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Array CGH analysis reveals deletion of chromosome 22q11 in CLL with normal karyotype and no fish alterations

2017; Wiley; Volume: 183; Issue: 1 Linguagem: Inglês

10.1111/bjh.14949

1365-2141

Francesca Mestichelli, Alessia Dalsass, Silvia Ferretti, Elisa Camaioni, Mario Angelini, Sabrina Mei, Valerio Pezzoni, Fosco Travaglini, Emanuela Troiani, Stefano Angelini, Piero Galieni,

Glycosylation and Glycoproteins Research

Chronic lymphocytic leukaemia (CLL) has a variable clinical course. The presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes (Kujawski et al, 2008). Chromosomal aberrations examined by classical karyotype analysis or Fluorescent In Situ Hybridization (FISH) can be detected in ≥80% of CLL patients. Array Comparative Genomic Hybridization (aCGH) enabled the identification of other genomic aberrations in CLL cells with potential pathogenetic relevance (Mraz et al, 2013). Genomic aberrations have increasingly gained importance as prognostic markers in CLL (Döhner et al, 2000). Recently, a number of additional studies have clearly demonstrated the feasibility of using aCGH as a clinical tool to identify genomic alterations of prognostic importance in CLL (Gunn et al, 2008; Higgins et al, 2008). aCGH is able to identify a significant percentage of genomic abnormalities that escapes conventional cytogenetics and testing with a CLL FISH panel due to the limitations of these methods (Urbankova et al, 2014). However, approximately half of the 20% of cases found to be normal by aCGH, will have clonal cell populations below the 30% detection limit of this method (Gunn et al, 2008) A total of 174 diagnostic blood samples from patients with CLL were received for karyotype and FISH analysis at the U.O.C of Hematology of the C.G Mazzoni Hospital of Ascoli Piceno. CLL diagnosis was based on standard immunophenotypic criteria. Of the 174 cases, 23 (13%) showed a normal karyotype at cytogenetic analysis and no FISH alterations. The cytogenetic study was performed on nuclei and metaphases after stimulation with a combination of CpG-oligonucleotide DSP30 and interleukin 2 (IL-2) (Dicker et al, 2006). Chromosome Banding Analysis (CBA) was used to analyse a minimum of 20 metaphases for each patient. Interphase FISH was performed using a comprehensive set of commercially available probes, as follows: TP53 Deletion (17p13), ATM Deletion (11q22), D13S25 (RB1) Deletion (13q14·3), MYB Deletion (6q23) (Cytocell, Cambridge, UK), CEP-12 (Chromosome 12) (Abbott, Chicago, IL, USA), XL IGH plus Break Apart Probe (MetaSystems, Milan, Italy). A minimum of 100–200 interphase nuclei were evaluated per probe for each patient. Using Oligonucleotide-based aCGH we detected copy number changes in the tumour genomes of 23 CLL cases. Total DNA isolated from CLL samples was fragmented, labelled and hybridized to aCGH (CytoChipCancer 4x180K-Illumina, San Diego, CA, USA) according to the manufacturer's instructions and compared to human reference DNA. Each array was scanned by a DNA microarray scanner (Innopsys, Chicago, IL, USA). Data were subsequently analysed by BluFuse Multi V4·0 (Illumina, San Diego, CA, USA). All aberration-identified regions were checked manually and loss/gain of at least 3 probes was necessary to consider a region as aberrant. Of the 23 cases, 21 (91%) were successfully analysed by Oligonucleotide-based aCGH. We observed a submicroscopic deletion of chromosome 22q11 as the sole anomaly in 4/21 cases (19%). The clinical and biological characteristics of enrolled patients are summarized in Table 1. All patients with loss of 22q11 showed progressive disease and required treatment; the median time to treatment (TT) was 64·2 months. Howwever, 6 out of 17 cases (35%) without loss of 22q11 were also treated (TT: 38 months). Loss of 22q11 ranged in size from 0·68 Mb–0·49 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2 and PRAME genes. Of these four genes, only ZNF280A, ZNF280B and PRAME showed statistically significant difference in expression in del(22)(q11) cases compared to the non-deleted cases (Gunn et al, 2009). ZNF280A and ZNF280B encode closely related zinc finger proteins of 542 and 543 amino acids, respectively. To date, there is no direct evidence linking these two zinc finger proteins with human cancers. However, it is conceivable that alterations in the copy number of these genes could have a significant effect on gene regulation in CLL and, possibly, other human cancers (Gunn et al, 2009). PRAME encodes a 509-amino acid membrane and cytoplasm-associated protein that was first described as a tumour antigen in melanoma, which triggered autologous cytotoxic T-cell-mediated immune responses (Proto-Siqueira et al, 2006; Gunn et al, 2009). The authors suggested that the PRAME gene is the candidate tumour-suppressor localized in 22q11 (Gunn et al, 2009), because it was previously associated with the biology and aggressiveness of both solid tumours and myeloid haematological malignancies (Steinbach et al, 2002). Moreover, this genomic change had not been detected by standard cytogenetic and/or FISH analyses. Gunn et al (2009) suggested that the incidence of 22q11 deletions was second only to loss of the 13q14 region, found in approximately 50% of CLL cases; few and divergent data have been reported in literature regarding the prognostic impact of this genomic abnormality in CLL (Gunn et al, 2009; Mraz et al, 2013). Our results showed that a submicroscopic 22q11 deletion is a potentially significant genomic aberration in CLL and that this alteration is missed by current routine techniques. The number of analysed cases needs to be increased in order to confirm the data obtained and to correlate this alteration to the clinical course, so that the prognostic significance of these gene deletions can be evaluated and to determine how these alterations contribute to the pathogenesis of CLL. FM and AD equally contributed to this work. FM and AD contributed to the conception and design of the study by performing the majority of the experiments, analysing and interpreting data and writing the article. AD, FM, EC and SF performed the cytogenetic studies; VP and FT performed flow cytometry analysis; MA and SM performed the molecular biology studies; SA performed the statistical analysis; ET, and PG performed the clinical analysis and follow-up of the patients. PG supervised data collection and the clinical management of patients. The authors have no competing interests.