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RESEARCH ARTICLE
Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: A systematic reappraisal of classic cytogenetic data Panagiotis Baliakas,1,2 Michalis Iskas,1 Anne Gardiner,3 Zadie Davis,3 Karla Plevova,4 Florence Nguyen-Khac,5 Jitka Malcikova,4 Achilles Anagnostopoulos,1 Sharron Glide,3 Sarah Mould,3 Kristina Stepanovska,4 Martin Brejcha,6 Chrysoula Belessi,7 Frederic Davi,5 Sarka Pospisilova,4 Anastasia Athanasiadou,1 Kostas Stamatopoulos,1,2,8* and David Oscier3 The significance of chromosomal translocations (CTRAs) and karyotype complexity (KC) in chronic lymphocytic leukemia (CLL) remains uncertain. To gain insight into these issues, we evaluated a series of 1001 CLL cases with reliable classic cytogenetic data obtained within 6 months from diagnosis before any treatment. Overall, 320 cases were found to carry 1 CTRAs. The most frequent chromosome breakpoints were 13q, followed by 14q, 18q, 17q, and 17p; notably, CTRAs involving chromosome 13q showed a wide spectrum of translocation partners. KC (3 aberrations) was detected in 157 cases and significantly (P < 0.005) associated with unmutated IGHV genes and aberrations of chromosome 17p. Furthermore, it was identified as an independent prognostic factor for shorter time-to-first-treatment. CTRAs were assigned to two categories (i) CTRAs present in the context of KC, often with involvement of chromosome 17p aberrations, occurring mostly in CLL with unmutated IGHV genes; in such cases, we found that KC rather than the presence of CTRAs per se negatively impacts on survival; (ii) CTRAs in cases without KC, having limited if any impact on survival. On this evidence, we propose that all CTRAs in CLL are not equivalent but rather develop by different processes and are associated with distinct clonal behavior. C 2013 Wiley Periodicals, Inc. Am. J. Hematol. 89:249–255, 2014. V
䊏 Introduction Chronic lymphocytic leukemia (CLL) exhibits remarkable clinical heterogeneity that is linked to and likely reflects the underlying biological and genetic heterogeneity [1,2]. Therefore, not paradoxically, great efforts have been made towards the identification of biological markers that predict the tendency for disease progression at the time of diagnosis, thus assisting in accurate risk stratification and rational treatment design [3–5]. Amongst a plethora of markers proven to be prognostically relevant in CLL, perhaps the most powerful and widely used relate to the patients’ cytogenetic profile [3–7]. A turning point in CLL biology and prognostication came with the realization that >80% of cases carry gross cytogenetic alterations detected by fluorescence in situ hybridization (FISH) analysis and, more importantly, that as few as four lesions, namely deletions of chromosomes 11q, 13q, and 17p; and trisomy of chromosome 12 can be used to create a robust prognostic algorithm [8]. For instance, patients carrying chromosome 17p deletions leading to dysregulated p53 function have significantly worse outcomes compared to the cases with isolated chromosome 13q deletion. Subsequently, mutations of the TP53 gene were also shown to associate with clinical aggressiveness and chemorefractoriness [9,10]. For this reason, screening for del(17p) and TP53 gene mutations is strongly recommended before treatment initiation for both clinical trials and, importantly, standard clinical practice [11]. Very recently, the genetic risk stratification algorithm of CLL has been refined by the integration of FISH findings with mutational analysis for genes found to be recurrently mutated in CLL by high-throughput sequencing approaches, in particular, NOTCH1, SF3B1, and BIRC3 [12]. The pace at which novel mutations are identified is ever-increasing, thus underscoring the fact that the underlying genetic complexity of CLL is high and still incompletely characterized [13–15].
Additional Supporting Information may be found in the online version of this article. 1
Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece; 2Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; 3Department of Haematology, Royal Bournemouth Hospital, Bournemouth, United Kingdom; 4Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 5Hematology Department and University Pierre et Marie Curie, H^opital Pitie-Salpe`trie`re, Paris, France; 6Department of Hematology, J.G. Mendel Cancer Center Novy Jicin, Czech Republic; 7Hematology Department, Nikea General Hospital, Piraeus, Greece; 8Institute of Applied Biosciences, CERTH, Thessaloniki, Greece
Conflict of interest: Nothing to report. *Correspondence to: Kostas Stamatopoulos. Institute of Applied Biosciences, Center for Research and Technology Hellas, 57001 Thessaloniki, Greece. E-mail:
[email protected] Received for publication: 19 September 2013; Revised: 14 October 2013; Accepted: 21 October 2013 Am. J. Hematol. 89:249–255, 2014. Published online: 26 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.23618 C 2013 Wiley Periodicals, Inc. V
doi:10.1002/ajh.23618
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Baliakas et al.
With hindsight, evidence for the turbulent genetic landscape of CLL was already available by classic cytogenetic studies from the early 1990s [6,16–18]. Here it should be noted that, though considerably more laborious and hence superseded by FISH for routine diagnostics, classic cytogenetic analysis offers the opportunity to globally assess the karyotype of the malignant clone that is beyond the scope of FISH screening with probes for selected chromosomal regions [19– 22]. Indeed, such studies have revealed various numerical and structural aberrations as well as karyotype complexity (KC) that go unrecognized by the commonly used FISH approaches. Importantly, these findings have been confirmed by more recent studies using new high resolution technologies [23–28]. Indeed, genomic complexity has been identified as an adverse prognostic indicator, however, the mechanisms underlying the poor outcome associated with complexity remain undefined. In contrast to other B-cell malignancies, CLL is generally considered as conspicuous for the lack of recurrent chromosomal translocations (CTRAs). Against this, CTRAs have been reported in all classic cytogenetic studies, although their true frequency and significance are still a matter of debate [6,16–18,29–31]. Factors confounding the interpretation of published classic cytogenetic results relate to different cohort sizes, possible selection biases, methodological discrepancies between studies, as well as the timing of the analysis, i.e., whether it was conducted at diagnosis or at later time-points during the course of the disease, thus potentially reflecting clonal evolution [29–31]. Furthermore, limited data exists regarding correlations of certain types of CTRAs with other biological parameters, including KC, as well as clinical course and eventual outcome. For instance, observations that CTRAs per se are associated with a poor outcome [29] have not been confirmed by other studies [30]. Finally, although newer technologies, especially arrays, have led to the identification of novel copy number alterations and refined regions of loss/amplification, only paired-end sequencing, RNA sequencing, and translocation capture sequencing have the capacity to detect translocations [32–34] and little data in CLL exists using these techniques. In order to address the biological and clinical significance of CTRAs and their relationship to genomic complexity in CLL, we systematically investigated a large multi-institutional cohort with available classic cytogenetic data. We document that all CTRAs in CLL are not equivalent but rather they can be assigned to two broad categories. In particular, CTRAs present in the context of KC, often with involvement of chromosome 17p aberrations, occur mostly albeit not exclusively in CLL with unmutated IGHV genes; in such cases, KC rather than the presence of CTRAs per se negatively impacts on survival. In contrast, CTRAs in cases without KC have limited, if any, impact on survival.
䊏 Patients and Methods Patient group The study group included 1,001 patients with CLL from collaborating institutions in Czech Republic, France, Greece, and the UK. The diagnosis of CLL was established according to the guidelines of the International Workshop Chronic Lymphocytic Leukemia/National Cancer Institute (IWCLL/NCI) [1]. Classic cytogenetic analysis was performed either at diagnosis or within 6 months from diagnosis and before the initiation of any treatment (median time from diagnosis to karyotype analysis: 1.6 months). The study was approved by the local Ethics Review Committee of the participating institutions. Demographic, clinical, and biological data for the patient cohort are summarized in Supporting Information Table I.
Classic cytogenetic and FISH analysis For metaphase induction, 106 per mL peripheral blood mononuclear cells were cultured using two different protocols. In particular, until 2008, cells were cultured for 72 and 96 hr in Roswell Park Memorial Institute (RPMI) 1640 medium with 20%
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fetal calf serum with phorbol-12-myristate-13-acetate (TPA) at 50 ng/mL; colcemid (0.01 lg/ml) was added 45 min before harvest. Since 2008, metaphases were also obtained after cell culture in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and interleukin 2 (IL-2) (200 U/mL); after 72 hr, colcemid (0.015 lg/ml), was added for another 4 hr before chromosome preparation. Hypotonic treatment was with 0.075 M KCl, fixation of chromosomes was accomplished with 3v methanol:1v glacial acetic acid solution. Chromosome preparation and staining was done according to standard protocols. A minimum of 20 mitotic cells were examined. Karyotypes were classified according to the International System for Human Cytogenetic Nomenclature (ISCN) 2009. Karyotypes obtained before 2009 were re-classified following ISCN 2009 [35]. Interphase FISH analysis was conducted using probes for those cytogenetic regions commonly abnormal in CLL. Specifically probes co-hybridized for (del)11q23 were LSI ATM and CEP11; for del(13q14), D13S319 and LSI13q34; for del(17p13), LSI TP53 and CEP17. For trisomy 12, CEP12 was hybridized alone. All probes were from Vysis (Downers Grove, IL) and used in accordance with the manufacturer’s recommended procedures. Preparations were counterstained with 4,6-diamidino-phenyl-indole (DAPI) and a minimum of 200 interphase nuclei were examined.
Definitions Translocations were characterized as: (i) balanced, when the exchange of genetic material resulted in no apparent gain or loss of genetic material; and, (ii) unbalanced when the exchange of chromosome material was unequal, resulting in extra or missing genetic material. A karyotype was defined as complex when 3 chromosomal aberrations were observed (structural and/or numerical). Cases carrying a certain chromosomal aberration detected either by FISH or classic cytogenetic analysis were considered positive regarding the involvement of this particular aberration.
PCR amplification, sequence analysis and sequence interpretation of IGHV–IGHD–IGHJ rearrangements Amplification of IGHV–IGHD–IGHJ rearrangements was performed as previously described [36,37]. Sequence data were analyzed using the international IMGTV database [38] and the IMGT/V-QUEST tool [39] (http://www.imgt.org). R
Statistical analysis Descriptive statistics were used for the presentation of data in terms of frequency distributions (discrete variables) and mean, median values (quantitative variables). Time-to-first-treatment (TTFT) was measured from the date of diagnosis to the date of first treatment. Survival curves were plotted using the Kaplan–Meier method. Bivariate differences in survival distributions were studied with the use of Log-rank test. Multivariate Cox regression models were implemented for the study of the simultaneous effect of factors on survival outcomes taking into account the
TABLE I. Clinicobiogical features of cases carrying chromosomal translocations (CTRAs) versus those cases without CTRAs Cases with CTRAs, n (%) 320
Cases without CTRAs, n (%) 681
Clinical stage A B C Not available
197 (82%) 26 (11%) 18 (7%) 79
526 (84%) 72 (11%) 27 (5%) 56
ns ns
Gender Male Female
204 (64%) 116 (36%)
450 (66%) 231 (34%)
ns ns
IGHV genes Mutated Unmutated Not available
90 (63%) 52 (37%) 178
357 (64%) 204 (36%) 120
ns ns
Karyotype complexity 3 Abnormalities 5 Abnormalities
113 (35%) 33 (10%)
44 (6.5%) 7 (1%)
