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Genome-wide DNA profiling better defines the prognosis of chronic lymphocytic leukaemia Chronic lymphocytic leukaemia (CLL) is the most common form of adult leukaemia in the Western world (Dighiero & Hamblin, 2008; Hallek et al, 2008). The disease has a very heterogenous clinical course and survival after diagnosis can range from months to decades. For many years the disease was considered as a chronic non-curable malignancy that required only a few diagnostic tools and palliative chemotherapy was limited to chlorambucil with or without corticosteroids. However, fast and significant therapeutic progress has been achieved in the past decade by combining different agents; indeed fludarabine, bendamustine and three monoclonal antibodies, alemtuzumab, rituximab and ofatumumab have Andrea Rinaldi,1* Michael Mian,1,2* Ivo Kwee,1,3* Davide Rossi,4 Clara Deambrogi,4 Afua A. Mensah,1 Francesco Forconi,5 Valeria Spina,4 Emanuele Cencini,5 Daniela Drandi,6 Marco Ladetto,6 Rita Santachiara,7 Roberto Marasca,7 Valter Gattei,8 Franco Cavalli,1 Emanuele Zucca,1 Gianluca Gaidano4 and Francesco Bertoni1 1Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland, 2Division of Haematology, Azienda Ospedaliera S. Maurizio, Bolzano/Bozen, Italy, 3Dalle Molle Institute for Artificial Intelligence (IDSIA), Manno, Switzerland, 4Division of Haematology, Department of Clinical and Experimental Medicine and IRCAD, Amedeo Avogadro University of Eastern Piedmont, Novara, 5Division of Haematology, Department of Clinical Medicine and Immunological Sciences, University of Siena and AOUS, Siena, 6Haematology, University of Turin, Turin, 7Division of Haematology, Department of Oncology and Haematology, University of Modena and Reggio Emilia, Modena, and 8Clinical and Experimental Onco-Haematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano (PN), Italy Received 30 March 2011; accepted for publication 31 May 2011 Correspondence: Francesco Bertoni, MD, Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland (IOSI), via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. E-mail: frbertoni@mac.com *These authors contributed equally to this study. Summary The integration of molecular and clinical information to tailor treatments remains an important research challenge in chronic lymphocytic leukaemia (CLL). This study aimed to identify genomic lesions associated with a poor outcome and a higher risk of histological transformation. A mono- institutional cohort of 147 cases was used as the test series, and a multi- institutional cohort of 176 cases as a validation series. Genomic profiles were obtained using Affymetrix SNP 6.0. The impact of the recurrent minimal common regions (MCRs) on overall survival was evaluated by univariate analysis followed by multiple-test correction. The independent prognostic significance was assessed by multivariate analysis. Eight MCRs showed a prognostic impact: gains at 2p25.3-p22.3 (MYCN), 2p22.3, 2p16.2-p14 (REL), 8q23.3-q24.3 (MYC), losses at 8p23.1-p21.2, 8p21.2, and of the TP53 locus. Gains at 2p and 8q and TP53 inactivation maintained prognostic significance in multivariate analysis and a hierarchical model confirmed their relevance. Gains at 2p also determined a higher risk of Richter syndrome transformation. The prediction of outcome for CLL patients might be improved by evaluating the presence of gains at 2p and 8q as novel genomic regions besides those included in the ‘standard’ fluorescence in situ hybridization panel. Keywords: leukaemia, prognostic factors, Richter’s syndrome, transforma- tion, Affymetrix. research paper First published online 12 July 2011 doi:10.1111/j.1365-2141.2011.08789.x ª 2011 Blackwell Publishing Ltd, British Journal of Haematology, 154, 590–599 recently been approved by American and/or European regu- latory agencies for the treatment of CLL (Hallek, 2009). New insights into CLL molecular genetics have identified several features that may predict prognosis (Krober et al, 2002; Stevenson & Caligaris-Cappio, 2004; Hallek, 2008; Hallek et al, 2008; Dal-Bo et al, 2009; Malcikova et al, 2009; Rossi et al, 2009a,b; Zenz et al, 2010; Chiorazzi & Ferrarini, 2011). Nevertheless, major efforts are still required for the identifi- cation of the biological and genetic features that may allow a rational application of the new therapies aimed at increased curability for poor risk patients and that spare toxicity for the good risk cases. Here, we present a study of two independent large series of CLL samples analysed with microarray-based comparative genomic hybridization (arrayCGH), which iden- tified novel genomic lesions associated with a poor outcome and transformation to Richter syndrome, possibly allowing a more precise definition of the prognostic groups in CLL. Materials and methods Tumour panel Chronic lymphocytic leukaemia samples from two large consecutive series were investigated. A first mono-institutional cohort consisted of samples with more than 60% of CD19+ cells and was used as the test series. A second multi- institutional cohort of unselected consecutive CLL cases was used as the validation series. All samples were collected at diagnosis. The study was approved by the Bellinzona ethical committee. CLL diagnosis and management were based on the National Cancer Institute (NCI) Working group criteria (Hallek et al, 2008). Cases of monoclonal B-cell lymphocytosis were discarded. Baseline clinical and molecular characteristics were collected as previously described (Rossi et al, 2009a,b). TP53 inactiva- tion was defined as the presence of TP53 somatic mutations and/or del(17p13) by fluorescence in situ hybridization (FISH) or del(17p13) by arrayCGH. FISH was defined as favourable in the presence of a normal FISH result, or del(13q14.3) or +12, whilst unfavourable FISH was defined as the presence of del(11q22-q23) or del(17p13) (Rossi et al, 2009b). ArrayCGH DNA samples were obtained as previously reported (Rossi et al, 2009a; Rinaldi et al, 2011). DNA integrity was verified by electrophoresis of 50 ng on a 1% agarose gel (Rinaldi et al, 2011). Genomic profiles were obtained using Affymetrix Human Mapping GeneChip 6.0 arrays (Affymetrix, Santa Clara, CA, USA) and data mining was performed as previously described (Mian et al, 2011). Initial array intensity data was obtained using Affymetrix GeneChip Command Console Software (AGCC) with 270 Caucasian HapMap samples as a reference dataset for normalization (Mian et al, 2011). Affymetrix Power Tools (APT 1.10.2) were used to obtain genotype data and raw copy number (CN) and segmentation was performed with the circular binary segmentation algo- rithm using default settings (Olshen et al, 2004) on log 2-transformed data (Mian et al, 2011). After normalization of each profile to a median log 2-ratio of zero, thresholds for loss and gain were defined as six times the median absolute deviation symmetrically around zero with an associated P < 0Æ001 after Bonferroni multiple test correction (Mian et al, 2011; Rinaldi et al, 2011). Thresholds for homozygous deletions and amplifications were established heuristically because the low frequency of these events impeded the statistical computation of boundaries (Mian et al, 2011). Profiles were considered as poor quality and discarded from further analyses if they showed severe over-segmentation as evaluated by two independent investigators. Segments containing <10 probes and/or with more than 80% of overlap with known copy number variations (CNV, Database of Genomic Variants, http://projects.tcag.ca/variation) were discarded. Minimal common regions (MCR) were calculated in the test set alone according to the algorithm by Lenz et al (2008). Four distinct types of MCR were defined: short recurrent abnormality (SRA), long recurrent abnormality (LRA), abnormal chromosome arm (ACA) and abnormal whole chromosome (AWC). A maximum segment size of 25 Mb and gap size of 500 kb for SRA analysis, and a minimal segment size of 15 Mb and gap size of 10 Mb for LRA analysis were applied (Lenz et al, 2008). MCR frequencies were calculated as the fraction of samples bearing the same aberration contained in the MCR without regard to its MCR type. MCRs containing genes encoding the immunoglobulin heavy chain (IGHV) genes and the kappa and lambda light chains were discarded since CN changes in these regions probably represent physiological rearrangements occurring in B-cells. Validation of arrayCGH data DNA losses at 8p, and DNA gains at 2p and 8q were validated by FISH and by genomic real-time polymerase chain reaction (PCR). FISH analysis was performed by staining the nuclei of peripheral white blood cells used for CLL diagnosis. Nuclei were counterstained with 4¢,6¢-diamidino-2-phenylindole (DAPI) and antifade, and signals were visualized using an Olympus BX51 microscope (Olympus Italia, Milan, Italy). For each probe, at least 400 interphase cells with well-delineated fluorescent spots were examined. Validation by FISH of gains at 2p was performed using an LSI MYCN probe (Abbott, Rome, Italy), as previously described (Deambrogi et al, 2010). Validation by FISH of gains at 8q was performed using LSI IGHV/MYC/CEP8 and MYC break-apart probes (Abbott). Depending on the extent of the deletions and on the availability of appropriate biological material, validation of losses affecting 8p was done by FISH using the LSI LPL probe (Abbott) (8p21.3) or by real-time PCR on genomic DNA using the TaqMan Copy Number Assay Hs03275475_cn (Applied Genomic Lesions and Outcome in CLL ª 2011 Blackwell Publishing Ltd, British Journal of Haematology, 154, 590–599 591 Biosystems, Rotkreuz, Switzerland) (8p22). Real-time PCR reactions were run in quadruplicate in 96-well plates with 5 ng of DNA per reaction in a StepOnePlus instrument and analysed with CopyCaller software v.1.0 (Applied Biosystems, Carlsbad, CA, USA). Statistical analyses Overall survival (OS) was measured from the date of diagnosis to the last follow-up or death by any cause. Univariate analysis was performed with the log-rank test in order to evaluate the impact of the genetic aberrations on OS. The Benjamini- Hochberg-method (q-value) was used to control false positive rate. MCR showing an unadjusted P < 0Æ05 and a q-value < 0Æ10 and occurring in at least five cases in the test series underwent univariate analysis in an independent validation series. For MCR residing in a larger type of MCR (e.g., a SRA inside a LRA), with both MCR satisfying the significance criteria in the test series, only the smaller type of MCR was evaluated in the validation series, and patients carrying a lesion contained in both a SRA and in a LRA were counted for both types of MCR (Scandurra et al, 2010a; Rinaldi et al, 2011). MCRs with a P < 0Æ05 and a q-value < 0Æ1 in the validation series were considered as statistically significant. As the samples of the validation set had not been used to calculate MCR, each sample was classified as bearing or not bearing the MCR selected in the test series: an overlap of >85% of the probes reporting the specific aberration was used for SRA and LRA types, an overlap of >60% for ACA and AWC types. To identify MCR with an independent impact on survival, a Cox proportional hazard regression was applied with backward stepwise selection algorithm. The multivariate analysis was performed on the whole population of patients to maximize the statistical power (Janoueix-Lerosey et al, 2009). A hierarchical model for OS was built based on genetic features. Patients were classified into different genetic groups sorted by hazard ratios as obtained with a Cox regression analysis including the standard karyotype groups (Dohner et al, 2000) plus the presence of the novel parameters significant by multivariate analysis. Associations in two way tables were tested for statistical significance using either the v2 test or Fisher exact test (two-tailed), as appropriate. Statistical analyses were performed with Statistical Package for the Social Sciences software v.17.0.2 (SPSS, Chicago, IL, USA) and R statistical package (version 2.11.1) (R Development Core Team 2009). P values < 0Æ05 were considered statistically significant. Results DNA copy number profiles of two independent sets of CLL identifies genomic aberrations with an impact on outcome To identify DNA CN changes affecting the outcome of newly diagnosed CLL patients, we followed a test-validation approach (Fig S1). Recurrent genomic lesions were first identified across a test series of 158 CLL patients from a single institution and tested for their prognostic significance on the 147 cases with follow-up data. The statistically significant lesions were then evaluated in an independent validation group of 176 consecutive CLL patients, collected from three additional institutions. The most important clinical characteristics at the time of diagnosis were compa- rable in both series and reflected the general CLL population (Table I). Also, no differences could be observed in terms of the percentage of patients needing treatment (81/147, 55%, vs. 100/176, 57%, P = n.s.) or for patients receiving a fludarabine-containing regimen (51/147, 35%, vs. 51/176, 29%, P = n.s.). Three hundred and sixty-seven MCRs were evaluated for their prognostic impact on OS in the test series (Fig S2; Table SI). Univariate analysis followed by multiple test correction identified 14 MCRs with a statistically significant impact on OS. Eight MCRs confirmed the prognostic impact on OS in the validation group (Table II): 2p25.3-p22.3 gains (MYCN), 2p22.3 gains, and 2p16.2-p14 (REL) gains, del(8p23.1-p21.2), 8p21.2 loss, 8q23.3-q24.3 gains (MYC), and, as expected (Rossi et al, 2009a; Zenz et al, 2010), losses of the TP53 region [del(17p13.3-p11.2), del(17p12-p11.2)]. Lesions detected by arrayCGH at 2p, 8q were confirmed by FISH in 7/7 cases tested: gains affecting 2p and 8q were validated using locus-specific probes for MYCN in five cases and for MYC in two cases, respectively. Deletions affecting 8p were confirmed in 12/12 cases tested: by FISH using a commercially available probe targeting 8p21.3 in four cases and by real-time PCR using primers for 8p22 in eight cases. In one of the patients bearing these lesions, it was possible to show that gains affecting 2p were of somatic origin, not being detected in the matched normal DNA analysed by arrayCGH. Table I. Biological and clinical characteristics of the test CLL series and of the validation CLL series. Test series Validation series P-value Median follow-up 60 months 61 months n.s. Median age (range), years 71 (43–92) 62 (21–94) <0Æ001 No. (%) No. (%) Age >60 years 125/147 (85) 112/176 (64) <0Æ001 Gender, male 83/147 (56) 110/176 (62) n.s. Binet, stage A 107/147 (73) 126/175 (72) n.s. Mutated IGHV genes 91/147 (62) 111/176 (63) n.s. Favourable FISH panel [del(13q14Æ3)/normal/+12] 112/142 (79) 140/169 (82) n.s. Unfavourable FISH panel [del(11q22-q23)/del(17p13)] 30/142 (21) 29/169 (17) n.s. Mutated TP53 16/142 (11) 5/32 (16) n.s. CD38, >30% 44/147 (30) 69/168 (41) 0Æ04 ZAP70, > 20% 36/129 (28) 67/146 (46) 0Æ02 ns, not significant; FISH, fluorescence in situ hybridization. A. Rinaldi et al 592 ª 2011 Blackwell Publishing Ltd, British Journal of Haematology, 154, 590–599 Gains at chromosome 2p and 8q are independent prognostic markers Due to the relatively low frequency of the lesions on 2p and on chromosome 8, and because both clinical series were compa- rable regarding their clinical characteristics, we pooled the two series together to increase statistical power. The lesions showing the highest level of statistical significance at univariate analysis, namely del(8p23.1-p21.2), gains at 2p16.2-p14 and 8q23.3-q24.3 (Fig 1, Table II) were evaluated for their inde- pendent prognostic significance in a Cox regression model adjusted for age, gender, Binet stage, IGHV mutational status, and TP53 inactivation (Table III). Gains occurring at 2p16.2- p14 and 8q23.3-q24.3 maintained their independent prognos- tic significance on OS. The two lesions also remained significant in a model including age, gender, Binet stage, CD38 + >30%, ZAP70 > 20%, del(8p23.1-p21.2), TP53 inac- tivation, and the presence, as detected by FISH, of del(13q14.3), del(11q22-q23), and trisomy 12. Gains at 2p, gains at 8q and losses at 8p are associated with different clinical and biological features Patients with 2p gains presented significantly more often with unmutated IGHV genes (85% vs. 35%; P < 0Æ001), increased serum lactate dehydrogenase (LDH; 50% vs. 21%; P = 0Æ030) and a higher rate of transformation to Richter syndrome (22% vs. 5%; P = 0Æ021). The presence of 2p gains was associated with concomitant del(13q14.3) (MIR15A/MIR16-1) (77% vs. 48%; P = 0Æ027), del(11q23) (ATM) loss (21% vs. 6%; P = 0Æ027), and TP53 inactivation (31% vs. 11%; P = 0Æ032). None of the cases with gains at 2p had concomitant trisomy 12 (P > 0Æ05). The presence of 8q23.3-q24.3 gains was significantly more frequent in patients with del(17p13) (TP53) (44% vs. 5%; P = 0Æ001), del(8p) (44% vs. 2%; P < 0Æ001), gains of the long arm of chromosome 3 (44% vs. 3%; P < 0Æ001) and inversely associated with ZAP70 positivity (0% vs. 38%; P = 0Æ038). Del(13q14.3), del(11q23) and trisomy 12 were equally distrib- uted in patients with and without 8q gains. Deletions at 8p were associated with 8q23.3-q24.3 gains (MYC) (40% vs. 1%; P < 0Æ001) and del(17p13) (TP53) (80% vs. 10%; P < 0Æ001). Trisomy 12 and del(13q14.3) (MIR15A/ MIR16-1) occurred at the same frequency in patients with and without 8p deletions, whilst del(11q23) (ATM) was never observed in patients with del(8p21). Gains at 2p, gains at 8q and losses at 8p better define CLL prognostic groups The impact of the detection of lesions on chromosomes 2 and 8 was evaluated within the CLL prognostic groups defined by IGHV mutational status, Binet stage, age and FISH. Table IV shows the prognostic groups that were better defined by the demonstration of additional genomic lesions. Gains at 8q23.3-q24.3 identified patients with a different outcome amongst those older than 60 years, in Binet stage A, with unmutated or mutated IGHV genes, or with both favourable and unfavourable FISH (Fig S3). Similarly, the presence of 8p deletions was a prognosticator in CLL with Binet stage A, age older than 60, unmutated or mutated IGHV genes, and an unfavourable FISH (Fig S4). The presence of gains affecting 2p identified groups with poorer outcome amongst patients younger or older than 60, with Binet stage A, or mutated IGHV genes, or with both favourable and unfavourable FISH (Fig S5). Resistance to alkylating agents was observed in 3/3 patients with gains at 8q23.3-q24.3 (two with concomitant TP53 inactivation), in 2/2 with 8p deletions (one with concomitant TP53 inactivation) and in 6/8 with gains affecting 2p (two with concomitant TP53 inactivation). Fludarabine-resistance was observed in 3/4 patients with gains at 8q23.3-q24.3 (two with concomitant TP53 inactivation), in 4/6 with 8p deletions (all with concomitant TP53 inactivation) and in 3/4 with gains affecting 2p (one with concomitant TP53 inactivation). A hierarchical model (Fig 2) was built including gains at 2p or 8q and the genetic subgroups according to Dohner et al (2000), assigning patients to the worst prognostic group on the basis of the Cox regression analysis (Table SII). Table II. MCR with a significant impact on OS in the test CLL series, the validation CLL series and in the pooled CLL population. Lesion Start Size (bp) % Test series (n = 147) Validation series (n = 176) All (n