoprotein of the Ig superfamily expressed atthe surface of tumor cells metastasizing in bone marrow(27) and is believed to induce matrix metalloproteinasesproduction (28). The Galectin-1 (alias LGALS1) geneencoding a 14-kDa lectin, is overexpressed in numeroustumors including lymphomas (29) and CLL, and is
Fig. 4. Identification of gene signatures characterizing 13q14 classes.A, dendrogram of the 22 CLL samples clustered according to theexpression profiles of the genes located at 13q14. B, expression profiles ofthe NMF CLL group I versus group II for the 76 probe sets selected by aSAM 2-class analysis. Information on chromosomes 11, 13, and 17deletions, chromosome 12 trisomy (þ, positive; –, negative; /, biallelicdeletion), CD38 (þ, �30%; –, <30%), ZAP-70 expression (þ, �30%; –,<30%), and IgVH mutational status (þ, mutated; –, unmutated) is includedalongside the patient ID. The color scale bar represents the relativechanges in gene expression normalized by the standard deviation, and thecolor changes in each row represent gene expression in relation to themean across the samples.
Integrative Genomics Analysis of 13q14-Deleted CLL
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implicated in abnormal mechanisms of cell adhesion,induction of apoptosis, and tumor angiogenesis (30,31). PAK2 belongs to the p21-activated kinase family,which are well-known regulators of cytoskeletal remodel-ling, cell motility, proliferation, and apoptosis (32).Finally, 2 other upregulated genes, b-parvin (PARVB)and vimentin (VIM), have been reported to play a criticalrole in transducing signals from integrins to the actincytoskeleton and intracellular signaling proteins (33).Low levels of PARVB correlate with low adhesion to col-lagen (34), whereas increased levels reduce the activatingphosphorylation of AKT (35), causing propensity to apop-tosis. Upregulation of VIM is thought to provide a selectiveadvantage to tumor cells following signaling cues frommesenchymal and epithelial extracellular matrixes (36).Notably, a modulation of genes thought to act as regulatorsof tumor invasion and integrin-mediated cell motility andadhesion has been recently described in CLL, in particularduring disease progression (37, 38).
In conclusion, our data may represent a valid contribu-tion to the definition of the genomic profile of CLL. Inparticular, we provide evidence of 2 clearly distinguishable
molecular subtypes among CLL patients with 13q14 dele-tion thatmay contribute toward the better understanding ofthe pathogenetic and clinical relevance of this lesion in CLL.
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Grant Support
This study was supported by grants from AIRC to A.N. (IG 4659)and F.M. (RG 6432 cofinanced by AIRC, CARICAL, Fondazione ‘AmeliaScorza’ and Provincia di Cosenza); AIL Sezione Milano; Fondazione‘Amelia Scorza’ ONLUS, Cosenza; Progetti Strategici–Ricerca FinalizzataMinistero Italiano della Salute "RFPS20063339960" (to G.C.) and"RFPS2006340196" (to F.M. and M.F.); and FIRB (Grant RBIP06LCA9 toM.F.). The Progetto Ordinario Ricerca Finalizzata Ministero Italianodella Salute-2007 (to G.C.), Progetto Compagnia San Paolo (to G.C.),and the Fondazione Internazionale Ricerche Medicina Sperimentale(FIRMS) provided financial and administrative assistance. L.A. and S.M.were supported by fellowships from the Fondazione Italiana Ricerca sulCancro (FIRC).
Received 01/19/2010; revised 06/25/2010; accepted 07/24/2010;published OnlineFirst 10/14/2010.
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Integrative Genomics Analysis of 13q14-Deleted CLL
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Research. on October 18, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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2010;16:5641-5653. Published OnlineFirst October 14, 2010.Clin Cancer Res Laura Mosca, Sonia Fabris, Marta Lionetti, et al. with 13q14 DeletionSubgroups of B-Cell Chronic Lymphocytic Leukemia Patients Integrative Genomics Analyses Reveal Molecularly Distinct
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Research. on October 18, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst October 14, 2010; DOI: 10.1158/1078-0432.CCR-10-0151